Discovery and analgesic evaluation of 8-chloro-1,4-dihydropyrido[2,3- b ]pyrazine-2,3-dione as a novel potent d -amino acid oxidase inhibitor

Article abstract of DOI:10.1016/j.ejmech.2016.04.017

A series of 5-azaquinoxaline-2,3-dione derivatives were synthesized and evaluated on d-amino acid oxidase (DAAO) inhibition as potential α-hydroxylactam-based inhibitors. The potent inhibitory activities in vitro suggested that 5-nitrogen could significantly enhance the binding affinity by strengthening relevant hydrogen bond interactions. The analgesic effects of intrathecal and systemic injection of 8-chloro-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione, a representative molecule of 5-azaquinoxaline-2,3-dione, were investigated in rodents. This research not only confirmed the analgesic effect of the DAAO inhibitors but provided a new class of chemical entities with oral application potential for the treatment of chronic pain and morphine analgesic tolerance.

Full text of DOI:10.1016/j.ejmech.2016.04.017

European Journal of Medicinal Chemistry 117 (2016) 19e32
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery and analgesic evaluation of 8-chloro-1,4-dihydropyrido[2,3-
b]pyrazine-2,3-dione as a novel potent -amino acid oxidase inhibitor
D
Dongsheng Xie 1, Jun Lu 1, Jin Xie, Junjun Cui, Teng-Fei Li, Yan-Chao Wang, Yuan Chen,
Nian Gong, Xin-Yan Li, Lei Fu*, Yong-Xiang Wang**
School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 5-azaquinoxaline-2,3-dione derivatives were synthesized and evaluated on D-amino acid
Received 3 September 2014
Received in revised form
5 April 2016
Accepted 6 April 2016
Available online 9 April 2016
oxidase (DAAO) inhibition as potential a-hydroxylactam-based inhibitors. The potent inhibitory activities
in vitro suggested that 5-nitrogen could significantly enhance the binding affinity by strengthening
relevant hydrogen bond interactions. The analgesic effects of intrathecal and systemic injection of 8-
chloro-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione, a representative molecule of 5-azaquinoxaline-2,3-
dione, were investigated in rodents. This research not only confirmed the analgesic effect of the DAAO
inhibitors but provided a new class of chemical entities with oral application potential for the treatment
of chronic pain and morphine analgesic tolerance.
Keywords:
D-amino acid oxidase
DAAO inhibitors
© 2016 Elsevier Masson SAS. All rights reserved.
5-Azaquinoxaline-2,3-diones
Analgesic effects
8-Chloro-1,4-dihydropyrido[2,3-b]pyrazine-
2,3-dione
1. Introduction
Furthermore, DAAO inhibition has also been implicated with
morphine analgesic tolerance [25,26] and hyperalgesia [30]. DAAO
D
-serine has been demonstrated to be antipsychotic by acti-
inhibitors thus represent a new drug discovery opportunity for the
treatment of chronic pain and morphine analgesic tolerance. In
search of potent DAAO inhibitors with novel structures for phar-
macological research on pains, herein we reported the synthesis
and evaluation of a series of 5-azaquinoxaline-2,3-dione de-
rivatives as a new family of noncarboxylic DAAO inhibitors.
vating the glycine site of the N-methyl-D-aspartic acid (NMDA)
receptor in the central nervous system [1e4]. However, high doses
of -serine required for clinical efficacy may cause nephrotoxicity,
which is generated by -amino acid oxidase (DAAO)-mediated
metabolism of -serine in kidneys [5e7]. Inhibition of peripheral
DAAO may be an effective means of delivering -serine to the brain
D
D
D
D
As
potential
a
-hydroxylactam-based
inhibitors,
5-
[8e10]. Due to the promising therapeutic application in schizo-
phrenia, great interest has been aroused in discovery of novel and
potent DAAO inhibitor molecules [8,11e17]. In addition, researchers
azaquinoxaline-2,3-diones exhibited significantly improved po-
tency (IC50 ¼ 100e500 nM for 10 derivatives) over our previously
reported quinoxaline-2,3-diones [31]. The newly synthesized
compound 16 (8-chloro-1,4-dihydropyrido[2,3-b]pyrazine-2,3-
dione) with higher potency (IC50 ¼ 100e200 nM against porcine,
rat and human DAAO) dose-dependently relieved formalin-
induced tonic pain, effectively blocked spinal nerve ligation
induced mechanical allodynia and heat hyperalgesia, and signifi-
cantly prevented and reversed morphine analgesic tolerance in
rodents.
have also investigated the roles of D-serine and DAAO in painful
responses [18e22]. In recent years, the analgesic effects of the
structurally different DAAO inhibitors have been reported in animal
models such as formalin-induced tonic pain [21e26], neuropathic
pain [27,28] and bone cancer pain [29], with a proposed mechanism
related to the inhibition of spinal hydrogen peroxide [24,25].
2. Chemistry
* Corresponding author.
** Corresponding author.
5-Azaquinoxaline-2,3-dione derivatives were prepared from
appropriate 2,3-diaminopyridines by condensation with diethyl
E-mail addresses: leifu@sjtu.edu.cn (L. Fu), yxwang@sjtu.edu.cn (Y.-X. Wang).
1
The two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2016.04.017
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

20
D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
oxalate [32]. However, most 2,3-diaminopyridines needed to be
synthesized due to their commercial limitation. The general syn-
thesis procedure for 5-azaquinoxaline-2,3-diones was outlined in
Scheme 1. 2-Aminopyridines (a) without protection of the amino
could be nitrified, and rearrange in nitro-sulfuric acid to give 2-
amino-3-nitropyridines (c) and 5-nitroisomers at room tempera-
ture or above. Nevertheless, in most cases, the intermediates (b)
which is readily isolated in early stage of the nitration at low
temperature (ꢀ5 to 0 ꢁC), could be more effectively and completely
converted to nitropyridines just in concentrated sulfuric acid.
When 5-position of 2-aminopyridines was occupied, the 3-
nitroisomer products were exclusively obtained. Otherwise, 3-
nitroisomers had to be separated from mixed isomers in various
yields from 2% to 50%. 2-Amino-3-nitropyridines (c) were chemi-
cally reduced with stannous chloride or zinc powder/acetic acid, or
hydrogenated with Pd/C to give 40e100% yield of 2,3-
diaminopyridines (d), which are unstable during long exposure to
air and light. They should be taken into next reaction without pu-
rification or immediately once purified after column chromatog-
raphy. The cyclization of 2,3-diaminopyridines were carried out in
excess of diethyl oxalate under reflux to afford 40e70% yield of 5-
azaquinoxaline-2,3-diones (e) as grey to brown solid. To avoid the
data deviation in enzyme assay, the colored solid products must be
strictly purified by first decolourization with activated charcoal and
then recrystallization in water and DMF or DMSO.
Six and 8-alkoxyl-5-azaquinoxaline-2,3-dione derivatives’
preparation was illustrated in Scheme 2. 2-Amino-3-nitro-6-
chloropyridine, an intermediate for compound 8, was first con-
verted to 6-alkyloxy-2-amino-3-nitropyridines by treatment in
DMF with corresponding sodium alkoxides, which could also be
produced in situ using corresponding alcohols and sodium hydride.
Next steps followed the general procedure in Scheme 1 to give 6-
alkoxyl-5-azaquinoxaline-2,3-dione derivatives. In the same way,
8-alkoxyl derivatives were prepared from 2-amino-3-nitro-4-
chloropyridine, an intermediate in preparation of compound 16.
7,8-Dihalogen-5-azaquinoxaline-2,3-dione derivatives were
prepared from 2-amino-4-halogenpyridine (Scheme 3). Halogena-
tions were carried out in CH3CN or DMF using NCS or NBS, and
60e80% yield of 2-amino-4,5-dihalogenpyridines were obtained by
column chromatography [33,34]. The next nitration and rear-
rangement were carried out in one pot. Zinc powder in acetic acid
was employed to reduce 2-amino-3-nitropyridine intermediates,
otherwise, hydrogenation by Pd/C or even stannous chloride led to
seriously dehalogenated products. Finally, the condensation of 2,3-
diamino-4,5-dihalogenpyridine and diethyl oxalate were carried
out under reflux for 6 h to give 7,8-dihalogen-5-azaquinoxaline-
2,3-diones.
3. Results and discussion
3.1. Discovery of 5-azaquinoxaline-2,3-diones as novel
acid oxidase inhibitors
D-amino
As noncarboxylic DAAO inhibitors 3-hydroxyquinolin-2-one
[14] and newly discovered scaffolds in recent years, such as 1-
hydroxybenzoimidazol-2-one [15], 3-hydroxypyridin-2-one [16],
and 4-hydroxypyridazin-3-one [16] shared
a
common
pharmacophored -hydroxylactam, by which a hydrogen bond
a
(HB) net with DAAO residues including Arg283, Tyr 228 and Gly313
are constructed (Fig. 1).
Due to the possible amide-iminol tautomerism, we reported
that quinoxaline-2,3-dione and its derivatives were potential
hydroxylactam-based DAAO inhibitors [31]. Docking simulation
demonstrated that quinoxaline-2,3-dione could bind to the active
site of DAAO in a similar manner to that of 3-hydroxyquinolin-2-
one in crystalline complex (Fig. 2A), which suggested that
bonlactam moiety could also function as -hydroxylactam one,
a-
a-car-
a
constructing corresponding HB net with DAAO. Regrettably, vary-
ing substitution on the benzene ring of quinoxaline-2,3-dione
endowed these molecules with moderate DAAO inhibition
(IC50 > 5
azaquinoxaline-2,3-dione
dihydropyrido[2,3-b]pyrazine-2,3-dione exhibited marked DAAO
M). The improved potency of this
m
M) [31]. To our surprise, in random screening, 5-
derivative, i.e., 8-methyl-1,4-
inhibitory property (IC50 ¼ 0.63
m
5-azaquinoxaline-2,3-dione derivative seemed hardly to be
explained only by molecular docking simulation because of its
similarity to quinoxaline-2,3-dione in binding mode (Fig. 2B). We
proposed that the weak potencies of quinoxaline-2,3-diones may
be caused by the similar enolizability of di-lactam, and introduction
of 5-nitrogen may be able to significantly break the similar enoli-
zation state of di-lactam and make it more favorable to form a-
hydroxylactam pharmacophore. Furthermore, 4-amino (ortho po-
sition of pyridine) owing to the increased bond polarity may prefer
to donate its hydrogen atom and form a stronger HB with carbonyl
group of Gly313. Thus, 5-azaquinoxaline-2,3-dione derivatives are
much likely to be a novel family of the potent DAAO inhibitors.
As expected, the inhibitory activities of 5-azaquinoxaline-2,3-
diones on porcine kidney DAAO (pkDAAO) are widely superior to
the corresponding quinoxaline-2,3-diones (Table 1). Some newly
synthesized
derivatives
exhibited
potent
efficacy
(IC50 ¼ 100e500 nM) in vitro (compounds 7, 13, 16, 17, 27e29 and
31e33). Limited by the restricted space around DAAO active site,
small substituents such as methyl, trifluoromethyl and halogen
atoms are favorable for potent inhibitory activity. Larger groups
than methyl even if ethyl or methoxyl are detrimental to the
Scheme 1. General synthesis route of 5-azaquinoxalinedinones. Reagents and conditions: (i). 98% H2SO4, fuming HNO3, ꢀ5 to 50 ꢁC, 3 h; (ii). 98% H2SO4, fuming HNO3, ꢀ5 to 0 ꢁC,
15 min; (iii). 98% H2SO4, 0e50 ꢁC, 2 h; (iv). H2, 10% PdeC, EtOAc/MeOH (V/V ¼ 8/1), RT, overnight or SnCl2, EtOH, reflux, 3e5 h; (v). (COOEt)2, reflux, 4 h.

