Dihydrobenzoxazinone derivatives as aldose reductase inhibitors with antioxidant activity

Article abstract of DOI:10.1016/j.bmc.2020.115699

Dihydrobenzoxazinone based design and synthesis produced two series of compounds as aldose reductase (ALR2) inhibitor candidates. In particular, phenolic residues were embodied into the compounds for the combination of strengthening the inhibitory acitvity and antioxidant ability to retard the progression of diabetic complications. Most of the derivatives with styryl side chains exhibited excellent activities on selective ALR2 inhibition with IC₅₀ values ranging from 0.082 to 0.308 μM, and {8-[2-(4-hydroxy-phenyl)-vinyl]-2-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl}-acetic acid (3a) was the most potent. More significantly, most of dihydrobenzoxazinone compounds revealed not only good inhibitory effect on ALR2, but also showed powerful antioxidant activity. Notably, phenolic compound 3a was even comparable to the well-known antioxidant Trolox, confirming that the C8 p-hydroxystyryl substitution was key structure of lowering oxidative stress. Therefore, these results provided an achievement of multifunctional ALR2 inhibitors possessing capacities for both ALR2 inhibition and as antioxidants.

Full text of DOI:10.1016/j.bmc.2020.115699

Journal Pre-proofs
Dihydrobenzoxazinone derivatives as aldose reductase inhibitors with antioxi-
dant activity
Huan Chen, Xin Zhang, Xiaonan Zhang, Zhenya Fan, Wenchao Liu, Yanqi
Lei, Changjin Zhu, Bing Ma
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S0968-0896(20)30529-0
https://doi.org/10.1016/j.bmc.2020.115699
BMC 115699
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Bioorganic & Medicinal Chemistry
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Please cite this article as: H. Chen, X. Zhang, X. Zhang, Z. Fan, W. Liu, Y. Lei, C. Zhu, B. Ma,
Dihydrobenzoxazinone derivatives as aldose reductase inhibitors with antioxidant activity, Bioorganic &
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115699
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Graphical Abstract
Dihydrobenzoxazinone derivatives as aldose
reductase inhibitors with antioxidant activity
Leave this area blank for abstract info.
Huan Chen, Xin Zhang, Xiaonan Zhang, Zhenya Fan, Wenchao Liu, Yanqi Lei, Changjin Zhu*, Bing Ma*.
Beijing Institute of Technology, No. 5, Zhongguancun South Street, 100081 Beijing, China

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Dihydrobenzoxazinone derivatives as aldose reductase inhibitors with antioxidant
activity
Huan Chen a, Xin Zhanga, Xiaonan Zhanga, Zhenya Fana, Wenchao Liua, Yanqi Leia, Changjin Zhua,*, Bing
Ma a,*.
aSchool of Chemistry and Chemical Engineering, Beijing Institute of Technology, No. 5, Zhongguancun South Street, 100081 Beijing, China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Dihydrobenzoxazinone based design and synthesis produced two series of compounds as aldose
reductase (ALR2) inhibitor candidates. In particular, phenolic residues was embodied into the
compounds for the combination of strengthening the inhibitory acitvity and antioxidant ability to
retard the progression of diabetic complications. Most of the derivatives with styryl side chains
exhibited excellent activities on selective ALR2 inhibition with IC50 values ranging from 0.082 to
0.308 μM, and {8-[2-(4-hydroxy-phenyl)-vinyl]-2-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl}-
acetic acid (3a) was the most potent. More significantly, most of dihydrobenzoxazinone
compounds revealed not only good inhibitory effect on ALR2, but also showed powerful
antioxidant activity. Notably, phenolic compound 3a was even comparable to well-known
antioxidant Trolox, confirming that the C8 p-hydroxystyryl substitution was determinative
structure of alleviating oxidative stress. Therefore, these results provided an achievement of
multifunctional ALR2 inhibitors possessing both capacities for ALR2 inhibition and as
antioxidants.
Available online
Keywords:
Aldose reductase inhibitors
Antioxidant activity
Dihydrobenzoxazinone derivatives
Diabetic complications
2009 Elsevier Ltd. All rights reserved.
 Corresponding author. E-mail: mabing@bit.edu.cn (B. Ma), zcj@bit.edu.cn (C. Zhu)