D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
21
Scheme 2. Synthesis of compounds 9, 10 and 18e26. Reagents and conditions: (i). sodium alkoxide or alcohol/NaH, DMF, RT, 3 h; (ii). H2, 10% Pd/C, EtOAc/MeOH (V/V ¼ 8/1), RT,
overnight or SnCl2, EtOH, reflux, 3 h; (iii). (COOEt)2, reflux, 4 h.
Scheme 3. Synthesis of compounds 30e33. Reagents and conditions: (i). NCS or NBS, DMF or CH3CN, ꢀ20 ꢁC to RT, 16e24 h; (ii). 98% H2SO4, fuming HNO3, ꢀ5 to 50 ꢁC, 3 h; (iii). Zn
powder, AcOH, RT, 3 h; (iv). (COOEt)2, reflux, 6 h.
significant activation of 8-substitution, we believed that the 8-
substituents with proper size may promote the formation of HB
between the 4-amino and Gly313 by pushing the ligands nearer to
Gly313. With an attempt to gain additional interactions on the
“subpocket region” which was confirmed in the cases of 3-
hydroxypyridin-2-one [16], 4-hydroxypyridazin-3-one [16] and
Kojic acid inhibitors [17], we introduced side chains containing
phenyl group into 6 or 8-position of 5-azaquinoxaline-2,3-dione,
however these derivatives resulted in a complete loss of activity
(compounds 21e26).
For those 5-azaquinoxaline-2,3-diones with IC50 values below
300 nM against pkDAAO, we further tested them on DAAO origi-
nating from the spinal homogenates of rats (rsDAAO) and recom-
binant human DAAO (hDAAO). Noteworthly, 8-chloro-1,4-
dihydropyrido[2,3-b]pyrazine-2,3-dione (compound 16) exhibited
potent inhibitory activities (IC50 ꢂ 210 nM) on three forms of DAAO,
which are comparable to previously reported CBIO [8] and 3-
hydroxyquinolin-2-one [14] (Table 2).
Fig. 1. The complex of DAAO with 3-hydroxyquinolin-2-one (3G3E) and the repre-
sentative
a-hydroxylactam-based inhibitors. FAD (flavin adenine dinucleotide) and
3.2. In vivo blockade effects of pain and morphine analgesic
tolerance
residues are highlighted in yellow; the inhibitor and hydrogen bonds are highlighted in
green and red, respectively. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Due to potent inhibition on pkDAAO, hDAAO and especially
rsDAAO, compound 16 was therefore selected as a representative
molecule for further investigation on its analgesic effects in labo-
ratory mice or rats.
inhibitory activity (e.g. compounds 2/9/10 and 4/5/18/19/20). For
mono-substitution, the inhibitory activity of 8-substitution de-
rivatives excelled that of 6 or 7-substitution derivatives (e.g. com-
pounds 2/3/4 and 8/14/16), and halogen atoms such as chloro and
bromo at 8-position (compounds 16 and 17) afforded the most
potent efficacies, which reached the same level with the known
DAAO inhibitors 6-chlorobenzo[d]isoxazol-3-ol (CBIO) [8] and 3-
hydroxyquinolin-2-one [14]. When the 8-position was occupied
by methyl or halogen, various halogen atoms at the 7-position also
provided potent DAAO affinity (compounds 27e33). Given the
3.2.1. Differential effects of compound 16 given intrathecally on
formalin and NMDA-induced pain
We previously showed that DAAO inhibitors such as CBIO
blocked formalin-induced tonic pain [21e26]. To confirm com-
pound 16, as a novel DAAO inhibitor, also has such an effect, the
analgesic effect of compound 16 on the formalin test was studied

22
D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
Fig. 2. Molecular docking simulation of quinoxaline-2,3-dione and 8-methyl-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione with DAAO. FAD (flavin adenine dinucleotide) and resi-
dues are highlighted in yellow; the inhibitor and hydrogen bonds are highlighted in green and red, respectively. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
via direct spinal cord administration in rats chronically implanted
monofilaments and withdrawal latency to redial heat were
measured before and 0.5, 1, 2, 4 h after drug injection. Paw with-
drawal thresholds were transformed into a % maximum possible
effect (% MPE), which equals to 100 ꢃ [New threshold - baseline
with intrathecal cannulas. Five groups of rats (n ¼ 6 in each group)
received intrathecal bolus injection of normal saline (10
mL) or
compound 16 (0.1, 0.3, 1 or 3 mg) 30 min before formalin injection.
Subcutaneous injection of formalin in control rats receiving intra-
thecal injection of normal saline produced a characteristic bi-phasic
flinching responses consisting of an initial, rapidly decaying acute
phase (within 10 min after formalin injection) followed by a slowly
rising and long-lived (10e90 min) tonic phase as shown in Fig. 3A.
threshold]/[Contralateral threshold - baseline threshold] [38].
Compared with contralateral paws, spinal nerve ligation induced
marked mechanical and thermal hypersensitivity. Intrathecal in-
jection of compound 16 significantly increased both paw with-
drawal thresholds to mechanical stimuli (Fig. 4A) and withdrawal
latency to heat stimuli in ipsilateral paws (Fig. 4B) (P < 0.05, by two-
way ANOVA followed by post hoc Dunnett's test). At 1 h after in-
jection, the blockade effects for mechanical allodynia and heat
hyperalgesia were 54.4 5.2% and 80.8 11.3% MPE, respectively. In
contrast, compound 16 failed to influence paw withdrawal re-
sponses in contralateral paws.
Compared with the normal saline control, compound 16 up to 3 mg
did not prevent formalin-induced flinch response in the acute
phase, however it prevented formalin-induced pain in the tonic
phase in a dose-dependent manner (Fig. 3A). The areas under the
flinching response curve from 10 to 90 min (AUC10e90 min) were
calculated for doseeresponse analysis by best fitting. Maximum
inhibition (Emax) of formalin-induced tonic pain was 67.1% and half-
effective dose (ED50) was 0.25 mg (Fig. 3B).
It had been reported that a number of quinoxaline-2,3-diones
showed antagonism at the NMDA glycine site [36,37]. In order to
exclude the possibility that the analgesic property of compound 16
is due to its antagonism of the NMDA receptors, three groups of rats
3.2.3. Preventive and reversal effects of compound 16 given
intrathecally on morphine analgesic tolerance in the mouse hot-
plate test
To evaluate the preventive and reversal effects of compound 16
on morphine analgesia tolerance, three groups of mice (n ¼ 6 in
(n ¼ 6 in each group) received intrathecal injection of saline (10
the NMDA receptor antagonist MK-801 (0.1 g) or compound 16
(10 g) 30 min before intrathecal injection of 5 nmol NMDA. The
mL),
m
each group) received intrathecal injections of saline (10
m
L),
g),
twice daily with 12 h intervals for 7 days. On day 8, mice received a
single bolus intrathecal administration of 10 L saline or 10
compound 16 at 15 min before saline (10 L) or morphine (10 mg)
m
morphine (10 mg), and compound 16 (10 mg) plus morphine (10 m
pain behaviors were immediately measured for 10 min after NMDA
injection. In the saline-pretreated control rats, intrathecal injection
of NMDA produced immediate scratching, biting and licking re-
sponses, with measured pain duration approximately 200 s in
10 min. Pretreatment with intrathecal MK801 effectively inhibited
NMDA-induced pain response by approximately 80%. However,
compound 16 had no effects on NMDA-induced pain behaviors
(Fig. 3C).
m
mg
m
challenge. The latency of licking/biting/jumping was measured
before and 0.5, 1, 2 h after saline or morphine injection on day 1, 3,
5, 7 and 8. The areas under the tail-flicking or licking/biting/
jumping response curves over 2 h (AUC0e2 h) were calculated. As
shown in Fig. 5A, multiple bi-daily intrathecal injections of
morphine induced progressive and complete analgesic tolerance
during the 7-day administration; coadministration of compound 16
significantly attenuated the development of morphine analgesic
tolerance (P < 0.05 by two-way ANOVA followed by post-hoc stu-
dent-Newman-Keuls test). Furthermore, single bolus injection of
compound 16 significantly reversed the established morphine
tolerance on day 8 (P < 0.05 by two-way ANOVA followed by post-
hoc student-Newman-Keuls test) (Fig. 5B).
3.2.2. Analgesic effects of compound 16 given intrathecally on
spinal nerve ligation-induced mechanical allodynia and heat
hyperalgesia
To evaluate the effects of compound 16 on neuropathic pain,
spinal nerve ligated neuropathic rats 7 days after surgery were
divided into two groups (n ¼ 6 in each group): saline (10
mL) and
compound 16 (30 g). The paw withdrawal thresholds to van frey
m