1. Introduction
further reduction in the polyol pathway of glucose metabolism
is supposed to be an efficient way to remedy and delay the
development of diabetic complications.15
Diabetes mellitus (DM) is a metabolic abnormality caused
by different elements such as genetic factors, immune
disorders, microbial infections, and their toxins, free radical
toxins, and mental factors, leading to chronic complications,
including neuropathy, nephropathy, and retinopathy,
associated with long-term damage, dysfunction, and failure of
different organs.1,2 Accumulation of sorbitol in the polyol
pathway of glucose metabolism and oxidative stress under the
condition of high-level blood sugar are regarded as the major
pathogenesis of diabetic complications.3-5
Figure 2. Structures of representative aldose reductase inhibitors.
By restraining the abnormal accumulation of sorbitol and
decreasing the level of oxidative stress, ALR2 inhibitors
(ARIs) are attractive agents preventing and alleviating diabetic
complications.16-18 Since the early 1970s, a large variety of
synthetic ARIs have been developed, and some of them have
been evaluated in preclinical and clinical trials (Figure 2).19-24
There are three major categories according to their functional
groups; spirohydantoin derivatives, sulfonyl derivatives, and
carboxylic acid derivatives. Spirohydantoin inhibitors such as
sorbinil and fidarestat were effective but resulted in
hypersensitivity and hepatotoxicity as side effects.24,25 New
classes of sulfonyl ARIs including ARI-809 and M-16209 have
been reported for their excellent selectivity and potent
inhibitory activity both in vitro and in vivo.26,27 However, the
efficacy of sulfonyl derivative in clinic trials remained
unknown. Comparing most reported inhibitors, the carboxylic
acid derivatives emerged as potent ARIs, in which epalrestat
has been identified as a highly potent ARI with a favorable
pharmacokinetic profile and is the only commercial one for
treatment of diabetic complications.29,30 Aside from epalrestat,
other carboxylic acid ARIs have failed in clinical trials because
of pharmacokinetic drawbacks, adverse side effects, or low
efficacy. Poor selectivity over aldehyde reductase (ALR1, EC
1.1.1.2), which plays a detoxification role in specifically
metabolizing toxic aldehydes, is believed to be the crucial
reason for the side effects.31-33 Both ALR2 and ALR1 belong
to the aldo-keto reductase superfamily, sharing structural
homology with the difference at the C-terminal end of the
enzyme proteins, which is the region lining the hydrophobic
pocket of the active site called the ‘specificity pocket’ and
responsible for specificity of inhibitors between the two
enzymes.34,35
Figure 1. Metabolic pathway of glucose and pathogenesis of diabetic
complications.
Aldose reductase (ALR2, EC 1.1.1.21) that is a member of
the aldo-keto reductase superfamily is the first rate-determining
enzyme of the polyol pathway (Figure 1).6,7 Due to a relatively
low affinity of ALR2 for glucose, only a small amount of
substrate is metabolized through the polyol pathway. Most of
the glucose is phosphorylated by hexokinase to form glucose
6-phosphate for glycolysis and the pentose phosphate pathway,
which is a metabolic pathway parallel to glycolysis.8,9 Under
hyperglycemia conditions, however, hexokinase is rapidly
saturated, and excess glucose is then metabolized by the polyol
pathway, in which ALR2 reduces glucose to sorbitol utilizing
NADPH as a cofactor.10,11 The strong polarity of sorbitol
hinders easy penetration through membranes and subsequent
removal from tissues by diffusion.12 Since sorbitol is formed
faster than it is converted to fructose, it will be accumulated
directly through the stimulated polyol pathway, and eventually
leading to osmotic imbalance, cell swelling, and membrane
permeability changes.2 Also, both the exhaustion of NADPH
and the disturbance of the NADH/NAD+ ratio alter cellular
redox potentials. This disorders impair the activity of enzymes
such as nitric oxide synthase (NOS) and glutathione reductase,
and further leading to cellular oxidative stress, as a
consequence of the imbalance between increased production of
radical oxygen species (ROS) and reduced intracellular
antioxidant defense.13 Furthermore, connected with the polyol
pathway activation, the increased level of fructose enhances the
intracellular formation of advanced glycation end products
which results in further generation of ROS.14 Obviously, both
osmotic stress and oxidative stress induced by enhancement of
ALR2 are responsible for cellular damage associated with
diabetic complications. Therefore, the ALR2 inhibition and
Thus, the design and synthesis of multifunctional ARIs may
be a new efficient strategy to selectively inhibit ALR2 and to
simultaneously retard the process of oxidative stress. Synthetic
benzoxazinones have demonstrated potential for the
development of new medications for treating diseases, and
some of them showed interesting activity of antioxidant.36-39
Besides, benzoxazinone derivatives were identified as potential
neuroprotective agents possessing the antioxidant effect
comparable to α-tocopherol.38 Notably, isopropyl-substituted
analogs have been tested in ALR2 inhibitory assay but showed

2.2. ALR2/ALR1 inhibitory activity
weak activity probably due to the improper side chain (IC50
>
1 μM).39 In the present study, a new series of substituted 3,4-
dihydro-(2H)-1,4-benzoxazin-2-one derivatives were designed
to verify their ALR2 inhibition, selectivity, and antioxidant
effect.
All benzoxazinone derivatives were screened for their
inhibitory effect on ALR2 isolated from rat lenses. Only
significantly active compounds coming from the test for the
ALR2 inhibition were then subjected to estimate for their
inhibitory activity against ALR1 isolated from rat kidneys, in
order to evaluate their selectivity for ALR2. Epalrestat was
utilized as a positive ARI to ensure validity. The results were
summarized in Table 1.
2. Result and discussion
2.1. Chemistry
The syntheses of all title compounds with an acetic acid
substituent at N4 position and a variety of aromatic residues at
C8 position of dihydrobenzoxazinone scaffold followed the
general route outlined in Scheme 1. (8-Bromo-2-oxo-2,3-
dihydro-benzo[1,4]oxazin-4-yl)-acetic acid (2a) as a key
intermediate was obtained by the N-alkylation of 2-amino-6-
bromophenol with 2-chloroacetic acid and then intramolecular
condensation. 2b without substituent at the C8 position of the
scaffold was prepared from 2-aminophenol and 2-chloroacetic
acid by a similar method. Different styrenes were readily
attached to the C8 position of 2a by Heck crossing coupling to
give C8-styryl derivatives (3a-j). Subjected to Suzuki coupling
reaction with the corresponding phenylboronic acid, C8-phenyl
compounds (4a-c) were then obtained.
In the ALR2 inhibition, compounds with the C8 side chain
were more efficient (3a-j, 4a-c) than the compound having no
such side chains (2b). There were two series of C8-substituted
compounds obtained according to the spacer length between
the C8 position and the aromatic ring of the side chain. The first
series of compounds contained styryl residues (3a-j) possessed
a two-atom length spacer, while in the other series (4a-c)
phenyl rings were directly attached to the C8 position.
Compounds in the first series showed good inhibitory activity
with IC50 values varying from 0.082 to 0.308 μM, which was
much stronger than the counterparts in the second series with
IC50 values ranging between 0.774 and 3.34 μM. Of all,
compound 3a embodied with the C8-vinylphenolic group
showed the most potent inhibitory effect (IC50 = 0.082 μM)
which is close to that of epalrestat, the positive control.
Besides, phenolic compound 3b also had excellent activity
(IC50 = 0.188 μM), demonstrating that the introduction of
phenolic group could reinforce the inhibitory activity. Among
the styryl series, the inhibitory activity of all compounds was
increased by substituents of alkyl, heteroalkyl, hetero, and
halogen at the aromatic residue of the styryl side (3a-i > 3j).
Interestingly, with increased hydrophobicity of the alkyl group
at C8-styryl residues, compounds were more active against
ALR2 (3d > 3c > 3j). In addition, the compounds with
electronic-donating groups or electronic-withdrawing groups
at the aromatic ring of the C8-styryl were active in the ALR2
inhibition, implying a positive effect by altering the electron
density of C8 aromatic rings.
3a R = p-OH-Ph
1
O
3b R = m-OMe,p-OH-Ph
1
3c R = p-Me-Ph
1
OH
O
3d R = p-tBu-Ph
1
N
O
3e R = p-OMe-Ph
1
3f R = p-NO -Ph
1
2
iii
3g R = p-CF -Ph
1
3
3h R = p-F-Ph
1
3i R = biphenyl
1
R
1
3j R = Ph
O
1
OH
O
3
NH
N
O
2
O
i
Cl
OH
OH
Br
Br
O
1a
2a
OH
O
iv
N
O
4a R = Ph
2
4b R = p-OMe-Ph
2
4c R = p-CHO-Ph
R
2
2
The synthetic products with potent activity of ALR2
inhibition (IC50 < 0.200 μM), as well as compounds 3j and 2b
for comparisons, were tested for their inhibition against ALR1
in order to evaluate their selectivity. Most of these compounds
were inactive, demonstrating their inclination of the ALR2
inhibition. Of them, compound 3a had the highest selectivity
towards ALR2 over ALR1 with the selectivity index (SI) of
60.58, revealing that 3a was superior to eparlestat. Notably, the
SI values were decreased by alkyl substitution yet increased by
polar residues.
O
4
OH
O
OH
NH
N
O
ii
O
Cl
OH
2
1b
2b
Scheme 1. Reagents and conditions: (i) NaOH, H2O 100 °C, 4 h, 42%; (ii)
NaOH, H2O 100 °C, 3 h, 56%; (iii) styrene, Pd(OAc)2, P(o-tolyl)3, Et3N,
DMF, 110 °C, 32~41%; (iv) C6H5B(OH)2, K2CO3, Pd(OAc)2, i-PrOH, H2O,
100 °C, 12~26%.

Table 1. Enzyme inhibition activity of dihydrobenzoxazinone derivatives
Substit.
Comp.
IC50 for ALR2 (μM) a
IC50 for ALR1 (μM) a
Selectivity index b
R
p-OH-Ph
m-OMe,p-OH-Ph
p-Me-Ph
p-tBu-Ph
p-OMe-Ph
p-NO2-Ph
p-CF3-Ph
p-F-Ph
X
3a
3b
3c
3d
3e
3f
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
-
0.082±0.011
0.188±0.017
0.220±0.097
0.170±0.064
0.208±0.082
0.262±0.069
0.138±0.047
0.167±0.018
0.281±0.018
0.308±0.067
3.34±0.71
4.992±0.345
4.498±0.507
60.58
23.96
0.669±0.333
3.93
3g
3h
3i
3.268±0.214
4.280±0.219
23.68
25.66
biphenyl
Ph
3j
2.812±0.097
9.11
4a
4b
4c
2b
Ph
p-OMe-Ph
p-CHO-Ph
H
-
0.774±0.054
1.05±0.31
-
-
1.932±0.421
0.045±0.010
13.72±1.552
1.198±0.034
7.10
Epalrestat
26.70
a n = 3.
b Defined as IC50[ALR1] : IC50[ALR2].
good selectivity in ALR2 inhibitory (3a-b, 3g, 3h) were tested for
DPPH radical scavenging activity, and compounds 3j, 4a and 2b
served as comparisons. Derivatives 3a-b, 3h, 3j, which were better
than Trolox, showed excellent DPPH radical scavenging activity
(Table 2). Among them, compound 3b had attractive antioxidant
properties, achieving 18.22% of inhibition rate at a concentration
of 1 μM. It should also be pointed out that 3a-b were more
effective than 3j, suggesting that the phenolic subunit might be a
structural determinant of DPPH free radical scavenging activity.
What is more, compounds 3a-b, 3g, 3h, and 3j also exhibited
better antioxidant activity than that 4a and 2b did, representing that
styryl side chains contributed to the elimination of free radicals.
2.3. DPPH radical scavenging activity
Oxidative stress plays a vital role in the pathological process of
diabetic complications. To decrease the level of oxidative stress
and alleviate the disorder, ARIs with antioxidant capacities were
desirable. Thus, the antioxidant functions of the compounds being
active in the ALR2 inhibition were investigated.
The free radical scavenging capacity of the compounds was
measured by a model reaction with the stable free radical of 1,1-
diphenyl-2-trinitrophenylhydrazine (DPPH), and 6-hydroxy-
2,5,7,8-chroman-2-carboxylic acid (Trolox) served as a reference
compound. Several dihydrobenzoxazinone derivatives which had
Table 2. DPPH radical scavenging activity of dihydrobenzoxazinone derivatives
Comp.
Substit.
% of inhibition of control value a

R
X
100 μM
10 μM
5 μM
1 μM
3a
3b
3g
3h
3j
p-OH-Ph
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
-
93.25±1.32
93.09±2.96
91.34±0.61
92.64±0.31
86.50±2.33
91.35±0.60
92.83±2.13
94.62±0.29
87.55±1.19
84.98±4.53
89.18±0.81
90.04±0.31
84.18±1.03
36.5±1.66
65.61±1.64
82.61±3.96
57.36±1.10
57.77±0.75
44.30±1.03
57.14±1.06
43.63±3.11
13.24±2.31
23.14±1.73
47.82±1.34
14.72±1.62
18.22±4.81
9.92±0.79
12.55±1.62
12.03±1.86
6.12±0.79
7.17±1.19
10.17±1.79
m-OMe,p-OH-Ph
p-CF3-Ph
p-F-Ph
Ph
4a
2b
Ph
H
-
Trolox
a n = 3
2.4. Lipid peroxidation suppression
Compounds 3a-b, 3h, and 3j that were already active in the
DPPH radical scavenging activity were evaluated in the
suppression of the lipid peroxidation. Compounds 3j and 2b
served as comparisons. Consistent with the conclusion of the
DPPH radical scavenging assay, inhibitory data of the lipid
peroxidation showed that tested compounds bearing styryl side
chains (3a-b, 3h, 3j) were active in the indicative
heterogeneous assay (Table 3). The activities of these
suppressors were equivalent to that of Trolox. Of them,
compounds 3a-b had appreciable antioxidant properties and
were more effective than the other tested compounds. This
indicated the importance of the phenolic substituents in the
derivatives for their antioxidant activity.
Generating from the reaction of thiobarbituric acid with
malondialdehyde (MDA), thiobarbituric acid reactive
substances (TBARS) are regarded as an index of lipid
peroxidation.
Induced
by
radical
oxidation,
the
lipoperoxidation product of MDA will finally result in dramatic
damage to cellular membranes and proteins. In order to further
identify whether the compounds described above can act as the
antioxidants through a different mechanism in an oxidative
pathway, we also examined the effect of compounds on the
hydroxyl radical-dependent lipoperoxidation in rat brain
homogenate induced by the oxidant system Fe(III)/ascorbic
acid.40,41
Table 3. Inhibition data of lipid peroxidation for benzoxazinone derivatives
Substit.
MDA inh. % a
100μM
Comp.
R
X
3a
3b
3h
3j
p-OH-Ph
CH=CH
CH=CH
CH=CH
CH=CH
-
81.70±1.22
80.72±0.92
74.84±0.46
75.82±1.22
72.22±1.67
75.49±2.40
m-OMe,p-OH-Ph
p-F-Ph
Ph
2b
H
Trolox
a n = 3
2.5. Scavenging activity of superoxide anion radicals