D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
23
Table 1
Inhibitory effects of 5-azaquinoxaline-2,3-diones on porcine kidney
D
-amino acid oxidase (pkDAAO).
Compound
Structure
IC50
(m
M)a
Compound
Structure
IC50 (m
M)a
CBIO
0.21
16
0.18
3-hydroxyquinolin-2-one
quinoxaline-2,3-dione
0.10
33.6
17
18
0.22
85.7
1
2
3
5.7
19
20
21
>100
>100
17.7
36.7
>1000
4
5
0.63
2.4
22
23
>300
>300
6
7
22.9
0.52
20.3
24
25
>300
>300
8
9
26
27
>300
>100
>100
0.15
10
11
28
29
0.26
0.40
>1000
>1000
12
30
0.65
(continued on next page)

24
D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
Table 1 (continued )
Compound
13
Structure
IC50
(m
M)a
Compound
Structure
IC50 (m
M)a
0.46
31
0.48
14
15
6.7
32
33
0.24
0.53
12.4
a
The pyruvate production method was adopted [35]; all of the readings are means of triplicates, repeated two to three times.
nociception (Fig. 6A), but inhibited tonic pain in a dose-dependent
manner (P < 0.05 by one-way ANOVA followed by post-hoc stu-
dent-Newman-Keuls test) (Fig. 6B), with an Emax of 49% inhibition
and an ED50 of 1.8 mg/kg (Fig. 6C). Oral gavage of compound 16
(10 mg/kg) blocked formalin-induced tonic pain by approximate
30%, which was comparable to that of 3 mg/kg but significantly less
than that of 10 mg/kg given subcutaneously, suggesting that com-
pound 16 has an apparent oral bioavailability of approximately 30%
(vs. subcutaneously).
Table 2
Inhibitory effects of partial 5-azaquinoxalinedinones on recombinant human D-
amino acid oxidase (hDAAO), porcine kidney DAAO (pkDAAO) and rat spinal DAAO
(rsDAAO).
Compound
Structure
IC50 (nM)a
pkDAAO
rsDAAO hDAAO
210 (188) 90 110
CBIO
3-hydroxyquinolin-2-one
16
104
180
94 (215) 43 (4)
4. Conclusions
In search of novel and potent DAAO inhibitors for pharmaco-
logical studies we synthesized and evaluated a series of 5-
azaquinoxalinedinone derivatives. As a novel family of non-
carboxylic DAAO inhibitor, 5-azaquinoxaline-2,3-diones exhibited
significantly improved potency over quinoxaline-2,3-diones. Some
newly synthesized derivatives were potent (IC50 of 100e500 nM) to
inhibit pkDAAO in vitro. These results supported our speculation
that introduction of 5-nitrogen into quinoxaline-2,3-dione could
significantly break the similar enolization of di-lactam and make it
more favorable to interact with guanidyl of Arg283, and 4-amino,
owing to the increased bond polarity prefers to donate its hydrogen
atom and form a stronger HB with carbonyl of Gly313.
We intensively studied the analgesic effects of compound 16,
which exhibited comparable DAAO inhibitory potency to the
existing inhibitors such as CBIO and 3-hydroxypyridin-2-one on
pkDAAO, rsDAAO and hDAAO. Given intrathecally, compound 16
can effectively and specifically block spinal nerve ligation-induced
mechanical allodynia and heat hyperalgesia in neuropathic rats,
prevent and reverse morphine analgesic tolerance, and relieve
formalin-induced tonic pain without influencing the NMDA re-
ceptor activity. Moreover, compound 16 could also attenuate
formalin-induced tonic pain by subcutaneous injection or oral
gavage. This research not only confirmed the analgesic effects of
DAAO inhibitors based on a new scaffold, but provided a new class
of chemical entities with oral application potential for the treat-
ment of chronic pain and morphine analgesic tolerance.
140
210
230
17
220
2240
27
28
32
150
260
240
150
340
4160
7990
1130
1380
a
5-Azaquinoxalinedinones with IC50 < 300 nM on pkDAAO were chosen; The
pyruvate production method was adopted [35] to measure the DAAO enzymatic
activity; all of the readings are means of triplicates, repeated two to three times; The
data in brackets are reported by literatures [8,14].
3.2.4. Analgesic effects of compound 16 given subcutaneously and
by oral gavage on formalin-induced pain
5. Experimental
In order to determine whether compound 16 has a systemic
exposure and oral bioavailability, we finally evaluated subcutane-
ous injection and oral gavage of compound 16 on formalin-induced
pain in mice. Six groups of mice (n ¼ 6 in each group) received
subcutaneous injection of saline (10 mL/kg), compound 16 (0.3, 1, 3
and 10 mg/kg) and one group of 6 fasted mice received gavage of
compound 16 (10 mg/kg) 30 min before formalin challenge. Sub-
cutaneous injection of compound 16 did not prevent acute
5.1. Biology
5.1.1. Drugs and reagents
NMDA, MK-801 and 3-hydroxypyridin-2-one were purchased
from Sigma (Sigma, St Louis, MO, USA). CBIO (5-chloro-benzo[d]
isoxazol-3-ol) was manufactured from Maybridge (Cornwall, U.K.)
and was a kind gift from Dr. Kenji Hashimoto at the Chiba

D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
25
Fig. 3. Differential effects of intrathecal injection of compound 16 on formalin- and NMDA-induced pain. A. Effects on formalin-induced pain by intrathecal injection. Rats received
intrathecal injection of normal saline (10 L) or compound 16 (0.1, 0.3, 1, or 3 g) 30 min before subcutaneous injection of 50 L of 5% formalin. Nociceptive behavior was quantified
by counting the number of the formalin-injected paw flinches in 1-min epochs; B. Dose response analysis on formalin-induced tonic pain; C. Effects on NMDA-induced pain by
intrathecal injection. Rats received intrathecal injection of normal saline (10 L), compound 16 (10 g) or MK-801 (0.1 g) 30 min before intrathecal NMDA (5 nmol). The pain
m
m
m
m
m
m
*
behaviors were immediately measured for 10 min after NMDA injection. Data are presented as means S.E.M. (n ¼ 6 in each group). denotes statistically significant difference
(P < 0.05 by one-way ANOVA followed by post-hoc Student-Newman-Keuls test), compared to the saline control group.
University Center for Forensic Mental Health, Japan. Porcine kidney
DAAO (pkDAAO), recombinant human DAAO (hDAAO) and FAD
(flavin adenine dinucleotide) were purchased from SERVA (Hei-
delberg, New York, USA), Sigma (Sigma, St Louis, MO, USA) and
Novoprotein (Shanghai, China), and formalin and morphine hy-
drochloride injection were purchased from Sinopharm Group
Chemical Reagent Co. (Shanghai, China) and Shenyang First Phar-
maceutical Co. (Shenyang, Liaoning, China), respectively. All test
drugs were freshly dissolved in sterile normal saline solution
(Sinopharm Group Chemical Reagent Co., Ltd.) with pH adjusted to
7.3e7.5 by 1 N NaOH solution as needed. All enzymes were freshly
dissolved in TriseHCl (pH ¼ 8.2).
the keto acid method [35]. DAAO inhibitors were assayed for their
effects on rsDAAO, pkDAAO and hDAAO. In the control homoge-
nates incubated for 5 min for pkDAAO and 60 min for both rsDAAO
and hDAAO, the a-keto acid produced by D-alanine was determined
by measurement of pyruvic acid. All enzymes and FAD were dis-
solved in 0.1 M TriseHCl, pH ¼ 8.2 (final concentration: 0.04 mg/mL
for pkDAAO, 0.3 mg/mL for rsDAAO and 0.05 mg/mL for hDAAO, and
0.2 mg/mL for FAD). Fifty microliters of
tration: 1 mM for both pkDAAO and hDAAO, and 6 mM for rsDAAO)
was added to 25 L of DAAO inhibitors solution (concentration as
follow) and enzyme solution (50 L of pkDAAO and rsDAAO, while
25 L of FAD should be added to 25 L of hDAAO). The mixture
above was incubated (700 rpm) at 37 ꢁC for 5 or 60 min. Tri-
chloroacetic acid (25%; 50 L) was then added to the assay mixture,
mixed, and centrifuged (14,000 rpm, 5 min). The supernatant
(50 L) was mixed with 50 L of 1 mM 2,4-dinitrophenylhydrazine
(in 1 N HCl) and incubated (700 rpm) at 37 ꢁC for 10 min. Finally,
D-alanine (final concen-
m
m
m
m
m
5.1.2. Animals
Male Wistar rats (180e250 g) and Swiss mice (18e22 g) were
obtained from the Shanghai Experimental Animal Institute for
Biological Sciences (Shanghai, China). The animals were housed in a
temperature- and humidity-controlled environment on a 12-h
light/dark cycle (lights on 7:00 AM). Food and water were freely
available. Animals were acclimated to the laboratory environment
for 3e5 days before entering the study. Experimental study groups
were assigned randomly, and the researcher was blind for behavior
testing. The research protocols were approved by the Animal Care
and Welfare Committee of Shanghai Jiao Tong University and fol-
lowed the animal care guidelines of the National Institutes of
Health. Efforts were made to reduce the number of animals used,
and to minimize their sufferings.
m
m
100 mL of 1.5 M sodium hydroxide was added, mixed and incubated
(700 rpm) at 37 ꢁC for 10 min. The absorbance was read at 450 nm
on an ELx800 Universal Microplate Reader (BioTek Instruments,
Winooski, VT) against a blank sample consisting of the same ho-
mogenates without
mogenates was quantified against the standard curve of pyruvic
acid (from 100 to 800 M; R2 > 0.99).
D-alanine. The activity of DAAO in the ho-
m
5.1.4. Rat intrathecal catheterization and injection
A 18-cm polyethylene catheter (PE-10: 0.28 mm i.d. and
0.61 mm o.d., Clay Adams, Parsippany, NJ, USA) with a volume of
5.1.3. DAAO enzymatic activity assay
13
mL was inserted into the rat lumbar level of the spinal cord as
The enzymatic activities of DAAO were determined according to
described elsewhere under inhale isoflurane anesthesia (4% for