Superoxide anion radicals are involved in the onset of
various diseases such as cancer, cardiovascular diseases,
immune system decline, and diabetes. Under diabetic
conditions, various sources of superoxide are activated,
including xanthine oxidases, NADPH oxidases, uncoupling of
nitric oxide synthase, and mitochondrial fuel oxidative
processes. As a precursor of ROS, superoxide species are
overproduced and accumulated, resulting in damage to cellular
components.
Phenolic styryl compounds 3a-b had been proved to be
provided with excellent antioxidant effect in both the tests of
DPPH radical and MDA. Similarly, they exhibited good
activity suppressing superoxide anion radicals, and were
comparable to a reference compound of Trolox (3a ≈ 3b ≈
Trolox) (Table 4). Unfortunately, the activities of compounds
3h, 3j, and 2b were much lower than that of Trolox. Again,
these results are explicit that compounds with phenolic styryl
group had potential in the removal of superoxide radicals,
preventing the overproduction of ROS.
Table 4. Superoxide anion radicals scavenging activity of dihydrobenzoxazinone derivatives
Substit.
Comp.
O2●- inh. activity (U/uM) a
R
X
3a
3b
3h
3j
p-OH-Ph
CH=CH
CH=CH
CH=CH
CH=CH
-
1.07±0.01
1.02±0.03
0.79±0.15
0.81±0.04
0.73±0.03
1.13±0.01
m-OMe,p-OH-Ph
p-F-Ph
Ph
2b
H
Trolox
a
n = 3
to the active site of ALR2 (Fig. 3). The carboxyl group of these
compounds were inserted deeply into the anion binding pocket
by hydrogen bonds interacting. However, the carboxyl group
of compounds 2b totally “flowed out” of the anion binding
pocket. Moreover, the lengths of hydrogen bonds suggest that
compounds 3a, 3d-e, and 3g-j bound to the active site much
more tightly than 4a indicating the good inhibitory activity of
the C8-styryl compounds. Besides, the lengths of hydrogen
bonds formed by 3a and 3j in the anion binding pocket suggest
that 3a might be closer to the activate site than 3j. This was
coincident with the results from the ALR2 inhibition, in which
3a was more potent than 3j. Surprisingly, according to docking
results displayed in Fig. 3 and Table 5, hydrogen bonds between
the carboxyl group of lidorestat and amino residues located in
anion site were weaker than that of styryl type compounds.
Moreover, the binding energy for 3a, 3d-e, 3g-j and 4a was
calculated (Table 5) to further evaluate their inhibitory
efficiency. Data for 2b was excluded since its carboxyl group
failed in the binding to the anion pocket. All of the binding free
energy for 3a, 3d-e, and 3g-j were lower than that for 4a,
illustrating better affinities of C8-styryl derivatives for ALR2.
By the combination of the DPPH radical clearance, scavenging
of the superoxide anion radicals, and the MDA suppression,
dihydrobenzoxazinone derivatives have been proved to work
as potent antioxidants even compared favorably with the
typical antioxidant agent of Trolox. These results also revealed
that phenolic substitutions of the derivatives (3a-b) were
essential for the ideal antioxidant property of these ARIs.
2.6. Molecular docking
To better understand the binding details in the ALR2
inhibition of C8-styryl dihydrobenzoxazinone derivatives,
molecular docking of compounds 3a, 3d-e, and 3g-j
possessing significant activity in the ALR2 inhibition were
performed. Compounds 4a and 2b that were less active in the
ALR2 inhibition were also docked. The conformation resulted
from the ligand of lidorestat was selected for the docking study
because structures of compounds we potently identified were
close to the ligand more than other ligands. When docking of
compounds into the human ALR2/NADP+/lidorestat complex
(PDB code: 1Z3N),42 3a, 3d-e, 3g-j and lidorestat bound well

The docking behaviors of derivatives with the ALR1-NADP+
complex (PDB code: 1HQT)44 was also investigated to illustrate
the selectiveness towards ALR2 over ALR1. The total energies of
compounds 3a, 3d, 3g-h, 3j and lidorestat were calculated and
presented in Table 6. Remarkably, consistent with the results in
ALR1 inhibitory experiment, 3a exhibited highest reranked score
among all the tested derivatives, proving the best selectivity of 3a
for ALR2. Besides, the energy value of ligand 3d was much lower
than that of 3j, explaining the result that alkyl group located in
aromatic ring of C8-substituents had a negative effect on the
selectivity.
Among these compounds, 3a had the lowest total energy value
demonstrating the highest binding affinity and inhibitory
activity. 3g also showed a significant interaction with ALR2.
These results were consistent with that derived from the ALR2
inhibition. Notably, both hydrogen bonds energies of 3d and
3e were lower than that of 3a, yet the binding affinity scores
were higher. We consumed that the interactions might be affected
by steric hindrance of the alkyl groups located in aromatic ring of
C8-substituents. Although lidorestat displayed excellent
inhibition against ALR2 (IC50 = 5 nM)43, the docking energy result
revealed that lidorestat stabilized weak hydrogen bond interaction
with the enzyme.
Table 5. MolDock and reranked score for dihydrobenzoxazinone ligands against ALR2
Total Energy
Ligand
MolDock Score (Kcal/mol)
-172.130
Rerank Score (Kcal/mol)
-143.031
Hydrogen bonds (Kcal/mol)
-8.726
3a
3d
-154.254
-101.134
-96.155
-142.519
-97.708
-97.966
-95.392
-70.004
-134.905
-13.402
-10.697
-6.578
-5.895
-4.274
-6.487
-6.705
-5.000
3e
-138.379
3g
-173.017
3h
-137.533
3i
3j
-157.412
-133.745
4a
-130.337
lidorestat
-174.725
Table 6. MolDock and reranked score for dihydrobenzoxazinone ligands against ALR1
Total Energy
Ligand
MolDock Score (Kcal/mol)
-124.241
Rerank Score (Kcal/mol)
-68.757
Hydrogen bonds (Kcal/mol)
3a
-6.192
-7.609
-3.867
-4.544
-7.778
-7.312
3d
-132.956
-106.772
-97.454
-83.952
-98.323
-109.160
3g
3h
-124.075
-120.769
3j
-119.012
lidorestat
-140.112

3. Conclusions
chain to improve these ALR2 inhibitors. In general, to strengthen
the efficacy of retarding the long-term complications of diabetes,
compounds were designed for a combination of ALR2 inhibition
In this paper, we designed and synthesized a series of
dihydrobenzoxazinone based derivatives by altering of the C8 side

activity and .antioxidant Interestingly, most of the synthetic series
3 with styryl residues showed a good inhibitory activity and
selectivity towards ALR2, and 3a was verified as the most potent.
More significantly, 3a-b and 3g-h not only readily inhibit ALR2
with remarkable selectivity but also exhibited excellent activities
in DPPH radical scavenging, lipid peroxidation suppression and
superoxide anion radical’s clearance. Notably, containing with
phenolic group in the C8-styryl side chain, 3a-b were even
superior to the conventional antioxidant Trolox. The results
derived from structure-activity relationship analysis combined
with further docking studies demonstrated that the substitution of
C8-styryl side chain had a positive effect on the inhibitory activity
and the selectivity, and introduction of p-hydroxyl to the aromatic
rings of the side chains reinforced both the inhibitory activity and
the antioxidant property. As a consequence, with a structural
50.34, 50.15 ppm. HRMS (ESI) m/z calcd for [M-H]- 206.0532,
found 206.0463 .
4.4. General procedure for preparation of (2-Oxo-8-styryl-2,3-
dihydro-benzo[1,4] oxazin-4-yl)-acetic acid (3)
Pd(OAc)2 (0.011 g, 0.05 mmol) and P(o-tolyl)3 (0.030 g, 0.10
mmol) were added to a solution of 2a (0.287 g, 1 mmol) in DMF
(5 mL) and Et3N (0.5 mL). After being stirred at room temperature
under argon for 20 min, the appropriate styrene (1.2 mmol) was
added. The reaction mixture was stirred at 110 °C for 14 h. After
the completion of the reaction, the mixture was poured in NaOH
solution and washed with 100 mL of ethyl acetate five times. The
aqueous phase was collected and 6M HCl solution was added until
pH at 2~3, then extracted by ethyl acetate three times to collect
organic layer, dried with MgSO4. After filtration and evaporation
of ethyl acetate, the residue was purified by silica gel column
chromatography with petroleum ether/ethyl acetate (3:1~1:1) to
give desirable products 3, yield 32~41%.
determinant
of
C8
p-hydroxystyryl
side
chain,
dihydrobenzoxazinone compound 3a could serve as a significant
lead for the achievement of multifunctional ALR2 inhibitors.
4. Experiment
4.4.1.
{8-[2-(4-Hydroxy-phenyl)-vinyl]-2-oxo-2,3-dihydro-
4.1. Chemistry
benzo[1,4]oxazin-4-yl}-acetic acid (3a)
Melting points were recorded on an X-4 microscopic melting
point apparatus and are uncorrected. All reactions were routinely
checked by TLC on silica gel Merck 60F254. The NMR spectra
were recorded on a Bruker Ascend 400 M spectrometer (400 MHz
Yield: 0.111 g (34%); brown solid; m.p. 165-167 °C; purity:
98.17%, 1H NMR (400 MHz, DMSO-d6) δ 13.12 (s, 1H), 9.66 (s,
1H), 7.58 (m, 3H), 7.41(d, J = 2.1 Hz, 1H) 7.04 (m, 2H), 6.89 (d,
J = 7.4 Hz, 1H), 4.74 (s, 2H), 4.51 (s, 2H). ; 13C NMR (100 MHz,
DMSO-d6) δ 169.94, 164.92, 158.01, 142.29, 130.65, 129.75,
129.49, 128.42, 126.74, 122.93, 120.61, 118.48, 116.12, 114.27,
52.23, 43.35 ppm. HRMS (ESI) m/z calcd. for [M-H]- 324.0950 ,
found 324.0889 .
1
for H NMR, 100 MHz for 13C NMR) and chemical shifts were
given in δ units (ppm) relative to internal standard TMS and refer
to DMSO-d6 solutions. HRMS (ESI) was performed using an
AGILENT LC/MS. Analysis of sample purity was performed on a
Hitachi D-2000 Elite HPLC system. HPLC conditions were the
following: Inertsil ODS-2 250 mm × 10 mm, 5 mm column;
mobile phase: CH3CN (0.1% TFA) /CH3OH = 1/1, for 10 min;
room temperature; flow rate: 1 mL min-1 ; detection at λ 254 nm.
All final compounds in biological assays have a purity of ≥95%.
4.4.2.
{8-[2-(3-Methoxy-4-hydroxy-phenyl)-vinyl]-2-oxo-2,3-
dihydro-benzo[1,4]oxazin-4-yl}- acetic acid (3b)
Yield: 0.128 g (36%); yellow solid; m.p. 156-157 °C; purity:
97.31%, 1H NMR (400 MHz, DMSO-d6) δ 12.79 (s, 1H), 9.32 (s,
1H), 7.29 (m, 1H), 7.21 (m, 3H) 7.09 (m, 2H), 6.74 (d, J = 7.4 Hz,
2H), 6.63 (d J = 2.3 Hz, 1H), 4.16 (s, 2H), 4.13 (s, 2H), 3.68 (s,
1H). ; 13C NMR (100 MHz, DMSO-d6) δ 169.82, 165.00, 148.31,
147.42, 131.15, 129.70, 129.10, 126.80, 122.97, 118,76, 114.27,
110.53, 67.55, 60.22, 56.11 ppm. HRMS (ESI) m/z calcd for [M-
H]- 354.1056, found 354.0998 .
4.2. Procedure for preparation of (8-Bromo-2-oxo-2,3-dihydro-
benzo[1,4]oxazin -4-yl)-acetic acid (2a)
A mixture of 6-bromo-2-aminophenol (8.82 g, 0.049 mol),
chloroacetic acid (9.8 g, 0.1 mol), NaOH (6 g, 0.15 mol), in 100
mL water was stirred at 100 °C for 4 h under argon. A solution of
NaOH (6 M, aq) was added dropwise to the reaction in order to
maintain pH at 7–8. The reaction mixture was cooled and
concentrated HCl was added until the pH of the solution was
strongly acidic. Solid brown deposits were filtered and purified by
silica gel column chromatography with petroleum ether/ethyl
acetate (3:1) to give the desired compound 2a, yield 42%.
4.4.3. [2-Oxo-8-(2-p-tolyl-vinyl)-2,3-dihydro-benzo[1,4]oxazin-
4-yl]-acetic acid (3c)
Yield: 0.132 g (41%); yellow solid; m.p. 125-127 °C; purity:
95.88%, 1H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 7.43 (d,
J = 7.4 Hz, 2H), 7.41-7.20 (m, 5H) 7.09 (m, 1H), 6.74 (d, J = 7.4
Hz, 1H), 4.15 (s, 2H), 4.12 (s, 2H), 2.46 (s, 3H). ; 13C NMR (100
MHz, DMSO-d6) δ 171.40, 164.87, 138.79, 137.95, 134.59,
129.77, 124.98, 120.32, 116.83, 112.62, 50.51, 50.15, 21.33 ppm.
HRMS (ESI) m/z calcd for [M-H]- 322.1158, found 322.1093.
4.2.1. (8-Bromo-2-oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl)-acetic
acid (2a)
Yield: 5.86 g (42%); brown solid, 1H NMR (400 MHz, DMSO-
d6) δ 12.91 (s, 1H), 7.51 (s, 1H), 6.93 (m, 1H), 6.86 (d,J = 7.8 Hz,
1H), 4.20 (s, 2H), 4.16 (s, 2H).
4.4.4.
{8-[2-(4-tert-Butyl-phenyl)-vinyl]-2-oxo-2,3-dihydro-
benzo[1,4]oxazin-4-yl}-acetic acid (3d)
Yield: 0.128 g (35%); pink solid; m.p. 142-143 °C; purity:
96.21%, H NMR (400 MHz, DMSO-d6) δ 13.21-12.83 (s, 1H),
4.3. Procedure for preparation of (2-Oxo-2,3-dihydro-
benzo[1,4]oxazin-4-yl)-acetic acid (2b)
Compound 2b was prepared by similar method with 2-
aminophenol, yield 56%.
1
7.51 (d, J = 7.4 Hz, 2H), 7.48 (d, J = 7.4 Hz, 2H), 7.41-7.32 (m,
3H) 7.11 (m, 1H), 6.71 (d, J = 7.4 Hz, 1H), 4.13 (s, 2H), 4.10 (s,
2H), 1.89 (s, 9H). ;13C NMR (100 MHz, DMSO-d6) δ 171.41,
164.87, 151.10, 138.80, 134.61, 130.97,124.98, 120.53, 116.82,
112.65, 50.56, 50.16, 34.84, 31.52 ppm. HRMS (ESI) m/z calcd
for [M-H]- 364.1627, found 364.1554.
4.3.1. (2-Oxo-2,3-dihydro-benzo[1,4]oxazin-4-yl)-acetic acid
(2b)
Yield: 5.68 g (56%); pink solid; m.p. 166-168 °C; purity:
1
98.92%, H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H), 7.21-
4.4.5.
{8-[2-(4-Methoxy-phenyl)-vinyl]-2-oxo-2,3-dihydro-
7.18 (d, J = 7.8 Hz, 2H), 6.91 (m, 1H), 6.89 (d, J = 7.8 Hz, 1H),
4.19 (s, 2H), 4.14 (s, 2H) ; 13C NMR (100 MHz, DMSO-d6) δ
171.33, 165.04, 141.55, 134.72, 125.44, 119.85, 116.81, 113.56,
benzo[1,4]oxazin-4-yl}-acetic acid (3e)
Yield: 0.135 g (40%); white solid; m.p. 170-171 °C; purity:
95.46%, 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H), 7.52 (d,