26
D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
Fig. 4. Analgesic effects of compound 16 given intrathecally on mechanical allodynia
and heat hyperalgesia. A. Analgesic effect on mechanical allodynia; B. Analgesic effect
on heat hyperalgesia. Neuropathic rats 7 days after spinal nerve ligation received single
intrathecal administration of saline (10 mL) or compound 16 (30 mg). The contralateral
Fig. 5. Preventive and reversal effects of compound 16 on morphine analgesic toler-
ance in the hot-plate test. A. Preventive effect. Intrathecally cannulated rats received
and ipsilateral hind limb withdrawal thresholds to mechanical stimuli and withdrawal
latency to heat stimulus were measured at 0.5, 1, 2 or 4 h after intrathecal adminis-
intrathecal injections of saline (10
pound 16 (10 g) and morphine (10
Reversal effect. On day 8, rats received a single intrathecal administration of saline
(10 L) or compound 16 (10 g) 15 min before saline or morphine (5 g)) challenge
mL), morphine (10
mg) or the combination of com-
*
tration. Data are presented as means S.E.M. (n ¼ 6 in each group). denotes sta-
m
mg), twice daily with a 12 h-interval for 7 days. B.
tistically significant difference (P < 0.05 by two-way ANOVA test) compared with the
normal saline control.
m
m
m
30 min prior to the hot-plate test. Data are presented as means S.E.M. (n ¼ 6 in each
*
group). denotes statistically significant difference (P < 0.05, by two-way ANOVA
followed by post-hoc Student-Newman-Keuls test), compared to the morphine group.
induction and 1% for maintenance) run by an anesthesiameter (Ugo
Basile Gas Anesthesia System, Comerio, Italy). Two days after re-
covery from anesthesia, the placing of the catheter in the spinal
5.1.6. The mouse hot-plate test
cord was verified by administering 4% lidocaine (10
mL followed by
For the hot-plate test, pain reflexes in response to thermal
stimulus were measured using YLS-6B Intelligence Hot Plate
Analgesia Meter (Shandong Academy of Medical Sciences Device
Co., Shandong, China). The surface of the hot-plate was heated to a
15 L of saline for flushing) with a 50- L micro injector (Shanghai
m
m
Anting Micro-Injector Factory, Shanghai, China). Only rats that had
no motor impairment following intrathecal catheter were consid-
ered for the study; only rats that developed immediate bilateral
paralysis of hind limbs following intrathecal administration of
lidocaine were selected for the study.
constant temperature at 55
0.1 ꢁC, as measured by a built-in
digital thermometer with an accuracy of 0.1 ꢁC and verified by a
surface thermometer. Mice were placed on the hot-plate, which
was surrounded by a clear acrylic cage, and the Start/Stop button on
a timer was activated. The latency to respond with either a hind-
paw lick or flick, biting, or jump (whichever came first) was
measured to the nearest 0.1 s by deactivating the timer when the
response was observed. Trials were terminated if the animals did
not respond within the 40 s cut-off period to avoid tissue damage.
5.1.5. The rat and mouse formalin test
Animals were acclimated individually to the observation cage
for 30 min prior to testing. The formalin test was performed as
previously described by injecting 50 mL (for rats) or 10 mL (for mice)
of 5% formalin in 0.9% saline subcutaneously on the dorsal side of
the right hindpaw and the animal was immediately placed in a
transparent polycarbonate box. For rats, nociceptive behavior was
manually quantified by counting the number of the formalin-
injected paw flinches in 1-min epochs and measurements were
taken at 10-min intervals beginning immediately after formalin
injection and ending 90 min later. For mice, the duration of noci-
ceptive behaviors (licking/biting) was manually quantified in the
pooled durations at 0e5 min and 20e40 min, which were
considered as the acute nociception and tonic pain, respectively.
5.1.7. The rat spinal nerve ligation-induced neuropathic pain model,
and mechanical allodynia and heat hyperalgesia measurement
Spinal nerve ligation was used to induce a peripheral neuropa-
thy and pain hypersensitivity. The unilateral ligation of 2 spinal
nerves (L5 and L6) was performed under inhale isoflurane anes-
thesia (4% for induction and 1% for maintenance) run by an anes-
thesiameter (Ugo Basile Gas Anesthesia System, Comerio, Italy). The
unilateral ligation of 2 spinal nerves (L5 and L6) was described in
detail earlier. The left L5 and L6 spinal nerves were isolated and

D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
27
Fig. 6. Effects of systemic administration of compound 16 on formalin-induced pain. A. Effect on acute nociception. B. Effect of on tonic pain. C. Dose-response analysis on tonic
pain. Mice received subcutaneous injection of normal saline (10 mL/kg) and compound 16 (0.3, 1, 3, 10 or 30 mg/kg), and compound 16 (10 mg/kg) for gavage 30 min before paw 5%
formalin (10 mL) challenge. Cumulated biting duration from 0 to 5 min and 20e40 min after formalin injection represents acute phase nociception and tonic phase pain, respectively.
Data are presented as means S.E.M. (n ¼ 6 in each group). a and b denote statistically significant difference (P < 0.05 by one-way ANOVA followed by post-hoc Student-Newman-
Keuls test), compared to the saline control group and compound 16 (10 mg/kg, subcutaneously) group, respectively.
tightly ligated with 6e0 silk thread. After ligation, the wound was
sutured and the rats were allowed to recover. Of the nerve-ligated
animals, only those with marked unilateral allodynia to mechanical
stimulation (hind limb withdrawal thresholds in the operated
side < 8 g) and with no major impairment were included in the
study.
In order to evaluate mechanical allodynia, animals were accli-
mated for at least half an hour to the test environment, namely a
plexiglass box on a metal grid (0.5 ꢃ 0.5 cm). The hind paw
withdrawal threshold (PWT) was measured by a 2450 CE Elec-
tronic Von Frey hair (IITC Life Science Inc, Woodland Hill, CA, USA).
An electronic hand-held transducer with a No.15 monofilament
was applied perpendicularly to the medial, surface of the hind
paws with the force increasing until the rat suddenly withdrew or
licked the hind paw. The lowest force producing a withdrawal
response was considered the threshold; this was based on three
repeated measurements with a 10-min interval and the mean of
these threshold values for each hind paw at each time point was
used.
5.2. Chemistry
5.2.1. General method
Unless otherwise noted all materials were obtained from com-
mercial suppliers and used without further purification. Organic
extracts were routinely dried over anhydrous sodium sulfate. If
necessary silica gel column chromatography (200e300 mesh) were
performed for purification of intermediate using PE/EtOAc or DCM/
MeOH as mobile phase. NMR spectra were recorded on a Bruker
400 MHz spectrometer for 1H NMR and 100 MHz for 13C NMR.
High-resolution mass spectroscopy (HRMS) values were recorded
on a Waters Q-Tof Premier system. The purities of the tested
compounds were determined to be above 95% by Agilent 1200
HPLC system (4.6 mm ꢃ 250 mm column, 5
mm, 5%, 10% or 20%
MeOH to 95% MeOH in H2O in 20 min at 1.0 mL/min and UV
detection at 254 nm or 210 nm).
5.2.2. General procedure for synthesis of 5-azaquinoxaline-2,3-
diones
For assessment of heat hyperalgesia, rats were put in a plex-
iglass box on the elevated glass surface. Following an adaption
period of 30 min at least, radiant heat was applied to the plantar
medial surface of each hind paw. The hind paw withdrawal la-
tency (PWL) was measured by the 390G Plantar Test Analgesia
Meter (IITC Life Science Inc.). A cut-off time was 20 s to minimize
tissue damage upon the stimulation. The paw withdrawal latency
was defined as the time from the onset of radiant heat applica-
tion to the withdrawal of hind paws. Both hind paws were tested
independently for three times with a 10-min interval between
trials. Each test was calculated as a mean of three repeated
measurements.
Step 1. To 25e30 mL 98% H2SO4 was added portionwise
10.0 mmol substituted 2-aminopyridine at ꢀ5 ꢁC. After the solid
material was dissolved 0.6 mL fuming HNO3 was added dropwise
over 5 min maintaining internal temperature ꢂ0 ꢁC. The mixture
was allowed to warm to 50 ꢁC over 2 h, and then stirred for another
1 h at this temperature before poured onto 150 g crushed ice. The
aqueous solution was basified to pH ¼ 9 with ammonia and
extracted with ethyl acetate. The combine extracts were dried,
concentrated and purified by column chromatography to give 2-
amino-3-nitropyridine. Alternative method: To 25e30 mL 98%
H2SO4 was added portionwise 10.0 mmol substituted 2-
aminopyridine at ꢀ5 ꢁC. After the solid material was dissolved,

28
D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
0.6 mL fuming HNO3 was added dropwise over 5 min maintaining
internal temperature ꢂ0 ꢁC. The mixture was stirred at this tem-
perature for 10 min before poured onto 150 g crushed ice. Ammonia
was used to adjust pH ¼ 3e4, and the resulted white solid was
filtered, washed with water and 10 mL 50e75% ethanol, and dried
under vacuum to give 2-nitraminopyridine. To 25e30 mL 98%
H2SO4 was added portionwise 2-nitraminopyridine above obtained
at 0 ꢁC. The mixture was allowed to warm to 50 ꢁC over 1 h, and
then stirred for another 1 h at this temperature before poured onto
150 g crushed ice. The aqueous solution was basified to pH ¼ 9 with
ammonia and extracted with ethyl acetate. The combine extracts
were dried, concentrated and purified by column chromatography
to give 2-amino-3-nitropyridine.
Step 2. To a solution of 2-amino-3-nitropyridine in mixed sol-
vent of ethyl acetate and methanol (V/V ¼ 8:1) was added 10e15%
(m/m) 10% Pd/C. This mixture was vacuumed and backfilled with
hydrogen and stirred overnight at room temperature before
filtration. The filtrate was concentrated to give crude 2.3-
diaminopyridine, which could be purified by column chromatog-
raphy. Alternative method: To a solution of 10.0 mmol 2-amino-3-
nitropyridine in 20 mL ethanol was added 30.0 mmol SnCl2. This
mixture was heated to reflux for 3e5 h before treatment with 2 M
NaOH. The mixture was extracted with ethyl acetate, dried and
concentrated to give 2,3-diaminopyridine.
(400 MHz, DMSO-d6)
7.95 (1H, d, J ¼ 4.8 Hz, H-6), 11.48 (1H, brs, NH-1), 12.27 (1H, brs,
NH-4); 13C NMR (100 MHz, DMSO-d6)
16.62, 120.35, 120.79,
d
2.37 (3H, s, CH3), 6.99 (1H, d, J ¼ 4.8 Hz, H-7),
d
132.84, 138.77, 141.79, 155.40, 155.62; HRMS-EI C8H7N3O2 calcd
[MþNa]þ 200.0436, found 200.0436.
5.2.2.5. 8-Ethyl-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(5).
The title compound was prepared from 2-amino-4-ethylpyridine
according to the general procedure as white solid (308 mg, 16.1%).
2-Amino-4-ethylpyridine was synthesized according to the litera-
ture [39]. IR (KBr)
(400 MHz, DMSO-d6)
n
2755, 1704, 1690, 1622, 1387, 832 cmꢀ1; 1H NMR
d
1.15 (3H, t, J ¼ 7.6 Hz, CH3), 2.79 (2H, q,
J ¼ 7.6 Hz, CH2), 7.02 (1H, d, J ¼ 5.2 Hz, H-7), 7.99 (1H, d, J ¼ 5.2 Hz,
H-6), 11.48 (1H, brs, NH-1), 12.27 (1H, brs, NH-4); 13C NMR
(100 MHz, DMSO-d6)
d 13.57, 22.32, 118.96, 119.54, 138.47, 138.80,
142.08, 155.40, 155.51; HRMS-EI C9H9N3O2 calcd [MþNa]þ
214.0592, found 214.0591.
5.2.2.6. 7-Trifluoromethyl-1,4-dihydropyrido[2,3-b]pyrazine-2,3-
dione (6). The title compound was prepared from 2-amino-5-
trifluoromethylpyridine according to the general procedure as
white solid (635 mg, 27.5%). IR (KBr)
1165, 1094 cmꢀ1 1H NMR (400 MHz, DMSO-d6)
J ¼ 2.0 Hz, H-8), 8.44 (1H, s, H-6), 12.13 (1H, s, NH-1), 12.71 (1H, s,
NH-4); 13C NMR (100 MHz, DMSO-d6)
118.74 (t), 120.04 (d),
n
3055, 1717, 1698, 1627, 1342,
;
d
7.63 (1H, d,
Step 3. A mixture of 2,3-diaminopyridine in diethyl oxalate
(10 mL/1 mmol phenyldiamine) was heated to reflux for 4 h before
cooled to room temperature. The resulted solid was filtered,
washed with ethyl acetate and 95% ethanol, decolorized with
activated charcoal and recrystallized in water and DMF or DMSO to
give 5-azaquinoxalinediones as white solid.
d
122.52, 124.18 (d), 138.99 (t), 142.95, 154.95, 156.39; HRMS-EI
C8H4F3N3O2 calcd [MþNa]þ 254.0153, found 254.0150.
5.2.2.7. 8-Trifluoromethyl-1,4-dihydropyrido[2,3-b]pyrazine-2,3-
dione (7). The title compound was prepared from 2-amino-4-
trifluoromethylpyridine according to the general procedure as
5.2.2.1. 1,4-Dihydropyrido[2,3-b]pyrazine-2,3-dione (1). The title
compound was prepared from 2,3-aminopyridine and diethyl ox-
white solid (178 mg, 7.7%). IR (KBr)
1137 cmꢀ1; 1H NMR (400 MHz, DMSO-d6)
H-7), 8.20 (1H, d, J ¼ 4.8 Hz, H-6), 11.63 (1H, brs, NH-1), 12.62 (1H, s,
NH-4); 13C NMR (100 MHz, DMSO-d6)
114.72 (q), 118.58, 119.32
n
1700, 1458, 1374, 1188,
d
7.40 (1H, d, J ¼ 4.8 Hz,
alate as white solid (527 mg, 75.0%). IR (KBr)
n
3215, 2741, 1697,
7.13 (1H, dd,
1625, 1465, 1381 cmꢀ1; 1H NMR (400 MHz, DMSO-d6)
d
d
J ¼ 8.0, 4.8 Hz, H-7), 7.46 (1H, dd, J ¼ 8.0, 1.6 Hz, H-6), 8.07 (1H, dd,
(m), 121.30, 124.03, 126.75, 141.43, 142.47, 155.28, 155.63; HRMS-EI
J ¼ 4.8,1.6 Hz, H-8),11.97 (1H, s, NH-1),12.32 (1H, s, NH-4); 13C NMR
C8H4F3N3O2 calcd [MþNa]þ 254.0153, found 254.0149.
(100 MHz, DMSO-d6) d 118.78,121.60,122.23, 139.01,142.02, 154.68,
155.79; HRMS-EI C7H5N3O2 calcd [MþNa]þ 186.0279, found
5.2.2.8. 6-Chloro-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(8).
186.0278.
The title compound was prepared from 2-amino-6-chloropyridine
according to the general procedure as white solid (176 mg, 8.9%).
5.2.2.2. 6-Methyl-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(2).
IR (KBr) n ;
3117, 1688, 1625, 1587, 1469, 1390 cmꢀ1 1H NMR
The title compound was prepared from 2-amino-6-methylpyridine
according to the general procedure as white solid (391 mg, 22.1%).
(400 MHz, DMSO-d6)
d
7.19 (1H, d, J ¼ 8.0 Hz, H-7), 7.46 (1H, d,
J ¼ 8.0 Hz, H-8), 12.05 (1H, s, NH-1), 12.51 (1H, s, NH-4); 13C NMR
IR (KBr)
(400 MHz, DMSO-d6)
7), 7.35 (1H, d, J ¼ 8.0 Hz, H-8), 11.93 (1H, brs, NH-1), 12.22 (1H, brs,
n
3176, 3109, 1682, 1591, 1479, 1391 cmꢀ1
;
1H NMR
(100 MHz, DMSO-d6) d 118.69, 121.66, 125.84, 139.49, 141.58, 154.95,
d
2.40 (3H, s, CH3), 6.98 (1H, d, J ¼ 8.0 Hz, H-
156.19; HRMS-EI C7H4ClN3O2 calcd [MþH]þ 198.0070, found
198.0066.
NH-4); 13C NMR (100 MHz, DMSO-d6)
d 23.08,118.03,119.15,122.89,
138.24, 150.70, 154.65, 155.91; HRMS-EI C8H7N3O2 calcd [MþNa]þ
5.2.2.9. 6-Methoxy-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione (9).
To a solution of 2-amino-3-nitro-6-chloropyridine (300 mg,
200.0436, found 200.0435.
1.73 mmol, an intermediate in preparation of 8) in 5 mL anhydrous
DMF was added sodium methoxide (187 mg, 3.46 mmol). This
mixture was stirred at room temperature for 3 h then poured onto
20 g crushed ice and extracted with ethyl acetate. The extracts were
dried, evaporated and purified by column chromatography on silica
gel (PE/AcOEt ¼ 5:1) to give 2-amino-3-nitro-6-methoxylpyridine
(246 mg, 84.1%) as yellow solid. Next steps followed the general
procedure, and the title compound was obtained as white solid
5.2.2.3. 7-Methyl-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
The title compound was prepared from 2-amino-5-methylpyridine
according to the general procedure as white solid (623 mg, 35.2%).
(3).
IR (KBr)
d6)
n
3041, 2774, 1694, 1386 cmꢀ1; 1H NMR (400 MHz, DMSO-
d
2.27 (3H, s, CH3), 7.26 (1H, d, J ¼ 1.6 Hz, H-6), 7.91 (1H, d,
J ¼ 1.6 Hz, H-8), 12.07 (2H, brs, NH-1 & 4); 13C NMR (100 MHz,
DMSO-d6)
d 17.32, 121.41, 122.57, 128.04, 137.07, 142.00, 155.00,
155.75; HRMS-EI C8H7N3O2 calcd [MþNa]þ 200.0436, found
(188 mg, 67.0% over 2 steps). IR (KBr)
n 3246, 3184, 1708, 1635, 1599,
200.0435.
1488, 1462, 1341, 1260, 1024, 839 cmꢀ1; 1H NMR (400 MHz, DMSO-
d6)
d
3.81 (3H, s, CH3), 6.56 (1H, d, J ¼ 8.4 Hz, H-7), 7.42 (1H, d,
5.2.2.4. 8-Methyl-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(4).
J ¼ 8.4 Hz, H-8), 11.82 (1H, s, NH-1), 12.25 (1H, s, NH-4); 13C NMR
The title compound was prepared from 2-amino-4-methylpyridine
according to the general procedure as white solid (420 mg, 23.7%).
(100 MHz, DMSO-d6) d 53.50, 104.56, 115.19, 126.61, 136.21, 154.35,
156.11, 158.55; HRMS-EI C8H7N3O3 calcd [MþNa]þ 216.0385, found
IR (KBr)
n
3048, 2745, 1682, 1623, 1386, 906 847, 721 cmꢀ1; 1H NMR
216.0382.

D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
29
5.2.2.10. 6-Ethoxy-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione (10).
The title compound was prepared from 2-amino-3-nitro-6-
chloropyridine and sodium ethoxide in a manner similar to 9 as
according to the general procedure as white solid (284 mg,
14.4%). IR (KBr)
3044, 2743, 1697, 1594, 1373, 1276 cmꢀ1; 1H NMR
n
(400 MHz, DMSO-d6)
d
7.29 (1H, d, J ¼ 5.6 Hz, H-7), 8.01 (1H, d,
white solid (160 mg, 44.9%). IR (KBr)
n
3174, 1712, 1688, 1594, 1467,
1.29 (3H, t, J ¼ 7.2 Hz,
J ¼ 5.6 Hz, H-6), 11.74 (1H, s, NH-1), 12.50 (1H, s, NH-4); 13C NMR
1261 cmꢀ1 1H NMR (400 MHz, DMSO-d6)
;
d
(100 MHz, DMSO-d6) d 119.18, 119.79, 126.98, 140.28, 142.13, 155.13,
CH3), 4.23 (2H, q, J ¼ 7.2 Hz, CH2), 6.53 (1H, d, J ¼ 8.4 Hz, H-7), 7.41
155.57; HRMS-EI C7H4ClN3O2 calcd [MþH]þ 198.0070, found
(1H, d, J ¼ 8.4 Hz, H-8), 11.81 (1H, s, NH-1), 12.21 (1H, s, NH-4); 13
C
198.0064.
NMR (100 MHz, DMSO-d6)
d 14.47, 61.58, 104.75, 115.05, 126.56,
136.16, 154.34, 156.11, 158.17; HRMS-EI C9H9N3O3 calcd [MþNa]þ
5.2.2.17. 8-Bromo-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione (17).
The title compound was prepared from 2-amino-4-bromopyridine
according to the general procedure as white solid (334 mg, 13.8%).
230.0542, found 230.0540.
5.2.2.11. Methyl 2,3-dioxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazine-
7-carboxylate (11). The title compound was prepared from methyl
6-aminonicotinate according to the general procedure as white
IR (KBr) n ;
3126, 3087, 3040, 2728, 1714,1697, 1589, 1384,1275 cmꢀ1
1H NMR (400 MHz, DMSO-d6)
d
7.43 (1H, d, J ¼ 5.2 Hz, H-7), 7.92
(1H, d, J ¼ 5.2 Hz, H-6), 11.37 (1H, s, NH-1), 12.46 (1H, s, NH-4); 13
C
solid (818 mg, 37.0%). IR (KBr)
1615, 1434, 1391, 1319, 1229 cmꢀ1
3.86 (3H, s, CH3), 7.86 (1H, s, H-6), 8.55 (1H, s, H-8), 12.08 (1H, s,
NH-1), 12.66 (1H, s, NH-4); 13C NMR (100 MHz, DMSO-d6)
52.33,
n
3233, 3062, 2846, 2757, 1724, 1691,
;
NMR (100 MHz, DMSO-d6) d 116.87, 121.20, 122.46, 139.86, 142.16,
1H NMR (400 MHz, DMSO-d6)
155.17, 155.50; HRMS-EI C7H4BrN3O2 calcd [MþH]þ 241.9565,
d
found 241.9561.
d
120.29, 121.67, 122.08, 142.54, 143.11, 154.51, 155.97, 164.69; HRMS-
5.2.2.18. 8-Methoxy-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(18). To a solution of 2-amino-3-nitro-4-chloropyridine (300 mg,
1.73 mmol, an intermediate in preparation of compound 16) in 5 mL
anhydrous DMF was added sodium methoxide (187 mg,
3.46 mmol). This mixture was stirred at room temperature for 3 h
then poured onto 20 g crushed ice and extracted with ethyl acetate.
The extracts were dried, evaporated and purified by column chro-
matography on silica gel (DCM/MeOH ¼ 40:1) to give 2-amino-3-
nitro-4-methoxylpyridine (255 mg, 87.3%) as yellow solid. The
next steps followed the general procedure and gave the title com-
EI C9H7N3O4 calcd [MþNa]þ 244.0334, found 244.0334.
5.2.2.12. 2,3-Dioxo-1,2,3,4-tetrahydropyrido[2,3-b]pyrazine-7-
carboxylic acid (12). A mixture of compound 11 (300 mg,
1.36 mmol), water (15 mL) and sodium hydroxide (0.1 g) was
heated to 60 ꢁC and stirred at this temperature for 1e2 h. The
mixture was cooled to with ice-water and adjusted to pH ¼ 5e6
with 2 M HCl. The resulted precipitate was filtered, washed with
water, and dried under vacuum to give the title compound as white
solid (243 mg, 86.5%). IR (KBr)
1414, 1399, 1219 cmꢀ1; 1H NMR (400 MHz, DMSO-d6)
H-6), 8.56 (1H, s, H-8), 12.09 (1H, s, NH-1), 12.63 (1H, s, NH-4); 13
n
3174, 3043, 2977, 1716, 1673, 1613,
pound as white solid (166 mg, 57.1% over 2 steps). IR (KBr)
2870, 2750, 1714, 1688, 1621, 1506, 1378, 1140, 981 cmꢀ1 1H NMR
(400 MHz, DMSO-d6)
3.93 (3H, s, CH3), 6.91 (1H, d, J ¼ 6.0 Hz, H-7),
8.00 (1H, d, J ¼ 6.0 Hz, H-6), 11.50 (1H, s, NH-1), 12.23 (1H, s, NH-4);
n 3028,
d
7.88 (1H, s,
;
C
d
NMR (100 MHz, DMSO-d6)
d 121.48, 121.56, 122.45, 142.22, 143.38,
154.57, 155.97, 165.71; HRMS-EI C8H5N3O4 calcd [MꢀH]ꢀ 206.0202,
13C NMR (100 MHz, DMSO-d6)
d 56.33, 102.87, 110.64, 139.22,
found 206.0199.
143.62, 152.30, 154.66, 155.97; HRMS-EI C8H7N3O3 calcd [MþNa]þ
216.0385, found 216.0384.
5.2.2.13. 7-Fluoro-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione (13).
The title compound was prepared from 2-amino-5-fluoropyridine
according to the general procedure as white solid (530 mg,
5.2.2.19. 8-Ethoxy-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione (19).
The title compound was prepared from 2-amino-3-nitro-4-
chloropyridine and sodium ethoxide in a manner similar to 18 as
29.3%). IR (KBr)
NMR (400 MHz, DMSO-d6)
H-8), 12.24 (2H, brs, NH-1 & 4); 13C NMR (100 MHz, DMSO-d6)
n
3150, 3042, 1708, 1683, 1621, 1458, 1380 cmꢀ1; 1H
d
7.30 (1H, d, J ¼ 8.4 Hz, H-6), 8.07 (1H, s,
white solid (144 mg, 40.3%). IR (KBr)
n ;
1697, 1623, 1386, 1139 cmꢀ1
1H NMR (400 MHz, DMSO-d6)
d
1.39 (3H, t, J ¼ 7.2 Hz, CH3), 4.21
d
109.58 (d),122.64 (d),128.74 (d),136.02 (d),153.95,154.70,155.30,
(2H, q, J ¼ 7.2 Hz, CH2), 6.89 (1H, d, J ¼ 5.6 Hz, H-7), 7.97 (1H, d,
156.40; HRMS-EI C7H4FN3O2 calcd [MþNa]þ 204.0185, found
J ¼ 5.6 Hz, H-6), 11.43 (1H, s, NH-1), 12.22 (1H, s, NH-4); 13C NMR
204.0184.
(100 MHz, DMSO-d6) d 14.17, 64.70, 103.34, 110.62, 139.27, 143.61,
151.55, 154.68, 155.98; HRMS-EI C9H9N3O3 calcd [MþNa]þ
5.2.2.14. 7-Chloro-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione (14).
The title compound was prepared from 2-amino-5-chloropyridine
according to the general procedure as white solid (433 mg,
230.0542, found 230.0542.
5.2.2.20. 8-Isopropoxy-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(20). The title compound was prepared from 2-amino-3-nitro-4-
chloropyridine, isopropanol and sodium hydride in a manner
21.9%). IR (KBr)
n 3190, 3137, 3051, 2753, 1716, 1608, 1445,
1392 cmꢀ1; 1H NMR (400 MHz, DMSO-d6)
d
7.44 (1H, d, J ¼ 2.0 Hz,
H-6), 8.09 (1H, d, J ¼ 2.0 Hz, H-8); 13C NMR (100 MHz, DMSO-d6)
similar to 18 as white solid (137 mg, 35.8%). IR (KBr)
n
2757, 1692,
1.33
d
121.22, 122.83, 124.32, 138.19, 139.69, 154.58, 155.53; HRMS-EI
1621, 1500, 1370, 1280 cmꢀ1 1H NMR (400 MHz, DMSO-d6)
;
d
C7H4ClN3O2 calcd [MþH]þ 198.0070, found 198.0069.
(6H, d, J ¼ 6.0 Hz, CH3), 4.82 (1H, m, CH), 6.91 (1H, d, J ¼ 5.6 Hz, H-
7), 7.95 (1H, d, J ¼ 5.6 Hz, H-6), 11.38 (1H, brs, NH-1), 12.21 (1H, brs,
5.2.2.15. 7-Bromo-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione (15).
The title compound was prepared from 2-amino-5-bromopyridine
according to the general procedure as white solid (620 mg, 25.6%).
NH-4); 13C NMR (100 MHz, DMSO-d6)
d 21.46, 71.46, 104.05, 111.20,
139.53, 143.51, 150.65, 154.68, 156.00; HRMS-EI C10H11N3O3 calcd
[MþNa]þ 244.0698, found 244.0700.
IR (KBr)
n ;
3199, 3138, 3045, 2751, 1715, 1603, 1446, 1389 cmꢀ1 1H
NMR (400 MHz, DMSO-d6)
d
7.55 (1H, d, J ¼ 1.6 Hz, H-6), 8.15 (1H, d,
5.2.2.21. 8-Phenoxy-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(21). The title compound was prepared from 2-amino-3-nitro-4-
chloropyridine and sodium phenolate in a manner similar to 18
J ¼ 1.6 Hz, H-8); 13C NMR (100 MHz, DMSO-d6)
d 112.36, 123.20,
123.80, 138.46, 141.83, 154.55, 155.58; HRMS-EI C7H4BrN3O2 calcd
[MþH]þ 241.9565, found 241.9567.
as white solid (76 mg, 17.2%). IR (KBr) n 1716, 1688, 1621, 1493, 1381,
1202 cmꢀ1; 1HNMR (400 MHz, DMSO-d6)
d
6.49 (1H, d, J ¼ 5.6 Hz,
5.2.2.16. 8-Chloro-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione (16).
The title compound was prepared from 2-amino-4-chloropyridine
H-7), 7.20 (2H, m, C6H5-20 & 60), 7.29 (1H, m, C6H5-40), 7.49 (2H, m,
C6H5-30 & 50), 7.94 (1H, d, J ¼ 5.6 Hz, H-6), 11.91 (1H, s, NH-1), 12.38

30
D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
(1H, s, NH-4); 13C NMR (100 MHz, DMSO-d6)
d
106.48, 112.50,
EI C15H13N3O3 calcd [MþNa]þ 306.0855, found 306.0856.
120.19, 125.30, 130.25, 140.57, 143.21, 150.35, 154.04, 154.76, 156.00;
HRMS-EI C13H9N3O3 calcd [MþNa]þ 278.0542, found 278.0544.
5.2.2.27. 7-Fluoro-8-methyl-1,4-dihydropyrido[2,3-b]pyrazine-2,3-
dione (27). The title compound was prepared from 2-amino-4-
methyl-5-fluoropyridine according to the general procedure as
5.2.2.22. 8-(Benzyloxy)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(22). The title compound was prepared from 2-amino-3-nitro-4-
chloropyridine, benzyl alcohol and sodium hydride in a manner
white solid (441 mg, 22.6%). IR (KBr)
1625, 1523, 1454, 1381, 1275 cmꢀ1
2.3 (3H, d, J ¼ 1.6 Hz, CH3), 8.04 (1H, s, H-6), 11.64 (1H, s, NH-1),
12.33 (1H, s, NH-4); 13C NMR (100 MHz, DMSO-d6)
8.73 (d),
n
3047, 2971, 2866, 2783, 1718,
;
1H NMR (400 MHz, DMSO-d6)
similar to 18 as white solid (105 mg, 22.5%). IR (KBr)
n
1694, 1621,
d
1501, 1426, 1379, 1275, 1129 cmꢀ1 1H NMR (400 MHz, DMSO-d6)
;
d
d
5.34 (2H, s, CH2), 6.98 (1H, d, J ¼ 6.0 Hz, H-7), 7.29e7.44 (3H, m,
120.27 (d), 121.30 (d), 128.21, 128.49, 135.39 (d), 154.38 (d), 154.85,
155.31; HRMS-EI C8H6FN3O2 calcd [MþH]þ 196.0522, found
196.0516.
C6H5-20, 40 & 60), 7.57 (2H, d, J ¼ 8.4 Hz, C6H5-30 & 50), 7.95 (1H, d,
J ¼ 6.0 Hz, H-6), 11.62 (1H, s, NH-1), 12.26 (1H, s, NH-4); 13C NMR
(100 MHz, DMSO-d6) d 70.46, 104.44, 111.46, 128.20, 128.43, 128.74,
136.23, 140.02, 143.83, 151.63, 155.23, 156.40; HRMS-EI C14H11N3O3
5.2.2.28. 7-Chloro-8-methyl-1,4-dihydropyrido[2,3-b]pyrazine-2,3-
dione (28). The title compound was prepared from 2-amino-4-
methyl-5-chloropyridine according to the general procedure as
calcd [MþNa]þ 292.0698, found 292.0697.
5.2.2.23. 8-Phenylethoxy-1,4-dihydropyrido[2,3-b]pyrazine-2,3-
dione (23). The title compound was prepared from 2-amino-3-
nitro-4-chloropyridine, 2-phenylethanol and sodium hydride in a
white solid (325 mg, 15.4%). IR (KBr)
1613, 1449, 1381 cmꢀ1; 1H NMR (400 MHz, DMSO-d6)
CH3), 8.11 (1H, s, H-6), 11.62 (1H, brs, NH-1), 12.43 (1H, brs, NH-4);
n
3040, 2861, 2771, 1704, 1687,
d
2.42 (3H, s,
manner similar to 18 as white solid (178 mg, 36.4%). IR (KBr)
n
2862,
13C NMR (100 MHz, DMSO-d6)
d 13.71, 121.47, 125.86, 130.31, 137.75,
2754, 1703, 1620, 1502, 1386, 1281, 1139 cmꢀ1; 1H NMR (400 MHz,
140.05, 155.07, 155.28; HRMS-EI C8H6ClN3O2 calcd [MþH]þ
DMSO-d6)
d
3.14 (2H, t, J ¼ 6.8 Hz, CH2CH2O), 4.35 (2H, t, J ¼ 6.8 Hz,
212.0227, found 212.0218.
CH2CH2O), 6.92 (1H, d, J ¼ 5.6 Hz, H-7), 7.20e7.25 (1H, m, C6H5-40),
7.31 (2H, t, J ¼ 7.2 Hz, C6H5-20 & 60), 7.40 (2H, d, J ¼ 7.8 Hz, C6H5-30 &
50), 7.95 (1H, d, J ¼ 5.6 Hz, H-6), 11.48 (1H, s, NH-1),12.23 (1H, s, NH-
5.2.2.29. 7-Bromo-8-methyl-1,4-dihydropyrido[2,3-b]pyrazine-2,3-
dione (29). The title compound was prepared from 2-amino-4-
methyl-5-bromopyridine according to the general procedure as
4); 13C NMR (100 MHz, DMSO-d6)
d 34.36, 69.73, 103.50, 110.64,
126.36, 128.29, 129.08, 138.10, 139.32, 143.60, 151.47, 154.75, 155.97;
white solid (279 mg, 10.9%). IR (KBr)
1435, 1379 cmꢀ1; 1H NMR (400 MHz, DMSO-d6)
8.19 (1H, s, H-6), 11.58 (1H, brs, NH-1), 12.40 (1H, brs, NH-4); 13C
n
3032, 2854, 2762, 1697, 1609,
HRMS-EI C15H13N3O3 calcd [MþNa]þ 306.0855, found 306.0855.
d
2.45 (3H, s, CH3),
5.2.2.24. 8-Phenylpropoxy-1,4-dihydropyrido[2,3-b]pyrazine-2,3-
dione (24). The title compound was prepared from 2-amino-3-
nitro-4-chloropyridine, 3-phenylpropanol and sodium hydride in
NMR (100 MHz, DMSO-d6) d 16.68, 116.67, 121.61, 131.80, 138.28,
142.51, 155.18, 155.31; HRMS-EI C8H6BrN3O2 calcd [MþH]þ
255.9722, found 255.9723.
a manner similar to 18 as white solid (153 mg, 29.8%). IR (KBr)
3027, 2945, 2872, 2756, 1708, 1694, 1623, 1507, 1391, 1276,
1134 cmꢀ1 1H NMR (400 MHz, DMSO-d6)
2.03e2.13 (2H, m,
n
5.2.2.30. 7,8-Dichloro-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(30). To a solution of 2-amino-4-chloropyridine (1.28 g, 10.0 mmol)
in DMF (40 mL) at ꢀ20 ꢁC was added NCS (2.67 g, 20.0 mmol). This
mixture was allowed to warm to room temperature and stirred for
24 h, and then poured into 300 mL ice-water and extracted with
ethyl acetate. The extracts were washed with 1 M NaOH and brine,
dried and evaporated. The residue was purified by column chro-
matography on silica gel to give 4,5-dichloropyridin-2-amine
(1.12 g, 69.0%).
;
d
CH2CH2CH2O), 2.85 (2H, t, J ¼ 7.6 Hz, CH2CH2CH2O), 4.14 (2H, t,
J ¼ 6.0 Hz, CH2CH2CH2O), 6.87 (1H, d, J ¼ 5.6 Hz, H-7), 7.16e7.22
(1H, m, C6H5-40), 7.25e7.33 (4H, m, C6H5-20, 30, 50 & 60), 7.96 (1H, d,
J ¼ 5.6 Hz, H-6), 11.59 (1H, s, NH-1), 12.26 (1H, s, NH-4); 13C NMR
(100 MHz, DMSO-d6)
d 29.98, 31.31, 68.31, 103.25, 110.66, 125.76,
128.26, 128.38, 139.37, 141.60, 143.60, 151.72, 154.88, 156.01; HRMS-
EI C16H15N3O3 calcd [MþNa]þ 320.1011, found 320.1012.
To 30 mL 98% H2SO4 was added portionwise 4,5-
dichloropyridin-2-amine (1.0 g, 6.13 mmol) at ꢀ5 ꢁC. After the
solid material was completely dissolved, 3.7 mL fuming HNO3 was
added dropwise over 5 min maintaining internal temperature
ꢂ0 ꢁC. The mixture was allowed to warm to 50 ꢁC over 2 h, and then
stirred for another 1 h at this temperature before poured onto 150 g
crushed ice. The aqueous solution was basified to pH ¼ 7.3 with
ammonia and extracted with ethyl acetate. The combined extracts
were dried, concentrated and purified by column chromatography
to give 4,5-dichloro-3-nitropyridin-2-amine (0.80 g, 62.6%).
To a solution of 4,5-dichloro-3-nitropyridin-2-amine (300 mg,
1.44 mmol) in 5 mL AcOH was added portionwise zinc dust
(472 mg, 7.2 mmol) over 20 min. After addition, the mixture was
stirred for another 3 h then added dropwise to 100 mL saturated
Na2CO3 and extracted with ethyl acetate. The combined extracts
were washed with brine, dried, evaporated and purified by column
5.2.2.25. 6-(Benzyloxy)-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(25). The title compound was prepared from 2-amino-3-nitro-6-
chloropyridine, benzyl alcohol and sodium hydride in a manner
similar to 9 as white solid (121 mg, 26.0%). IR (KBr)
n 3028, 2952,
1697, 1623, 1594, 1476, 1456, 1340, 1261 cmꢀ1 1H NMR (400 MHz,
;
DMSO-d6)
d
5.29 (2H, s, CH2), 6.61 (1H, d, J ¼ 8.4 Hz, H-7), 7.28e7.40
(3H, m, C6H5-20, 40 & 60), 7.44 (1H, d, J ¼ 8.4 Hz, H-8), 7.48 (2H, d,
J ¼ 7.2 Hz, C6H5-30 & 50), 11.85 (1H, brs, NH-1),12.28 (1H, brs, NH-4);
13C NMR (100 MHz, DMSO-d6)
d 67.25,104.91,115.42,126.72,127.85,
128.31, 128.34, 136.08, 136.92, 154.37, 156.15, 157.82; HRMS-EI
C
14H11N3O3 calcd [MþNa]þ 292.0698, found 292.0697.
5.2.2.26. 6-Phenethoxy-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(26). The title compound was prepared from 2-amino-3-nitro-6-
chloropyridine, 2-phenylethanol and sodium hydride in a manner
similar to 9 as white solid (89 mg, 18.1%). IR (KBr)
1703, 1681, 1600, 1482, 1464, 1340, 1269 cmꢀ1; 1H NMR (400 MHz,
DMSO-d6)
n
3028, 2945,
chromatography
(148 mg, 57.5%).
to
give
4,5-dichloropyridine-2,3-diamine
d
3.01 (2H, t, J ¼ 7.2 Hz, CH2CH2O), 4.38 (2H, t, J ¼ 7.2 Hz,
A mixture of 4,5-dichloropyridine-2,3-diamine (148 mg,
CH2CH2O), 6.54 (1H, d, J ¼ 8.4 Hz, H-7), 7.18e7.35 (5H, m, C6H5),
0.83 mmol) in 8 mL diethyl oxalate was heated to reflux for 6 h
before cooled to room temperature. The resulted solid was filtered
and washed with ethyl acetate. The solid product was decolored
with activated charcol and recrystallized in water and DMF to give
7.42 (1H, d, J ¼ 8.4 Hz, H-8), 11.83 (1H, s, NH-1), 12.24 (1H, s, NH-4);
13C NMR (100 MHz, DMSO-d6)
d 34.67, 66.61, 104.75, 115.22, 126.27,
126.63, 128.29, 128.97, 136.19, 138.21, 154.36, 156.13, 158.02; HRMS-

D. Xie et al. / European Journal of Medicinal Chemistry 117 (2016) 19e32
31
[4] J.T. Kantrowitz, A.K. Malhotra, B. Cornblatt, G. Silipo, A. Balla, R.F. Suckow,
C. D'Souza, J. Saksa, S.W. Woods, D.C. Javitt, High dose D-serine in the treat-
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acid oxidase is involved in D-serine-induced nephrotoxicity, Chem. Res.
Toxicol. 18 (2005) 1678e1682.
the title compound as white solid (51 mg, 26.5%). IR (KBr)
3040, 1727, 1704, 1592, 1372 cmꢀ1 1H NMR (400 MHz, DMSO-d6)
8.24 (1H, s, H-6), 11.86 (1H, s, NH-1), 12.60 (1H, s, NH-4); 13C NMR
n 3113,
;
d
(100 MHz, DMSO-d6)
d 121.36, 123.38, 125.00, 138.97, 140.33,
155.04, 155.09; HRMS-EI C7H3Cl2N3O2 calcd [MþNa]þ 253.9500,
found 253.9502.
[7] M. Orozco-Ibarra, O.N. Medina-Campos, D.J. Sanchez-Gonzalez, C.M. Martinez-
Martinez, E. Floriano-Sanchez, A. Santamaria, V. Ramirez, N.A. Bobadilla,
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administration of dizocilpine, Biol. Psychiatry 65 (2009) 1103e1106.
[10] S.M. Smith, J.M. Uslaner, L. Yao, C.M. Mullins, N.O. Surles, S.L. Huszar,
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N.J. Brandon, B. Jones, S. Venkatraman, M.B. Young, N. Sachs, M.A. Jacobson,
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5.2.2.31. 7-Bromo-8-chloro-1,4-dihydropyrido[2,3-b]pyrazine-2,3-
dione (31). To a solution of 2-amino-4-chloropyridine (1.28 g,
10.0 mmol) in anhydrous CH3CN (40 mL) was added portionwise N-
bromosuccinimide (1.96 g, 11.0 mmol). The reaction mixture was
stirred at room temperature for 24 h then poured into 100 mL ice-
water and extracted with ethyl acetate. The combined extracts
were washed with 1 M NaOH and brine, dried and evaporated. The
residue was purified by column chromatography on silica gel to
give 5-bromo-4-dichloropyridin-2-amine (1.53 g, 60.8%). Next
steps followed the procedure of 30 and gave the title compound as
white solid (63 mg, 12.5% over 3 steps). IR (KBr)
1587, 1398, 1367 cmꢀ1; 1H NMR (400 MHz, DMSO-d6)
H-6), 11.82 (1H, s, NH-1), 12.59 (1H, s, NH-4); 13C NMR (100 MHz,
n
3108, 3033, 1703,
acid oxidase inhibitor compound
8 [4H-thieno[3,2-b]pyrrole-5-carboxylic
d
8.30 (1H, s,
acid] and D-serine,, J. Pharmacol. Exp. Ther. 328 (2009) 921e930.
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P. Zaratin, In vitro and in vivo pharmacological profile of AS057278, a selective
d-amino acid oxidase inhibitor with potential anti-psychotic properties, Eur.
Neuropsychopharm. 18 (2008) 200e214.
DMSO-d6)
d 114.00, 121.88, 127.18, 139.80, 143.23, 155.49, 155.61;
HRMS-EI C7H3BrClN3O2 calcd [MþNa]þ 297.8995, found 297.8993.
[12] T. Sparey, P. Abeywickrema, S. Almond, N. Brandon, N. Byrne, A. Campbell,
P.H. Hutson, M. Jacobson, B. Jones, S. Munshi, D. Pascarella, A. Pike, G.S. Prasad,
N. Sachs, M. Sakatis, V. Sardana, S. Venkatraman, M.B. Young, The discovery of
fused pyrrole carboxylic acids as novel, potent D-amino acid oxidase (DAO)
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6.9%). IR (KBr)
NMR (400 MHz, DMSO-d6)
12.58 (1H, s, NH-4); 13C NMR (100 MHz, DMSO-d6)
n ;
3236, 1767, 1687, 1582, 1419, 1194, 693 cmꢀ1 1H
d
8.20 (1H, s, H-6), 11.49 (1H, s, NH-1),
117.63,
d
123.34, 126.14, 138.88, 140.48, 155.49, 155.56; HRMS-EI
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5.2.2.33. 7,8-Dibromo-1,4-dihydropyrido[2,3-b]pyrazine-2,3-dione
(33). The title compound was prepared from 2-amino-4-
bromopyridine in a manner similar to 31 as white solid (17 mg,
5.2%). IR (KBr)
(400 MHz, DMSO-d6)
(1H, s, NH-4); 13C NMR (100 MHz, DMSO-d6)
n ;
1703, 1577, 1387, 1355, 1260 cmꢀ1 1H NMR
d
8.27 (1H, s, H-6), 11.45 (1H, s, NH-1), 12.57
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d
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Acknowledgments
This study was supported by the National Natural Science
Foundation of China [No: 81072623], the Shanghai Innovation
Project (No: 11ZR1416400, 12JC1404800) and the Shanghai Indus-
trial and Translational Project (No: 12431900603). We thank Dr.
Kenji Hashimoto at the Chiba University Center for Forensic Mental
Health in Japan for his kind gift of CBIO.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.017.
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