J = 2.5 Hz, 2H), 7.32 (s, 3H) 7.12 (m, 1H), 6.96 (d, J = 7.4 Hz,
2H), 6.61 (d J = 2.3 Hz, 1H), 4.15 (s, 2H), 4.11 (s, 2H), 3.72 (s,
1H). ; 13C NMR (100 MHz, DMSO-d6) δ 174.41, 164.90, 159.72,
138.68 135.22, 130.84, 128.43, 125.88, 124.96, 119.00, 116.71,
114.75, 112.35, 55.64, 50.50, 50.16 ppm. HRMS (ESI) m/z calcd
for [M-H]- 338.1107, found 338.1038.
was added. The mixture was stirred at 100 °C for 30 h under an
atmosphere of argon, and then washed with ethyl acetate five
times. The aqueous solution was obtained and the pH of the
solution was adjusted at 2~3 by 6M HCl aqueous. The suspension
was extracted by ethyl acetate three times, dried with MgSO4, and
filtered and concentrated under reduced pressure. The crude
product was flashed by column chromatography(ethyl
acetate/petroleum ether 1:5~1:1) to provide the products 4, yield
12~26%.
4.4.6.
{8-[2-(4-Nitro-phenyl)-vinyl]-2-oxo-2,3-dihydro-
benzo[1,4]oxazin-4-yl}-acetic acid (3f)
Yield: 0.138 g (39%); white solid; m.p. 168-169 °C; purity:
97.33%, 1H NMR (400 MHz, DMSO-d6) δ 12.83 (s, 1H), 8.22 (s,
2H), 7.91 (d, J = 2.5 Hz, 1H), 7.58 -7.49 (m, 2H), 7.38 (s, 1H) 7.12
(m, 1H), 7.10 (d, J = 7.4 Hz, 2H), 6.65 (d, J = 2.3 Hz, 1H), 2.93 (s,
2H), 2.87 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 170.32,
162.78, 146.81, 144.36 143.18, 141.60, 139.31, 134.78, 134.67,
127.84, 124.60, 124.57, 120.09, 116.88, 113.68, 52.22, 50.05 ppm.
HRMS (ESI) m/z calcd for [M-H]- 353.0852, found 353.0788.
4.5.1 (2-Oxo-8-phenyl-2,3-dihydro-benzo[1,4]oxazin-4-yl)-acetic
acid (4a)
Yield: 0.045 g (16%); white solid; m.p. 141-143 °C; purity:
96.05%, 1H NMR (400 MHz, DMSO-d6) δ 12.89 (s, 1H), 7.76 (d,
J = 2.3 Hz, 1H), 7.54 (d, J = 7.5 Hz, 2H), 7.21 (d, J = 7.5 Hz, 2H),
6.88 (m, 2H), 4.22 (s, 2H), 3.87 (s, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 170.94, 167.44, 164.01, 137.07, 136.02, 132.18,
131.99, 129.79, 129.71, 129.65, 129.55, 129.14, 128.66, 50.55,
50.03 ppm. HRMS (ESI) m/z calcd for [M-H]- 282.0845, found
282.0782 .
4.4.7.
{2-Oxo-8-[2-(4-trifluoromethyl-phenyl)-vinyl]-2,3-
dihydro-benzo[1,4]oxazin-4-yl}-acetic acid (3g)
Yield: 0.121 g (32%); yellow solid; m.p. 213-215 °C; purity:
1
98.49%, H NMR (400 MHz, DMSO-d6) δ 13.24-12.81 (s, 1H),
4.5.2.
[8-(4-Methoxy-phenyl)-2-oxo-2,3-dihydro-
7.91 (d, J = 2.5 Hz, 1H), 7.85 -7.71 (m, 4H), 7.60 -7.42 (m, 2H),
7.33 -7.10 (m, 2H), 7.38 (s, 1H) 7.12 (m, 1H), 2.97 (s, 2H), 2.86
(s, 2H) ;13C NMR (100 MHz, DMSO-d6) δ 169.76, 164.90, 142.98,
129.79, 129.12, 127.87, 127.64, 127.54, 127.39, 127.35, 126.14,
125.66, 124.77, 123.07, 121.43, 60.22, 43.11 ppm. HRMS (ESI)
m/z calcd for [M-H]- 376.0875, found 376.0805.
benzo[1,4]oxazin-4-yl]-acetic acid (4b)
Yield: 0.037 g (12%); pink solid; m.p. 191-192 °C; purity:
95.30%, 1H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 7.62 (d,
J = 7.2 Hz, 2H), 7.52 (d, J = 7.5 Hz, 1H), 7.47 (d, J = 2.6 Hz, 1H),
7.21 (d, J = 7.2 Hz, 1H), 6.97 (m, 2H), 4.85 (s, 2H), 4.62 (s, 2H) ;
13C NMR (100 MHz, DMSO-d6) δ 169.19, 159.38, 151.97, 137.47,
130.95, 129.98, 129.89, 122.34, 121.02, 114.44, 114.26, 114.15,
65.31, 55.63 ppm. HRMS (ESI) m/z calcd for [M-H]- 312.0950,
found 312.0890 .
4.4.8.
{8-[2-(4-Fluoro-phenyl)-vinyl]-2-oxo-2,3-dihydro-
benzo[1,4]oxazin-4-yl}-acetic acid (3h)
Yield: 0.108 g (33%); yellow solid; m.p. 169-170 °C; purity:
1
97.10%, H NMR (400 MHz, DMSO-d6) δ 13.00-12.78 (s, 1H),
4.5.3.
[8-(4-Formyl-phenyl)-2-oxo-2,3-dihydro-
7.69 (d, J = 7.2 Hz, 3H), 7.15 -7.31 (m, 4H), 7.12 (t, J = 7.2 Hz,
1H), 7.12 (d, J = 2.5 Hz, 1H), 4.21 (s, 2H), 4.10 (s, 2H) ;13C NMR
(100 MHz, DMSO-d6) δ 171.39, 164.81, 138.86, 135.25, 129.05,
128.91, 124.99, 121.35, 116.06, 112.82, 97.89, 50.54, 50.15 ppm.
HRMS (ESI) m/z calcd for [M-H]- 326.0907, found 326.0850.
benzo[1,4]oxazin-4-yl]-acetic acid (4c)
Yield: 0.080 g (26%); white solid; m.p. 154-156 °C; purity:
97.33%,1H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 1H), 10.14 (s,
1H), 8.07 (d, J = 7.2 Hz, 2H), 7.86 (d, J = 2.6 Hz, 2H), 7.30 (m,
1H), 7.87 - 7.65 (m, 2H), 4.38 (s, 2H), 4.22 (s, 2H) ; 13C NMR (100
MHz, DMSO-d6) δ 193.30, 171.37, 164.92, 143.03, 138.44,
135.61, 130.46, 128.29, 121.02, 113.82, 50.53, 50.19 ppm. HRMS
(ESI) m/z calcd for [M-H]- 310.0794, [M+HCOO-]- 356.0770,
found 310.0752, 356.1169.
4.4.10.
[8-(2-Biphenyl-4-yl-vinyl)-2-oxo-2,3-dihydro-
benzo[1,4]oxazin-4-yl]-acetic acid (3i)
Yield: 0.123 g (32%); red solid; m.p. 212-213 °C; purity:
1
95.18%, H NMR (400 MHz, DMSO-d6) δ 13.22-12.81 (s, 1H),
7.82 - 7,69 (m, 7H), 7.51 (d, J = 7.2 Hz, 3H), 7.28 (d, J = 2.4 Hz,
2H), 7.21 (d, J = 2.3 Hz, 1H), 6.72 (d, J = 7.2 Hz, 1H), 4.33 (s,
2H), 4.15 (s, 2H) ;13C NMR (100 MHz, DMSO-d6) δ 171.40,
167.43, 164.84, 140.03, 138.90, 136.55, 135.28, 129.13, 127.50,
125.02, 121.48, 116.95, 50.55, 50.16 ppm. HRMS (ESI) m/z calcd
for [M-H]- 384.1314, found 384.1245.
4.6. Enzyme assays
The ALR2 inhibition activity was tested in a reaction mixture
containing 0.25 mL NADPH (0.10 mM), 0.25 mL sodium
phosphate buffer (pH = 6.2, 0.1 M), 0.1 mL enzyme extracted from
rat lens, 0.15 mL deionized water, and 0.25 mL D,L-
glyceraldehyde (10 mM) as substrate in a final volume of 1 mL.
Before adding to substrate, the reaction mixture was incubated at
30 °C for 5 min, then the substrate was added to start the reaction,
which was monitored for 4 min. The ALR1 inhibition activity was
performed at 37 °C in a reaction mixture containing 0.25 mL
NADPH (0.12 mM), 0.25 mL enzyme extracted from rat kidney,
0.25 mL sodium phosphate buffer (pH=7.2, 0.1 M), and 0.25 mL
sodium D-gluconate (20 mM) as substrate in a final volume of 1
mL. Before adding to sodium D-gluconate, the reaction mixture
was incubated at 37 °C for 4 min, then the substrate was added to
start the reaction, which was monitored for 4 min.
The inhibitory activities of the newly synthesized compounds
against ALR2 and ALR1 were assayed by adding 5 μL of the
inhibitor solution to the reaction mixture described above. All
compounds were dissolved in dimethyl sulfoxide (DMSO), and
the solutions were diluted with DMSO to desirable concentrations.
To correct for the nonenzymatic oxidation of NADPH, the rate of
NADPH oxidation in the presence of all of the reaction mixture
4.4.12.
(2-Oxo-8-styryl-2,3-dihydro-benzo[1,4]oxazin-4-yl)-
acetic acid (3j)
Yield: 0.127 g (41%); white solid; m.p. 176-177 °C; purity:
98.49%, 1H NMR (400 MHz, DMSO-d6) δ 12.51 (s, 1H), 7.53 (d,
J = 7.2 Hz, 2H), 7.48 - 7.40 (m, 2H), 7.38 - 7.31 (m, 2H), 7.22 (d,
J = 7.2 Hz, 2H), 7.10 (d, J = 2.3 Hz, 1H), 6.71 (d, J = 7.2 Hz, 1H),
4.31 (s, 2H), 4.22 (s, 2H) ;13C NMR (100 MHz, DMSO-d6) δ
171.40, 164.84, 138.89, 137.34, 135.25, 129.30, 127.06, 125.01,
121.40, 116.95, 112.83, 50.53, 50.15 ppm. HRMS (ESI) m/z calcd
for [M-H]- 308.1001, found 308.0941.
4.5. General procedure for preparation of (2-Oxo-8-phenyl-2,3-
dihydro-benzo[1,4]-oxazin-4-yl)-acetic acid (4)
To 50 mL flask, Pd(OAc)2 (0.035 g, 0.15 mmol), K2CO3 (0.415
g, 3 mmol), water (8 mL), appropriate phenylboronic acid (2.5
mmol) and 2a (0.287 g, 1 mmol) in isopropanol (2 mL) solution

components except the substrate was subtracted from each
experimental rate. The compounds were tested at concentrations
ranging from 100 μM to 10 nM. Most dose-response curves were
generated using at least three concentrations of the compound with
inhibitory activity between 20% and 80%, with three replicates at
each concentration, and the IC50 values were calculated by least-
square analysis of the linear portion of the log (dose) versus
response curves (r2 > 0.95). The experiments were performed in
triplicate.
Scavenging activity of superoxide anion radicals assay was
monitored by manufacturer’s instruction of inhibition and produce
superoxide anion assay kit (Nanjing Jiancheng Bioengineering
Institute, Nanjing, China). To a tube, reagent and compounds were
added by following order: reagent 1 (1 mL), title compound (0.05
mL), reagent 2 (0.1 mL), reagent 3 (0.1 mL), reagent 4 (0.1 mL).
Resulting mixture was incubated at 37 °C for 40 min, and then
chromogenic reagent (2 mL) was added into the tube. After
standing for 10 min, the solution was measured at 550 nm with a
spectrophotometer. The experiments were performed in triplicate.
Activity of inhibition against superoxide anion radicals production
4.7. Antioxidant assays
4.7.1. DPPH radical scavenging activity
Acontrol  A
sample 150 Csample (M )
(U/μmol) =
To investigate the antioxidant activity of the given compounds
in a homogeneous system, an experiment based on the stable free
radical DPPH scavenging rate was conducted. Briefly, 100 μL
methanol solution of testing compounds (or the reference
compound Trolox) with varies of concentrations was added into
the tube containing 1 mL of DPPH methanol solution (0.025
mg/mL) and 1.9 mL of methanol, to obtain final concentrations of
100, 10, 5 and 1 μM. The absorbance of the stable DPPH radical
in the methanol solution at 517 nm continually decrease in the
presence of the tested compounds, as measured. And the
percentage of DPPH radical scavenging was determined after
steady state by the equation as shown below. The experiments
were performed in triplicate.
Acontrol  Ablank
Control: ascorbic acid (150 mg/L)
Sample: title compound
Blank: distilled water
4.8. Docking studies
Docking was performed using Molegro Virtual Docker
(version 5.0) with the same protocol as described previously.45
Energy data was calculated by Molegro Virtual Docker.
Acknowledgments
Percentage
Asample  A
of
DPPH
scavenging(%)
=
This work was supported by the National Natural Science
Foundation of China (grant no. 21272025). Furthermore, the
authors would like to thank Analysis & Testing Center, Beijing
Institute of Technology (China) for technical support for
performing in the structural analysis of synthetic compounds.
(1
blank )100%
Acontrol
Sample: tested compound + DPPH + methanol
Blank: tested compound + methanol
Control: DPPH + methanol
4.7.2. Inhibition of lipid peroxidation
Fresh rat brain was isolated and crushed with ice-cold normal
saline to prepare 25% brain homogenate. The homogenate was
centrifuged for 10 min at a speed of 3000 rpm, and the supernatant
was used for biochemical analyses. The MDA concentration in the
supernatant was determined using a commercially available kit
based on thiobarbituric acid (TBA) reactivity. The reaction
mixture containing the title compounds (100 μM), FeCl3 (0.02
μM), ascorbic acid (0.1 μM) and brain homogenate supernatant
was incubated at 37 °C for 30 min. Then, after mixing TBA with
the reaction mixture and centrifuging, a supernatant was obtained.
MDA concentration tests in the supernatants were carried out
following the manufacturer’s instructions (Malondialdehyde assay
kit (TBA method), Nanjing Jiancheng Bioengineering Institute,
Nanjing, China). Briefly, the working mixture of the kit was
prepared by volume ratio of reagent 1 : reagent 2 : reagent 3 = 1:
30: 10. Supernatants (0.1 mL) were added into tubes containing
working mixture (4 mL). After boiled in 95°C for 40 min, the tubes
were iced and centrifuged (4000 rpm, 10 min). The developed red
product of the resulting reaction was measured at 532 nm with a
spectrophotometer. The experiments were performed in triplicate.
Percentage
of
MDA
clearance(%)
=
Asample  A
blank1 )100%
Acontrol  Ablank 2
(1
Sample: tested compound + brain homogenate
Blank1: tested compound + saline
Control: methanol + brain homogenate
Blank2: methanol + saline
4.7.3. Superoxide anion radicals scavenging activity

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Graphic Abstract
Dihydrobenzoxazinone derivatives revealed potent activities of aldose reductase inhibition
and antioxidant against diabetic complications.

Declaration of interests
√ The authors declare that they have no known
competing financial interests or personal
relationships that could have appeared to influence
the work reported in this paper.
☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests: