Multifunctional agents based on benzoxazolone as promising therapeutic drugs for diabetic nephropathy

Article abstract of DOI:10.1016/j.ejmech.2021.113269

Diabetic nephropathy (DN) is resulted from activations of polyol pathway and oxidative stress by abnormal metabolism of glucose, and no specific medication is available. We designed a novel class of benzoxazolone derivatives, and a number of individuals were found to have significant antioxidant activity and inhibition of aldose reductase of the key enzyme in the polyol pathway. The outstanding compound (E)-2-(7-(4-hydroxy-3-methoxystyryl)-2-oxobenzo[d]oxazol-3(2H)-yl)acetic acid was identified to reduce urinary proteins in diabetic mice suggesting an alleviation in the diabetic nephropathy, and this was confirmed by kidney hematoxylin-eosin staining. Further investigations showed blood glucose normalization, declined in the polyol pathway and lipid peroxides, and raised glutathione and superoxide dismutase activity. Thus, we suggest a therapeutic function of the compound for DN which could be attributed to the combination of hypoglycemic, aldose reductase inhibition and antioxidant.

Full text of DOI:10.1016/j.ejmech.2021.113269

European Journal of Medicinal Chemistry 215 (2021) 113269
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Multifunctional agents based on benzoxazolone as promising
therapeutic drugs for diabetic nephropathy
Xin Zhang, Huan Chen, Yanqi Lei, Xiaonan Zhang, Long Xu, Wenchao Liu, Zhenya Fan,
Zequn Ma, Zhechang Yin, Lingyun Li, Changjin Zhu*, Bing Ma**
School of Chemistry and Chemical Engineering, Beijing Institute of Technology, No. 5, Zhongguancun South Street, 100081, Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Diabetic nephropathy (DN) is resulted from activations of polyol pathway and oxidative stress by
abnormal metabolism of glucose, and no specific medication is available. We designed a novel class of
benzoxazolone derivatives, and a number of individuals were found to have significant antioxidant ac-
tivity and inhibition of aldose reductase of the key enzyme in the polyol pathway. The outstanding
compound (E)-2-(7-(4-hydroxy-3-methoxystyryl)-2-oxobenzo[d]oxazol-3(2H)-yl)acetic acid was iden-
tified to reduce urinary proteins in diabetic mice suggesting an alleviation in the diabetic nephropathy,
and this was confirmed by kidney hematoxylin-eosin staining. Further investigations showed blood
glucose normalization, declined in the polyol pathway and lipid peroxides, and raised glutathione and
superoxide dismutase activity. Thus, we suggest a therapeutic function of the compound for DN which
could be attributed to the combination of hypoglycemic, aldose reductase inhibition and antioxidant.
© 2021 Elsevier Masson SAS. All rights reserved.
Received 29 September 2020
Received in revised form
24 January 2021
Accepted 30 January 2021
Available online 7 February 2021
Keywords:
Drug design
Antioxidants
Inhibitors
Hypoglycemic
Diabetic nephropathy
1. Introduction
diabetic rodent models [8]. Particularly, ARIs have been studied for
approximately 40 years for the treatment of chronic diabetic
Diabetic nephropathy (DN) is a major cause of end-stage renal
disease (ESRD), and therapeutic options for preventing its pro-
gression are limited [1]. The number of DN patients are growing
with the sharply increasing diabetic mellitus, therefore much
research for DN treatment focuses on the development of new
agents including inhibitors of renin-angiotensin-aldosterone sys-
tem (RAAS) [2] and sodium-glucose co-tranporter-2 (SGLT-2) [3],
activators of adenosine monophosphate activated protein kinase
(AMPK) [4], and aldose reductase inhibitors (ARIs) [5]. Both RAAS
and SGLT-2 inhibitors have their individuals on market. However,
RAAS inhibition has a concomitant increased risk of hyperkalemia,
renal impairment, and hypotension (e.g. telmisartan, ramipril) [6].
SGLT-2 inhibitors pose a risk for urinary tract infections as they
promote glucosuria (e.g. dapagliflozin) [7]. The favorable renopro-
tective effects of AMPK activators are being verified constantly in
complications but no success is found on DN [9]. Epalrestat is
currently the only ARIs available for the therapy of peripheral
neuropathy in Japan and more recently in China and India. Yet
despite all that, ARIs may still be a compelling approach to the
therapy of diabetic complications especially DN and therefore more
powerful agents are expected to be developed.
Aldose reductase (ALR2, EC 1.1.1.21) is the first and rate-limiting
enzyme in the polyol pathway which plays a key role in trans-
duction of metabolic abnormalities leading to diabetic complica-
tions [10]. ALR2 catalyzes the NADPH-dependent reduction of
glucose to sorbitol, which in turn converted into fructose with
accompanied by NADþ reduction under catalysis of sorbitol dehy-
drogenase. In the case of hyperglycemia, the hexokinase and
glucokinase as normal pathways of glucose metabolism appear to
be saturated, and the polyol pathway can utilize a ratio of about 30%
of total glucose [11], but there is no corresponding increase in
sorbitol dehydrogenase (SDH), resulting in the raise of intracellular
sorbitol concentration, eventually leading to osmotic imbalance,
cell swelling, and membrane permeability changes [12]. The
oxidative stress related to polyol pathway may also play a pivotal
role in the development of DN because the sharp decrease in
NADPH causes changes in cellular redox potential and impairs the
activity of enzymes such as nitric oxide synthase (NOS) and
* Corresponding author.
** Corresponding author.
E-mail addresses: 1175852337@qq.com (X. Zhang), chenhuaninbuct@163.com
(H. Chen), 15313252939@163.com (Y. Lei), zhangxiaonan965@163.com (X. Zhang),
1938270368@qq.com (L. Xu), Liuwhhh@163.com (W. Liu), 729266587@qq.com
(Z. Fan), 834238277@qq.com (Z. Ma), 2261820165@qq.com (Z. Yin), 3220191170@
bit.edu.cn (L. Li), zcj@bit.edu.cn (C. Zhu), mabing@bit.edu.cn (B. Ma).
https://doi.org/10.1016/j.ejmech.2021.113269
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
Abbreviations
AGEs
GSH
GR
advanced glycation end products
glutathione
glutathione reductase
DN
diabetic nephropathy
ESRD
RAAS
SGLT-2
AMPK
ARIs
ALR2
ALR1
VEGF
SDH
end-stage renal disease
SOD
RH
Rox
superoxide dismutase
renin-angiotensin-aldosterone system
sodium-glucose co-tranporter-2
adenosine monophosphate activated protein kinase
aldose reductase inhibitors
aldose reductase
aldehyde reductase
vascular endothelial growth factor
sorbitol dehydrogenase
reduced generic cellular molecule
oxidized generic cellular molecule
1,10-carbonyldiimidazole
2,2-diphenyl-1-picrylhydrazyl
thiobarbituric acid reactive materials
malondialdehyde
glucose tolerance test
albumin to creatinine ratio
free fatty acids
CDI
DPPH
TBARs
MDA
GTT
ACR
FFA
UAER
urinary albumin excretion rate
glutathione reductase [13]. Moreover, ARIs could partially correct
hyperfitration and elevated urinary albumin excretion rate (UAER)
[14] via the vascular endothelial growth factor (VEGF) suppression
[15]. They also alleviated hyperglycemia-mediated mesangial
expansion [16], and glucose- and advanced glycation end products
(AGEs)-driven profibrotic effects [17]. Therefore, the AR inhibition
may reduce the progression rate of DN in humans.
In addition, abnormal metabolism of glucose generally causes
mitochondrial superoxide overproduction and the activation of a
series of oxidation pathways including increased intracellular for-
mation of AGEs [18], increased expression of the receptor for AGEs
and its activating ligands, and over activation of protein kinase C
isoforms (PKC) [19]. These changes result in cellular oxidative
stress, as a result of the imbalance between increased production of
reactive oxygen species (ROS) and reduced intracellular antioxidant
defense [20]. The oxidative stress could cause defective angiogen-
esis, and activate a number of proinflammatory pathways, which
are events involved in the formation of DN [21,22]. We thus propose
that the search for multi-agents acting in the ALR2 inhibition and
antioxidant may be a promising strategy [9].
We have recently developed several groups of ARIs based on
structures of quinoxaline and pyridopyrazine, and they were
proved to be potent in the ALR2 inhibition with the antioxidant
activity in vitro (Fig.1, and Table S1 in Supporting Information) [23].
Carboxylic acid and phenolic hydroxyl groups were found to play a
key role in the ALR2 inhibition and antioxidant [23]. However,
further studies for the most potent compound (E)-2-(3-(3,4-
dihydroxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetic acid (10) in the
diabetic mice only obtained mixed results from antioxidant ex-
aminations. Besides, it did not show any effect on blood sugar
(Fig. S1eS2, Supporting Information). For our continue effort to
discover excellent agents, we screened a number of structures
including quinoxaline and pyridopyrazine by molecular docking
studies and identified the benzoxazolone as core structure to build
desired compounds. Therefore, in the present work, we designed a
series of the benzoxazolone derivatives with carboxyl acid and
phenolic hydroxyl groups as new multifunctional ARIs, and found
that a number of compounds are more powerful in ALR2 inhibition
and antioxidant both in vitro and in vivo. Particularly, even thera-
peutic function in DN including hypoglycemic action was also
found in diabetic animal models.
2. Results and discussion
2.1. Chemistry
We synthesized a series of benzoxazolone derivatives bearing
different substituents as described in Scheme 1. To afford the esters
3 as key intermediates for generating various target compounds,
compound 1 was cyclized by 1,10-carbonyldiimidazole (CDI) and
then N-alkylation. Different phenyl groups were readily attached to
the C4eC7 position of the benzoxazolinone in 3 by Heck coupling to
construct the C4eC7 styryl side chains. Hydrolysis of 4 with lithium
hydroxide afforded the desired carboxylic acid 5 as ARI candidates.
In order to decrease the length of the C4eC7 side chain in the core
structure, the Suzuki coupling was employed to give the desired 9.
To distinguish between the carbon-carbon double bond and single
bond as spacers, 6 was prepared by the Pd/C hydrogenation of 4.
2.2. Polyol pathway and antioxidant in vitro
2.2.1. Inhibition of enzymes in vitro and docking simulation
A group of compounds including series 5, 7, and 9 were designed
(Scheme 1). All compounds were tested for potential inhibition
against ALR2 isolated from rat lens. To evaluate their selectivity for
ALR2 inhibition, their inhibitory activity against ALR1 isolated from
rat kidneys was also examined. The results are summarized in
Table 1.
Most of benzoxazolone derivatives with N3-acetic acid group
and C7-benzyl showed strong and selective inhibition against ALR2
with IC50 values ranging from 0.007 to 0.387
mM. Of the com-
pounds, 5i showed best inhibitory activity against ALR2 with an
IC50 value of 6.9 nM and a 15-fold enhancement both in potency
and selectivity with respect to epalrestat (Table 1). Further, the
kinetic evaluation of 5i revealed that this compound produced
noncompetitive pattern of inhibition with respect to D,L-
glyceraldehyde as a substrate and uncompetitive with respect to
NADPH as a cofactor at concentrations approximating the IC50
value (Fig. S3eS4, Supporting Information). Structure-activity
relationship analysis indicates that the styrene group at the posi-
tion C7 of the benzoxazolone core was crucial importance for the
Fig. 1. Aldose reductase inhibitors based on quinoxaline and pyridopyrazine.
2

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
Scheme 1. (a) N, N0-Carbonyldiimidazole, DMF, 65 ꢀC; (b) BrCH2CO2CH3, K2CO3, CH3CN, 65 ꢀC, 2 h; (c) styrene, Pd(OAc)2, P(o-tolyl)3, Et3N, DMF, 100 ꢀC; (d) LiOH, H2O, THF, rt, 2 h,
then 0.1 N HCl; (e) H2, 10% Pd/C, CH3OH, rt, 6 h; (f) C6H5B(OH)2, Na2CO3, Tetrakis(triphenylphosphine)palladium(0), DMF, H2O, 100 ꢀC.
building of potent ARIs based on the core structure when
comparing compounds 5a-c with 5e. Then, the potency was overall
enhanced by the addition of substituents to the C7-styrene ring (5f-
k, except 5j). The substituent effect of the phenyl ring in the C7 side
chain on the ALR2 activity reappeared in C7-phenyl series 9, in
which compounds 9b-f with different substituents on the phenyl
ring were more active than was the unsubstituted 9a, and the effect
was intensified with bigger sized substituents on the ring because
compounds 9e with 4-phenyl, 9i with 4e10-naphthyl, and 9j with
4e20-naphthyl had two-to six-fold activity higher than did 9a.
Besides, the spacer between the phenyl group and C7 of benzox-
azolone core had an obvious impact on the activity. C7-styryl de-
rivatives (5e, 5f, and 5h) were likely more potent in the inhibition
than C7-phenyl (9a, 9c, and 9d, and the single bond spacer was
negative for the inhibition activity as observed by the comparisons
of 5f with 7a, and 5i with 7b.
All of the tested compounds were found to be slightly active on
aldehyde reductase (ALR1) compared to ALR2, demonstrating their
selectivity for ALR2.
To investigate the possible binding mode of tested inhibitors,
the compound 5i possessing excellent activity of the ALR2 inhibi-
tion was studied by molecular docking performed on the human
ALR2/NADPþ/IDD594 complex (PDB entry code 1Z3N). The docking
results revealed that compound 5i was tightly bound in the active
site of ALR2 (Fig. 2), in which the carboxylate group formed three
hydrogen bonds with Tyr 48 (2.3 and 2.4 Å), His 110 (2.6 Å) and
engaged a stable electrostatic interaction with the positively
charged nicotinamide moiety of the NADPþ cofactor
(NeO ¼ 4.28 Å). In addition, the benzoxazolone core structure
formed two additional hydrogen bonds with the side chain of Trp
111 (2.5 and 2.4 Å) and a stable stacking interaction with the indole
ring of the Trp111 side chain. Further, the 3-methoxy,4-
hydroxystyryl ring of the C7 side chain of 5i also formed a
hydrogen bond with the side chain of Thr 113 (2.6 Å) and was well
paralleled to the indole ring of Trp 111 forming two stacking
interactions.
2.2.2. Antioxidant activity in vitro
Oxidative stress may play a vital role in the development of
diabetic nephropathy, and of our designed compounds only those
containing phenolic hydroxyl group were tested for their antioxi-
dant property. According to a previously reported method [24], the
model reaction with the stable free radical of 2,2-diphenyl-1-
picrylhydrazyl (DPPH) was applied and Trolox was employed as a
positive control. Five compounds (5g, 5i, 5k, 7b, and 9h) revealed
obvious antioxidant activity with DPPH radical scavenging rates
ranging from 22.7% to 61.7% at the concentration of 10 mM (Fig. 3a).
Among them, the compound 9h containing phenolic 3,4-
dihydroxyl groups was the most excellent indicating the account
of the antioxidant effect by the phenolic hydroxyl structure. It is
noteworthy that 5i had not only high DPPH radical scavenging rate
but high potency in the aldose reductase inhibition.
3

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
Table 1
Enzyme Inhibition Activity of benzoxazolone Derivatives.
Substituent
Compd
R1
R2
R3
R4
IC50 (nM)a for ALR2 IC50 (
m
M)a for ALR1 (IC50ALR1)/(IC50ALR2)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
7a
7b
9a
9b
9c
CH¼CHeC6H5
H
H
H
H
H
H
H
(5.5 1.0) ꢁ 104
225.7 52.6
65.7 19.4
46.5 10.1
54.7 16.3
10.6 3.7
34.8 5.3
33.8 10.2
6.9 2.1
363.1
17.4
2.57
575
1698
6.3
67.6
29.5
4.9
2884
0.79
173.8
15.5
446.7
14.4
1.05
32.4
6.2
173.8
72.4
6.6
45.7
8.9
6.6
77
39
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH¼CHeC6H5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH¼CHeC6H5
CH¼CHeC6H4-4-OH
12365
31042
594
1943
873
710
20140
17
1394
172
3004
1485
44
510
82
8198
847
17
2040
387
1261
46
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH¼CHeC6H5
CH¼CHeC6H4-4-OCH3
CH¼CHeC6H4-4-OH
CH¼CHeC6H3-3,4-diOCH3
CH¼CHeC6H3-3-OCH3, 4-OH
CH¼CHeC6H4-4-CF3
CH¼CHeC6H3-3,4-diOH
CH2eCH2eC6H4-4-OCH3
143.2 40.0
45.4 14.7
124.7 25.2
CH2eCH2eC6H4-3-OCH3, 4-OH 90.1 7.8
C6H5
C6H4-4-CF3
C6H4-4-OCH3
C6H3-3,4-diOCH3
C6H4-4-Ph
C6H4-4-C(CH3)
C6H3-2,4-diOCH3
C6H3-3,4-diOH
C6H4-4-10- Naphthyl
C6H4-4-20- Naphthyl
C6H4-4-CH2OH
148.7 15.8
9.7 1.7
23.8 0.9
63.5 15.8
75.4 7.4
21.2 3.6
85.5 17.3
386.9 110.0
22.4 5.3
23.0 3.2
24.5 3.9
108.1 39.0
9d
9e
9f
9g
9h
9i
3
9j
9k
epalrestat
30.9
5.0
a
IC50 (95% CL) values represent the concentration required to decrease enzymatic activity by 50%. Values were determined from dose eresponse curves generated using at
least three concentrations. The results for ALR2 or ALR1 activity assays are from three or one measurements.
The antioxidant activity of the synthetic compounds were
confirmed by the test of hydroxy radical dependent lip-
operoxidation in rat brain homogenate. Still, compound 5i per-
formed well-reduced production of thiobarbituric acid reactive
materials (TBARs) as shown in Fig. 3b. TBARs, employed as an index
of lipid peroxidation, are generated from the reaction of thio-
barbituric acid with lipoperoxidation product of malondialdehyde
(MDA), which is induced by radical oxidation and causes huge
damage to cellular membranes and proteins [25]. Comparing 5k
with 9h, and 5i with 7b, demonstrating the antioxidant role of the
hydroxystyryl structure as the mechanism observed for the anti-
oxidant curcumin [26].
It should be also noted at this point that the antioxidant activity
is considered as an important indication for the ability of com-
pounds to suppress the oxidative stress resulting from the forma-
tion of AGE stress and the activation of PKC, which are implicated in
the development of long-term diabetic complications [27]. Because
of the good activity of compound 5i in the ALR2 inhibition and the
antioxidant with negligible cytotoxicity (Fig. S5, Supporting Infor-
mation), it may provide an excellent leading structure for the
candidate in the therapy of diabetic nephropathy.
Fig. 2. Docking of 5i into the active site of ALR2. (a) The protein structure is shown in
ribbon and tube representation with selected residues labelled and shown in line
2.3. Polyol pathway and antioxidant in vivo
representation, ligand 11i is shown as stick models. The docked pose of 11i is shown in
We further established the activity of compound 5i in vivo in the
cyan (C), red (O), blue (N). Hydrogen bonds are shown as yellow dashed lines and
streptozocin (stz)-diabetic mice model, because of its best aldose
reductase inhibitory activity and antioxidant capacity in the above
stacking interaction are shown as red dashed lines. (b) Protein residues are in surface
representation.
4

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
Fig. 3. (a) DPPH radical scavenging activity. (b) Inhibition of lipid peroxidation in vitro.
Fig. 4. Renal cortical sorbitol levels in all groups at week-8 after diabetic mice receiving 5i, or metformin, or epalrestat intervention. Diabetic mice were orally administrated with
compound 5i with high dose (160 mg/kg) and low dose (80 mg/kg) for 8 weeks, epalrestat (80 mg/kg) and metformin (80 mg/kg). Data indicate mean S.E.M (n ¼ 12). *P < 0.05 and
**P < 0.01 ***P < 0.001 vs. stz-group.
in vitro assay.
5

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
2.3.1. The reduction of sorbitol in kidney by compound 5i
was evaluated by changes in the urine protein and in the kidney
micromorphology.
Examination of sorbitol in kidney cortex showed a decrease in
its accumulation by the 5i employment by 19% (P < 0.05) in low
dose group, by 18% (P < 0.05) in high dose group, and 24% (P < 0.01)
in 5i-intervened group compared with stz-group (Fig. 4). This
demonstrates clearly the ALR2 inhibition in vivo by the compound,
and it is consistent with the in vitro ALR2 inhibition. Epalrestat had
similar effect as the positive control whereas metformin did not
work. These results indicate the polyol pathway in the kidney
declined by ARIs of 5i and epalrestat.
As showing in Fig. 7a, the urine total protein excretion of
0.82 mg/mg in stz-group was detected at 4 weeks after diabetes,
and then it was 0.99 mg/mg at 6 weeks and 1.01 mg/mg at 8 weeks,
respectively, revealing a gradual growth with the administration
time. The urine total protein in the 5i-high-dose group was found at
0.76 mg/mg (93%, P > 0.05) at 4 weeks post-diabetes, and at
0.64 mg/mg (78%, P > 0.05) in the 5i-low-dose group. Both were
lower than that in the stz-group but did not reach a statistical
significance. However, at 6 weeks after the diabetes, the urine total
protein dropped to 0.50 mg/mg (50%, P < 0.05) in the 5i-low-dose
group and further to 0.36 mg/mg (36%, P < 0.05) in the 5i-high-dose
group showing a significant alleviation with an obvious dose
dependent pattern. At week-8, the total protein level was 0.48 mg/
mg (53%, P < 0.001) for the 5i-low-dose group and 0.58 mg/mg
(58%, P < 0.001) the 5i-high-dose group, and both levels were
significantly lower than their counterparts of stz-group. A slight
reduction in the total protein was observed for metformin-group by
6% at week-4, 41% (P < 0.05) at week-6, and 18% at week-8,
respectively. In contrast, the urine total protein in epalrestat-
group grew gradually over the administration time similar to the
pattern in stz-group, and it was elevated significantly at week-6 and
-8 compared to that at week-4. In addition, no significant difference
between epalrestat-group and stz-group was observed during the
administration time.
2.3.2. The antioxidant effect of compound 5i
To further understand protective mechanism of the compound
in kidney under high blood sugar, we tested antioxidant ability of 5i
by changes in the level of three markers including liver MDA, su-
peroxide dismutase (SOD) in kidney, and glutathione (GSH) in
kidney. By the daily administration of 5i, the level of the liver MDA
significantly fell 67% in low dose group of 5i, 45% in high dose
group, and 54% in 5i-intervened group compared to that in stz-
group (Fig. 5a). When compared to normal group, the low dose
group received 16% of reduction in the MDA, while the high dose
group and 5i-intervened group had raises of 39% and 15%, respec-
tively, implying that the MDA could be changed to around the
normal level. Stz-group reached 250% of the normal in the MDA
level. As compared to the stz-group, metformin decreased 43% of
the MDA and epalrestat 65% (Fig. 5a).
The mice treated with 5i gained significantly elevated SOD level
by 38% in the high dose group and by 177% in the low dose group.
The SOD level in the stz-group largely fell, reaching 48% of the
normal group (Fig. 5b).
For the GSH, the 5i-treated group with high dose achieved the
level as high by approximately 210% as did stz-group, and by
approximately 170% with low dose. 5i-intervened group was at the
level by approximately 150% (Fig. 5c). Compared with the normal
group, the GSH level in stz-group is decreased by about 46%. These
studies demonstrate that the treatment with 5i could suppress
polyol pathway-induced glutathione depletion, and provided
compelling evidences for the antioxidant of 5i in DN.
When mice having been positive in total urine protein were
administrated with 5i leading to the 5i-intervened group, their
total urine protein was decreased significantly at 2 weeks after the
administration, and the decrease was also observed at week-4
(Fig. 7b).
A significant reduction in albumin to creatinine ratio (ACR)
compared with stz-group was obtained by 79% for the 5i-high-dose
group, 67% for the 5i-low-dose-group, and 57% for the 5i-inter-
vened group at 8 weeks after diabetes (Fig. 7c). Consistent with the
urine total protein, albuminuria was significantly dropped by the 5i
treatment. Moreover, all three groups treated with 5i displayed no
significant differences in ACR with the normal-group (P > 0.05)
indicating the functional recovery again. ACR in metformin-group
decreased yet still higher insignificantly than normal-group
(P < 0.05) while it in epalrestat-group did not reach a statistical
difference with that in the stz-group.
Kidney sections from normal-group, stz-group, 5i-low-dose
group, 5i-high-dose group, and 5i-intervened group were exam-
ined by HE staining. Significantly inflammatory cell infiltration, and
exfoliation and necrosis of some renal tubular epithelial cells were
seen in kidney from the stz-group (Fig. 8a and b). The tubular
dilatation with a large cystic cavity and glomerular atrophy were
also obvious. On the other hand, no such kidney lesions were
observed in the 5i-low-dose group, and the kidney maintained
normally structure with well-defied renal tubules and glomerulus
(Fig. 8c). Only slight glomerular atrophy and inflammatory cell
infiltration were observed in the 5i-high-dose group, and the kid-
ney maintained totally normal structure with clearly visible renal
tubules and glomerulus (Fig. 8d). No any virtually pathological
changes in the 5i-intervened group were observed and the kidney
structure remained normal with clearly visible structures of the
renal tubules and glomerulus although renal tubular epithelial cells
looked like a few loose (Fig. 8e). Therefore, for the kidneys treated
with 5i retained normal structure inspite of slight inflammatory
cell infiltration and insignificant glomerular atrophy were present,
and they were remarkably different from those from the stz-group.
As a result, the renal injury could be alleviated by the employment
of 5i.
2.4. Blood sugar lowering effect of compound 5i
Blood glucose levels of diabetic mice were significantly dropped
by the administration of compound 5i. In high dose group, the fall
of 35% (P < 0.001) was observed at week-2, and then 23% (P < 0.05)
at week-4, 19% (P < 0.05) at week-6, and 20% (P < 0.05) at week-8,
respectively (Fig. 6a). In low dose group, the corresponding drop-
pings were 18% (P < 0.05), 24% (P < 0.05), 19% (P < 0.01), and 9%. In
the 5i-intervened group, a reduced blood glucose was observed at
second and fourth week after the administration as compared to
that at the beginning (Fig. 6b). Pharmacodynamics study for 5i
showed that blood glucose after the administration was keeping
lowered level than those in stz- and epalrestat-group during 5 h of
the investigation, and the improvement was significant in initial 2 h
(Fig. 6c). Then, glucose tolerance test (GTT) revealed that 5i-inter-
vened group had a higher tolerance to glucose than stz-group
(Fig. 6d). These demonstrated the ability of 5i in the normaliza-
tion of blood sugar while epalrestat had no such activity.
2.5. The reno-protective effect of compound 5i
Because of the excellent performance of the compound 5i in the
polyol pathway inhibition and the antioxidant both in vitro and
in vivo, and in the hypoglycemia, it was anticipated that the com-
pound could have therapeutic action on DN of mice. Therefore, the
therapy effect of the compound on the kidney under hyperglycemia
Due to the urgency of agents for DN treatment, we designed and
6

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
Fig. 5. Effects of compound 5i, epalrestat, and metformin on the oxidative status in diabetic mice. (a) Quantification of liver MDA; (b) The activity of kidney SOD; (c) Quantification
of kidney GSH. Values are expressed as mean S.E.M. of 12 mice in each group. *P < 0.05 and **P < 0.01 ***P < 0.001 vs. stz-group.
7

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
time, and were at similar levels of urine total protein and ACR
demonstrating that epalrestat was ineffective for the DN treatment
although early study reported that epalrestat as well as zopolrestat
had the effect of reducing urinary protein and anti-fibrosis [28,29].
Additionally, the detailed hematoxylin-eosin staining samples of
kidney showed a strong protect effect in the 5i treated groups with
mild inflammatory cell infiltration and significantly reduced
tubular dilation. However, obvious renal injury was observed in stz-
group with inflammatory cell infiltration, tubular dilatation, and
glomerular atrophy. Our findings suggest that the kidney protect
effect may attribute to the combination of antioxidant activity,
ALR2 inhibition, and blood sugar lowering by the 5i administration.
In a fact, 5i normalized significantly a group of multiple oxidant
stress markers in stz-model mice other than its antioxidant in vitro.
Contents of SOD and GSH in the kidney were greatly enhanced with
5i both in low dose and high dose, and MDA in liver was normalized
to a level as the same as the normal group. The mitochondrial
isoform of SOD degrades ROS to hydrogen peroxide [30], and GSH
could be catalyzed to reduce the hydrogen peroxide and organic
hydroperoxide [31]. Thus, it is reasonable that 5i scavenged ROS
and quenched lipid peroxides, and in turn alleviated oxidative
stress in the kidney. Epalrestat also exhibited the significant anti-
oxidant effect in the mice models although it was little poorer than
5i, probably because it was a metabolic antioxidant which was
reported to correspondingly normalize levels of oxidant metabo-
lites related to ALR2 [32]. GSH level in the epalrestat-group was
increased but lowered than that in 5i treated groups, and MDA
levels decreased similar to that in normal group. Metformin was
poorer in the antioxidant effect than epalrestat although also a few
of increased GSH and decreased MDA were identified, which were
supposed to be derived from blood lowering behavior. Neither
epalrestat nor metformin was observed for antioxidant activity
in vitro in the present study. These results collectively suggest the
outstanding antioxidant efficacy of 5i which was superior to epal-
restat and metformin.
ALR2 was inhibited in vivo effectively as the sorbitol level in 5i-
treated groups decreased, and this is close to the epalrestat group.
In combination with the fact that 5i has demonstrated obvious
kidney protection investigated by the decreases in the total urinary
protein and ACR other than epalrestat, we suggest that only the
ALR2 inhibition and the metabolic antioxidant may be not enough
to protect against DN.
In addition to the ALR2 inhibition and the antioxidant of the
compound 5i, the hypoglycemic effect was found for the com-
pound. The blood sugar level in model mice had been lowered with
5i during the oral administration for 8 weeks, and the efficacy was
even close to that of metformin, which was most effective at week-
8. 5i worked on the blood sugar significantly in 2 h after the oral
administration by the pharmacokinetic investigation. Further, it
improved the glucose tolerance capacity although the ability was
lower than that of metformin. However, metformin is unlikely to be
enough for the DN treatment because it obtained mixed results in
the urine protein reduction and is less used as a single treatment
drug for diabetic nephropathy while some studies supports met-
formin’s ability to decreased the urine albumin excretion rate [31].
5i reduced albuminuria superior than metformin. Therefore, only
hypoglycemic and antioxidant are still insufficient yet for protect-
ing kidney from hyperglycemic damage.
Taken together, our results demonstrate the effect of the com-
pound 5i on the renal protection in animal models stronger than
that of metformin probably due to the multiple function including
the blood sugar lowering, the ALR2 inhibition and the antioxidant.
Accordingly, we proposed a mechanism of 5i on the renal protec-
tion as illustrated in Fig. 9. First, the blood sugar was lowered with
5i resulting in anti-inflammatory in DN as chronic hyperglycemia
Fig. 6. Hypoglycemic effect in stz-induced diabetic mice. (a) The changes of blood
glucose in 8 weeks; (b) The changes of blood glucose in 4 weeks in 5i-intervened group
orally given with 5i (80 mg/kg) for 4 weeks after the significant rise of urine total
protein; (c) Hypoglycemic in pharmacokinetic; (d) Intraperitoneal glucose tolerance
tested in 120 min. Data were expressed as the mean
standard error of the mean
(n ¼ 12). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. stz-group.
identified the class of benzoxazolone derivatives as potent ARIs, of
which compound 5i exhibited strong bi-bioactivity in vitro in the
ALR2 inhibition and the antioxidant, and was then subjected to in a
daily oral gavage in stz-diabetic mice model. As a result, it gained
control over the progression of renal insufficiency under hyper-
glycemia because both the urine total protein excretion and ACR
were decreased by the 5i treatments. It is worthy to note the
decrease in the urine total protein from the 5i-intervened group
during the period of 4 weeks, and in particular it was normalized
significantly at week-2. In contrast, stz and epalrestat groups pro-
duced a steady rise in total urinary protein with the administration
8

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
Fig. 7. Improved kidney function in diabetic mice. (a) Urinary total protein in 4 weeks. (b) Urinary total protein in 4 weeks in 5i-intervened group. (c) Urine albumin excretion at 8
weeks after diabetes. Data are presented as the means standard deviation (n ¼ 12). *P < 0.05 and **P < 0.01 ***P < 0.001 vs. stz-group.
9

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
Fig. 8. Pathological changes in renal tissues of mice stained with hematoxylin-eosin imaged by light photomicrographs of kidney sections (Scale bars, 100 mm). (a) normal group,
histological kidney structure with well-defied renal tubules (red arrow) and glomerulus (yellow arrow). (b) stz-group, tubular dilatation with a large cystic cavity (yellow arrow),
renal tubules with epithelial cells exfoliated and necrotic (red arrow), glomerular atrophy (green arrow), and inflammatory cell infiltration (black arrow). (c) 5i-low-dose-group, a
normal histological kidney structure with no tubular dilatation (red arrow), no exfoliated and necrotic renal tubules epithelial cells, no glomerular atrophy (yellow arrow), and no
inflammatory cell infiltration. (d) 5i-high-dose-group, a totally normal histological kidney structure with no tubular dilatation (red arrow), no exfoliated and necrotic renal tubules
epithelial cells, slight glomerular atrophy (yellow arrow), and slight inflammatory cell infiltration (black arrow). (e) 5i-intervened group, a totally normal histological kidney
structure with no tubular dilatation, no glomerular atrophy (yellow arrow), no inflammatory cell infiltration, but with loose renal tubules epithelial cells (red arrow).
activates a series of oxidation pathways and then generates various
inflammatory cytokines [33]. The oxidation pathways include AGE,
PKC, and hexosamine, while inflammatory mediators contain nu-
including PKC and AGEs also occurred within this metabolic
network [9] because the decreased polyol pathway reduces these
related oxidations leading to a metabolic antioxidant [39]. In
addition, the oxidative stress in DN is suppressed by the elevation
of GSH and SOD activity with 5i, and even directly by scavenging
ROS [26].
clear factor kappa B (NF-kb), tumor necrosis factor a (TNF-a), and
inducible NO synthase (iNOS). Also, it may promote de novo syn-
thesis of free fatty acids (FFA) in the liver which produces FFA
metabolites that triggers inflammatory processes and ROS forma-
tion [34]. A number of therapeutic agents containing insulin [35],
metformin [36], and SGLT-2 inhibitors [37] used to achieve glyce-
mic control in diabetes mellitus all have anti-inflammatory prop-
erties reflecting a reduction of diabetic complications, particularly
at the kidney level, although they act through different pathways.
Further, the polyol pathway playing a critical role in the patho-
genesis of DN is halted by the ALR2 inhibition of 5i in DN. It results
in a decrease in intracellular sorbitol concentration and cell damage
[12], and prevents an important generation and metabolism of
fructose in the kidney cortex which correlates with the develop-
ment of renal injury and dysfunction [38]. Other co-drivers of DN
3. Conclusion
We designed a series of benzoxazolone derivatives, and they
almost exhibited significant aldose reductase inhibition and anti-
oxidant activity. Among them, the compound 5i that was
outstanding in vitro reduced urinary total protein and albumin
more effectively in diabetic mice models than metformin and
epalrestat, suggesting an alleviation effect on the kidney failure
under the high blood glucose condition. The latter two are typical
hypoglycemic agent and aldose reductase inhibitor, respectively, in
the market. The alleviation effect of 5i on renal damage under
10

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
Fig. 9. A schematic that summarizes the proposed mechanism of compound 5i on the renal protection under the high blood glucose condition. By the compound treatment,
decreases occurred in the blood sugar level, the ALR2 activity, and the ROS content (red arrows). Then, ROS is reduced, which includes the reduced lipid peroxidation, and the
enhanced SOD activity and GSH (green arrows). These events result in the reduction of inflammation and damage of kidney. AGE, advanced glycation end product; GSH, glutathione;
GR, glutathione reductase; SOD, superoxide dismutase; iNOS, inducible nitric oxide synthase; NF-
kB, nuclear factor kB; PKC, protein kinase C; ROS, radical oxygen species; TNF-a,
tumor necrosis factor ; RH, reduced generic cellular molecule; Rox, oxidized generic cellular molecule.
a
hyperglycemia was confirmed by hematoxylin-eosin staining of
kidney sections. Further investigation of the compound behavior in
the diabetic mice showed the normalization of blood glucose. We
also detected remarkable decreases in sorbitol of the key product of
the polyol pathway and in lipid peroxides, and strongly raised
glutathione level and superoxide dismutase activity. Thus, we
suggest a therapeutic function of 5i on diabetic kidney which could
be attributed to the combination of hypoglycemic, aldose reductase
inhibition and antioxidant.
specific ELISA kit was from Abcam (Cambridge, UK).
Melting points are uncorrected and were recorded on an X-4
microscopic melting point apparatus. The purity of acetic acid de-
rivatives was obtained by the following HPLC methods using a
Hitachi D-2000 Elite and Shimadzu LC-20A HPLC system (detection
at
l
254 nm) and Inertsil ODS-2250 mm ꢁ 10 mm, 5 mm column.
CH3CN (0.1% TFA)/CH3OH ¼ 75/25 was used as mobile phase at a
flow rate of 1 mL/min for 8 min at room temperature. All final
compounds in biological assays have a purity of ꢂ95%.
4. Experimental section
4.1.1. Pan assay interference compounds (PAINS) evaluation
According to a recently published editorial [40], all final com-
pounds were screened to get rid of PAINS and aggregators via the
public tool, such as False Positive Remover [41] and Aggregator
Advisor [42]. Most of them was filtered out as problematic structure
except 5k and 9h. In the case of 5k and 9h, the catechol partial
structure was identified as a PAINS, but catechol did not exhibit
obvious ALR2 inhibition (Table S1, Supporting Information).
Moreover, the structure-activity relationship analysis have clearly
shown a key role of the carboxyl acid group for our compounds in
the inhibition of aldose reductase and of phenolic hydroxyl groups
in the DPPH radical scavenging, for example, compared with acid 5i,
ester 4i showed less ALR2 inhibition and 5e having no phenolic
hydroxyl group was much weak in the DPPH radical scavenging
4.1. Chemistry
To routinely check all reactions, we used TLC technique on silica
gel Merck 60F254. The NMR spectra were recorded on a Bruker
Ascend 400 M spectrometer (400 MHz for 1H NMR, 100 MHz for 13
C
NMR) with Chemical shifts reported in
d units (ppm) relative to
internal standard TMS in DMSO‑d6 solutions. An AGILENT LC/MS
mass spectrometer was used to perform HEMS (ESI). The assay kits
for quantifications of total protein, urine protein, MDA, and creat-
inine were purchased from Nanjing Jiancheng Bioengineering
Institute (Nanjing, China). The kits for SOD activity and GSH level
were from Beyotime (Shanghai, China). The mouse albumin-
11

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
(Table S1eS2, Supporting Information). In addition, 5i was stable in
30-min assay of the ALR2 inhibition confirmed by HPLC (Fig. S3,
Supporting Information). These indicate the interference of impu-
rities could be excluded and no PAINS involved in the present study.
the mixture was purified by silica gel chromatography to give the
desired product 4.
4.1.4.1. Methyl (E)-2-(2-oxo-4-styrylbenzo[d]oxazol-3(2H)-yl)acetate
(4a). Yield: 132 mg (43%); white solid; 1H NMR (400 MHz,
4.1.2. General procedure for synthesis of benzo[d]oxazol-2(3H)-one
derivatives (2)
DMSO‑d6)
d
7.59 ppm (d, J ¼ 8.1 Hz, 2H), 7.47 ppm (d, J ¼ 8.0 Hz,1H),
7.43 ppm (d, J ¼ 7.2 Hz, 2H), 7.34 ppm (t, J ¼ 7.1 Hz, 3H), 7.21 ppm (t,
J ¼ 8.0 Hz, 1H), 7.12 ppm (d, J ¼ 15.9 Hz, 1H), 5.04 ppm (s, 2H),
3.54 ppm (s, 3H).
1,10-Carbonyldiimidazole (1.6 mmol) was added to a solution of
the 2-amino-6-bromophenol (1.0 mol) in DMF (3 mL), and the so-
lution was heated to 60 ꢀC for 2 h [43]. Then, the reaction mixture
was poured into water (15 mL) and extracted with ethyl acetate
(3 ꢁ 15 mL). The organic layer was collected, dried, filtered, and
evaporated in vacuo. The residue was recrystallized from ethyl
acetate to give desired product 2.
4.1.4.2. Methyl (E)-2-(2-oxo-5-styrylbenzo[d]oxazol-3(2H)-yl)ace-
tate (4b). Yield: 143 mg (46%); white solid; 1H NMR (400 MHz,
DMSO‑d6)
d
7.71 ppm (s, 1H), 7.59 ppm (d, J ¼ 7.6 Hz, 2H),
7.43e7.32 ppm (m, 5H), 7.32e7.24 ppm (m, 2H), 4.82 ppm (s, 2H),
3.75 ppm (s, 3H).
4.1.2.1. 4-bromobenzo[d]oxazol-2(3H)-one (2a). Yield: 98 mg (46%);
yellow solid; 1H NMR (400 MHz, DMSO‑d6)
7.32 ppm (dd, J ¼ 13.9, 8.1 Hz, 2H), 7.04 ppm (t, J ¼ 8.4 Hz, 1H).
d
12.15 ppm (s, 1H),
4.1.4.3. Methyl (E)-2-(2-oxo-6-styrylbenzo[d]oxazol-3(2H)-yl)ace-
tate (4c). Yield: 105 mg (34%); white solid; 1H NMR (400 MHz,
DMSO‑d6)
d
7.72 ppm (s, 1H), 7.59 ppm (d, J ¼ 7.6 Hz, 2H), 7.45 ppm
4.1.2.2. 5-bromobenzo[d]oxazol-2(3H)-one (2b). Yield: 121 mg
(d, J ¼ 8.2 Hz, 1H), 7.38 ppm (t, J ¼ 7.5 Hz, 2H), 7.32 ppm (d,
(50%); yellow solid; 1H NMR (400 MHz, DMSO‑d6)
d 11.95 ppm (s,
J ¼ 8.2 Hz, 1H), 7.27 ppm (s, 3H), 4.81 ppm (s, 2H), 3.73 ppm (s, 3H).
1H), 7.28 ppm (p, J ¼ 4.0 Hz, 1H), 7.19e7.03 ppm (m, 2H).
6-bromobenzo[d]oxazol-2(3H)-one (2c). Yield: 115 mg (48%);
4.1.4.4. Methyl
3(2H)-yl)acetate (4d). Yield: 129 mg (40%); white solid; 1H NMR
(400 MHz, DMSO‑d6) 9.57 ppm (s, 1H), 7.64 ppm (s, 1H), 7.39 ppm
(E)-2-(6-(4-hydroxystyryl)-2-oxobenzo[d]oxazol-
yellow solid; 1H NMR (400 MHz, DMSO‑d6)
d 11.81 ppm (s, 1H),
7.59 ppm (d, J ¼ 1.6 Hz, 1H), 7.33 ppm (dd, J ¼ 8.3, 1.6 Hz, 1H),
d
7.05 ppm (dd, J ¼ 8.3, 1.2 Hz, 1H).
(dd, J ¼ 11.2, 8.1 Hz, 3H), 7.28 ppm (d, J ¼ 8.1 Hz, 1H), 7.15 ppm (d,
J ¼ 16.3 Hz, 1H), 7.03 ppm (d, J ¼ 16.4 Hz, 1H), 6.77 ppm (d,
J ¼ 8.1 Hz, 2H), 4.79 ppm (s, 2H), 3.73 ppm (s, 3H).
4.1.3. General procedure for synthesis of methyl 2-(2-oxobenzo[d]
oxazol-3(2H)-yl)acetate derivatives (3)
A suspension of the appropriate 2 (1 mmol), K2CO3 (1 mmol),
and methyl bromoacetate (1.2 mmol) in CH3CN (10 mL) was
allowed to react at 65 ꢀC for 3 h and filtered. Then, the mixture was
concentrated to dryness in vacuo and recrystallized from ethyl
acetate to give desired product 3.
4.1.4.5. Methyl (E)-2-(2-oxo-7-styrylbenzo[d]oxazol-3(2H)-yl)ace-
tate (4e). Yield: 166 mg (54%); white solid; 1H NMR (400 MHz,
DMSO‑d6)
d
7.67 ppm (d, J ¼ 7.6 Hz, 2H), 7.47e7.43 ppm (m, 2H),
7.41 ppm (d, J ¼ 7.8 Hz, 2H), 7.34 ppm (t, J ¼ 3.6 Hz, 2H),
7.27e7.23 ppm (m, 2H), 4.83 ppm (s, 2H), 3.74 ppm (s, 3H).
4.1.3.1. Methyl
(3a). Yield: 205 mg (71%); white solid; 1H NMR (400 MHz,
DMSO‑d6)
2-(4-bromo-2-oxobenzo[d]oxazol-3(2H)-yl)acetate
4.1.4.6. Methyl
3(2H)-yl)acetate (4f). Yield: 157 mg (46%); yellow solid; 1H NMR
(400 MHz, DMSO‑d6)
(E)-2-(7-(4-methoxystyryl)-2-oxobenzo[d]oxazol-
d
7.44 ppm (dd, J ¼ 16.2, 8.2 Hz, 2H), 7.14 ppm (td, J ¼ 8.2,
d
7.61 ppm (d, J ¼ 8.4 Hz, 2H), 7.44e7.37 ppm
1.2 Hz, 1H), 4.92 ppm (s, 2H), 3.75 ppm (s, 3H).
(m, 2H), 7.27e7.18 (m, 2H), 7.18 (d, J ¼ 16.5 Hz, 1H), 6.98 ppm (d,
J ¼ 8.4 Hz, 2H), 4.82 ppm (s, 2H), 3.79 ppm (s, 3H), 3.73 ppm (s, 3H).
4.1.3.2. Methyl 2-(5-bromo-2-oxobenzo[d]oxazol-3(2H)-yl)acetate
(3b). Yield: 226 mg (79%); white solid; 1H NMR (400 MHz,
4.1.4.7. Methyl
3(2H)-yl)acetate (4g). Yield: 118 mg (36%); yellow solid; 1H NMR
(400 MHz, DMSO‑d6)
(E)-2-(7-(4-hydroxystyryl)-2-oxobenzo[d]oxazol-
DMSO‑d6)
d
7.37 ppm (dd, J ¼ 12.0, 8.0 Hz, 2H), 7.20 ppm (t,
J ¼ 8.0 Hz, 1H), 4.81 ppm (s, 2H), 3.73 ppm (s, 3H).
d
9.26 ppm (s, 1H), 7.34 ppm (d, J ¼ 6.9 Hz,
2H), 7.24 ppm (d, J ¼ 12.8 Hz, 2H), 7.18 ppm (t, J ¼ 7.7 Hz, 1H),
7.12 ppm (d, J ¼ 16.5 Hz, 2H), 7.06 ppm (d, J ¼ 8.2 Hz, 1H), 6.80 ppm
(d, J ¼ 8.1 Hz, 1H), 4.81 ppm (s, 2H), 3.86 ppm (s, 3H).
4.1.3.3. Methyl 2-(6-bromo-2-oxobenzo[d]oxazol-3(2H)-yl)acetate
(3c). Yield: 190 mg (66%); white solid; 1H NMR (400 MHz,
DMSO‑d6)
d
7.72 ppm (d, J ¼ 1.7 Hz,1H), 7.45 ppm (dd, J ¼ 8.4,1.7 Hz,
1H), 7.31 ppm (d, J ¼ 8.4 Hz, 1H), 4.80 ppm (s, 2H), 3.72 ppm (s, 3H).
4.1.4.8. Methyl (E)-2-(7-(3,4-dimethoxystyryl)-2-oxobenzo[d]oxazol-
3(2H)-yl)acetate (4h). Yield: 101 mg (27%); yellow solid; 1H NMR
4.1.3.4. Methyl 2-(7-bromo-2-oxobenzo[d]oxazol-3(2H)-yl)acetate
(400 MHz, DMSO‑d6)
d
7.20 ppm (d, J ¼ 8.3 Hz, 1H), 7.17e7.11 ppm
(3d). Yield: 211 mg (74%); white solid; 1H NMR (400 MHz,
(m, 1H), 7.04 ppm (d, J ¼ 7.9 Hz, 1H), 6.87e6.78 ppm (m, 2H),
6.75 ppm (d, J ¼ 11.9 Hz, 2H), 6.53 ppm (d, J ¼ 12.4 Hz, 1H),
4.78 ppm (s, 2H), 3.72 ppm (d, J ¼ 1.5 Hz, 6H), 3.45 ppm (s, 3H).
Chloroform-d)
d
7.30e7.26 ppm (m,1H), 7.11 ppm (d, J ¼ 8.5 Hz,1H),
7.04 ppm (d, J ¼ 1.9 Hz, 1H), 4.55 ppm (s, 2H), 3.82 ppm (s, 3H).
4.1.4. General procedure for synthesis of methyl (E)-2-(2-oxo-7-
styrylbenzo[d]oxazol-3(2H)-yl)acetate derivatives (4)
4.1.4.9. Methyl (E)-2-(7-(3-hydroxy-4-methoxystyryl)-2-oxobenzo[d]
oxazol-3(2H)-yl)acetate (4i). Yield: 118 mg (35%); yellow solid; 1H
The appropriate 3 (1 mmol), P(o-tolyl)3 (0.07 mmol), and
Pd(OAc)2 (0.03 mmol) was added to DMF (10 mL) and the solution
was stirred at room temperature under argon for 30 min. Then a
solution of alkene (1.5 mmol) and Et3N (0.30 g, 3 mmol) in
DMF(1 mL) was added. The resulting mixture was heated to 100 ꢀC
for 12 h. Once the reaction was complete, the mixture was poured
in water and extracted with ethyl acetate (3 ꢁ 15 mL). The organic
layer was collected, dried, filtered, and evaporated in vacuo. Then,
NMR (400 MHz, DMSO‑d6)
d
9.26 ppm (d, J ¼ 1.7 Hz, 1H), 7.38 ppm
(s, 1H), 7.35 ppm (d, J ¼ 7.0 Hz, 1H), 7.24 ppm (d, J ¼ 13.0 Hz, 2H),
7.21e7.15 ppm (m, 1H), 7.12 ppm (d, J ¼ 16.5 Hz, 1H), 7.06 ppm (d,
J ¼ 8.2 Hz, 1H), 6.80 ppm (d, J ¼ 8.0 Hz, 1H), 4.81 ppm (s, 2H),
3.86 ppm (s, 3H), 3.73 ppm (s, 3H).
4.1.4.10. Methyl (E)-2-(2-oxo-7-(4-(trifluoromethyl)styryl)benzo[d]
oxazol-3(2H)-yl)acetate (4j). Yield: 126 mg (33%); white solid; 1H
12

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
NMR (400 MHz, DMSO‑d6)
d
7.90 ppm (d, J ¼ 8.0 Hz, 2H), 7.75 ppm
1H NMR (400 MHz, DMSO‑d6)
d
7.67 (d, J ¼ 7.6 Hz, 2H), 7.44 (d,
(d, J ¼ 8.0 Hz, 2H), 7.51 ppm (d, J ¼ 9.8 Hz, 2H), 7.46 ppm (d,
J ¼ 4.1 Hz, 1H), 7.31e7.25 ppm (m, 2H), 4.83 ppm (s, 2H), 3.74 ppm
(s, 3H).
J ¼ 2.5 Hz, 1H), 7.41 (d, J ¼ 3.7 Hz, 2H), 7.40 (s, 1H), 7.35e7.32 (m,
1H), 7.32e7.26 (m, 1H), 7.23 (q, J ¼ 5.1, 4.3 Hz, 2H), 4.69 (s, 2H); 13
C
NMR (101 MHz, DMSO‑d6)
d 169.19, 154.26, 139.53, 137.06, 132.87,
131.94, 129.30, 128.75, 127.19, 124.63, 121.83, 121.68, 120.60, 108.90,
43.48 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ294.0772, found
294.0775.
4.1.4.11. Methyl (E)-2-(7-(3,4-dihydroxystyryl)-2-oxobenzo[d]oxazol-
3(2H)-yl)acetate (4k). Yield: 58 mg (17%); white solid; 1H NMR
(400 MHz, DMSO‑d6)
d 9.25 ppm (s, 1H), 9.08 ppm (s, 1H),
d
7.36 ppm (dd, J ¼ 7.8, 1.5 Hz, 2H), 7.28 ppm (d, J ¼ 16.4 Hz, 1H),
4.1.5.5. (E)-2-(7-(4-methoxystyryl)-2-oxobenzo[d]oxazol-3(2H)-yl)
7.24e7.14 ppm (m, 2H), 7.07e6.98 ppm (m, 1H), 6.97e6.90 ppm (m,
1H), 6.81e6.73 ppm (m, 1H), 4.87 ppm (s, 2H), 3.75 ppm (s, 3H).
acetic acid (5f). Yield: 115 mg (35%); yellow solid; mp: > 300 ꢀC;
purity: 96%; 1H NMR (400 MHz, DMSO‑d6)
d
7.61 ppm (d, J ¼ 8.2 Hz,
2H), 7.45e7.34 ppm (m, 2H), 7.22 ppm (dd, J ¼ 13.2, 5.2 Hz, 2H),
4.1.5. General procedure for synthesis of (E)-2-(7-(3-hydroxy-4-
methoxystyryl)-2-oxobenzo[d]oxazol-3(2H)-yl)acetic acid
derivatives (5)
7.19e7.12 ppm (m, 1H), 6.98 ppm (d, J ¼ 8.1 Hz, 2H), 4.68 ppm (s,
2H), 3.79 ppm (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 169.20,
159.91, 154.30, 139.31, 132.54, 131.88, 129.69, 128.59, 124.58, 121.33,
121.00, 119.39, 114.74, 108.37, 55.66, 43.45 ppm; HRMS (ESI) m/z
calcd for [M ꢃ H]ꢃ324.0877, found 324.0876.
A solution of the appropriate 4 (1 mmol) and saturated aq LiOH
(5 mL) in THF (5 mL) was stirred at room temperature until the
starting material disappeared. Then, the alkaline suspension was
acidified with 0.1 N HCl to pH 3 and extracted with ethyl acetate
(3 ꢁ 15 mL). The organic layer was collected, dried, filtered, and
evaporated in vacuo. The residue was recrystallized from ethyl
acetate to get the desired final product 5. 4.1.5.1 (E)-2-(2-oxo-4-
styrylbenzo[d]oxazol-3(2H)-yl)acetic acid (5a). Yield: 143 mg
(48%); white solid; mp: > 300 ꢀC; purity: 99%; 1H NMR (400 MHz,
4.1.5.6. (E)-2-(7-(4-hydroxystyryl)-2-oxobenzo[d]oxazol-3(2H)-yl)
acetic acid (5g). Yield: 106 mg (34%); white solid; mp: > 300 ꢀC;
purity: 99%; 1H NMR (400 MHz, DMSO‑d6)
d 9.88 ppm (s, 1H),
7.48 ppm (d, J ¼ 8.2 Hz, 2H), 7.41e7.27 ppm (m, 2H), 7.18 ppm (t,
J ¼ 7.9 Hz, 1H), 7.12e7.00 ppm (m, 2H), 6.80 ppm (d, J ¼ 8.2 Hz, 2H),
4.43 ppm (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 169.48, 158.35,
DMSO‑d6)
d
7.60 ppm (d, J ¼ 7.7 Hz, 2H), 7.52 ppm (d, J ¼ 8.1 Hz, 1H),
154.50, 139.24, 132.76, 132.34, 128.63, 128.12, 124.36, 120.97, 120.84,
118.47, 116.14, 108.20, 44.60 ppm; HRMS (ESI) m/z calcd for [M ꢃ
H]ꢃ310.0721, found 310.0732.
7.43 ppm (d, J ¼ 6.0 Hz, 1H), 7.42e7.37 ppm (m, 2H), 7.33 ppm (dd,
J ¼ 7.8, 3.9 Hz, 2H), 7.24e7.15 ppm (m, 2H), 4.89 ppm (s, 2H); 13C
NMR (101 MHz, DMSO‑d6)
d 169.74, 154.69, 142.80, 137.00, 132.99,
129.22, 128.67, 127.93, 127.22, 123.23, 122.45, 122.36, 121.20, 109.42,
45.76 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ294.0772, found
294.0765.
4.1.5.7. (E)-2-(7-(3,4-dimethoxystyryl)-2-oxobenzo[d]oxazol-3(2H)-
yl)acetic acid (5h). Yield: 102 mg (29%); white solid; mp:
150e152 ꢀC; purity: 98%; 1H NMR (400 MHz, DMSO‑d6)
d
13.30 ppm (s, 1H), 7.20 ppm (d, J ¼ 8.0 Hz, 1H), 7.14 ppm (td,
4.1.5.1. (E)-2-(2-oxo-5-styrylbenzo[d]oxazol-3(2H)-yl)acetic
(5b). Yield: 129 mg (44%); white solid; mp: > 300 ꢀC; purity: 98%;
1H NMR (400 MHz, DMSO‑d6)
7.70 ppm (s, 1H), 7.59 ppm (d,
acid
J ¼ 7.9, 1.6 Hz, 1H), 7.03 ppm (d, J ¼ 7.8 Hz, 1H), 6.88e6.78 ppm (m,
2H), 6.78e6.72 ppm (m, 2H), 6.53 ppm (d, J ¼ 12.2 Hz, 1H),
4.65 ppm (s, 2H), 3.72 ppm (s, 3H), 3.44 ppm (s, 3H); 13C NMR
d
J ¼ 7.7 Hz, 2H), 7.40 ppm (d, J ¼ 7.6 Hz, 2H), 7.37 ppm (d, J ¼ 4.5 Hz,
1H), 7.34 ppm (s, 1H), 7.31 ppm (d, J ¼ 7.8 Hz, 1H), 7.27 ppm (t,
J ¼ 3.3 Hz, 2H), 4.66 ppm (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
(101 MHz, DMSO‑d6) d 169.18, 154.15, 148.98, 148.60, 139.78, 133.42,
131.77, 129.19, 124.20, 122.78, 122.34, 120.70, 120.02, 112.23, 111.85,
108.71, 55.83, 55.40, 43.43 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ
354.0893, found 354.0978.
d
169.18, 154.52, 141.97, 137.40, 134.07, 132.20, 129.24, 128.66,
128.48, 128.12, 126.84, 122.67, 110.28, 106.6, 43.49 ppm; HRMS (ESI)
m/z calcd for [M ꢃ H]ꢃ294.0772, found 294.0766.
4.1.5.8. (E)-2-(7-(4-hydroxy-3-methoxystyryl)-2-oxobenzo[d]oxazol-
3(2H)-yl)acetic acid (5i). Yield: 149 mg (43%); yellow solid; mp: >
4.1.5.2. (E)-2-(2-oxo-6-styrylbenzo[d]oxazol-3(2H)-yl)acetic
(5c). Yield: 148 mg (50%); white solid; mp: > 300 ꢀC; purity: 96%;
1H NMR (400 MHz, DMSO‑d6)
7.71 ppm (s, 1H), 7.58 ppm (d,
acid
300 ꢀC; purity: 99%; 1H NMR (400 MHz, DMSO‑d6)
d 9.30 ppm (s,
1H), 7.39e7.30 ppm (m, 2H), 7.25 ppm (s, 1H), 7.20 ppm (t,
J ¼ 7.9 Hz, 1H), 7.13 ppm (d, J ¼ 8.2 Hz, 1H), 7.10 ppm (s, 1H),
7.05 ppm (d, J ¼ 8.2 Hz, 1H), 6.80 ppm (d, J ¼ 8.1 Hz, 1H), 4.54 ppm
d
J ¼ 7.6 Hz, 2H), 7.45 ppm (d, J ¼ 8.2 Hz, 1H), 7.38 ppm (t, J ¼ 7.5 Hz,
2H), 7.32 ppm (d, J ¼ 8.1 Hz, 1H), 7.27 ppm (s, 3H), 4.67 ppm (s, 2H);
(s, 2H), 3.86 ppm (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 169.36,
13C NMR (101 MHz, DMSO‑d6)
d
169.16, 154.42, 142.97, 137.49,
154.43, 148.38, 147.74, 139.24, 133.22, 132.13, 128.68, 124.48, 121.11,
121.00, 118.75, 116.07, 110.44, 108.19, 56.13, 44.04 ppm; HRMS (ESI)
m/z calcd for [M ꢃ H]ꢃ340.0827, found 340.0816.
132.87, 131.05, 129.19, 128.41, 128.23, 128.01, 126.82, 123.76, 110.01,
107.30, 43.47 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ294.0772,
found 294.0779.
4.1.5.9. (E)-2-(2-oxo-7-(4-(trifluoromethyl)styryl)benzo[d]oxazol-
3(2H)-yl)acetic acid (5j). Yield: 133 mg (37%); white solid; mp: >
4.1.5.3. (E)-2-(6-(4-hydroxystyryl)-2-oxobenzo[d]oxazol-3(2H)-yl)
acetic acid (5d). Yield: 97 mg (31%); yellow solid; mp: > 300 ꢀC;
300 ꢀC; purity: 98%; 1H NMR (400 MHz, DMSO‑d6)
d 7.87 ppm (d,
purity: 98%; 1H NMR (400 MHz, DMSO‑d6)
d
9.62 ppm (s, 1H),
J ¼ 8.1 Hz, 2H), 7.74 ppm (d, J ¼ 8.0 Hz, 2H), 7.57e7.41 ppm (m, 2H),
7.37 ppm (d, J ¼ 8.0 Hz,1H), 7.20 ppm (t, J ¼ 7.9 Hz,1H), 7.05 ppm (d,
J ¼ 7.7 Hz, 1H), 4.22 ppm (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
7.61 ppm (s, 1H), 7.40 ppm (d, J ¼ 8.2 Hz, 2H), 7.35 ppm (d,
J ¼ 8.3 Hz, 1H), 7.21 ppm (d, J ¼ 8.1 Hz, 1H), 7.13 ppm (d, J ¼ 16.4 Hz,
1H), 7.02 ppm (d, J ¼ 16.3 Hz, 1H), 6.77 ppm (d, J ¼ 8.1 Hz, 2H),
d
169.50, 154.46, 141.23, 139.79, 132.62, 130.89, 128.61, 128.29,
4.52 ppm (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d
169.27, 157.70,
127.73, 126.10, 126.07, 124.6 (q, J ¼ 157 Hz), 121.49, 119.74, 109.61,
49.06 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ362.0646, found
362.0635.
154.55, 142.99, 133.23, 130.79, 128.53, 128.19, 125.14, 123.06, 116.03,
109.95, 106.73, 44.11, 39.46 ppm; HRMS (ESI) m/z calcd for [M ꢃ
H]ꢃ310.0721, found 310.0725.
4.1.5.10. (E)-2-(7-(3,4-dihydroxystyryl)-2-oxobenzo[d]oxazol-3(2H)-
4.1.5.4. (E)-2-(2-oxo-7-styrylbenzo[d]oxazol-3(2H)-yl)acetic
acid
yl)acetic (5k). Yield: 53 mg (16%); yellow solid; mp: >300 ꢀC; pu-
(5e). Yield: 107 mg (36%); white solid; mp: > 300 ꢀC; purity: 97%;
rity: 96%; 1H NMR (400 MHz, DMSO‑d6)
d
7.36 ppm (dd, J ¼ 7.8,
13

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
1.5 Hz, 2H), 7.28 ppm (d, J ¼ 16.4 Hz, 1H), 7.24e7.14 ppm (m, 2H),
7.07e6.98 ppm (m, 1H), 6.97e6.90 ppm (m, 1H), 6.81e6.73 ppm (m,
2 mmol), and H2O (1 mL) in DMF (10 mL) allowed to react at 100 ꢀC
for 12 h. Upon completion, the mixture was poured in water and
extracted with ethyl acetate (3 ꢁ 15 mL). The organic layer was
collected, dried, filtered, and evaporated in vacuo. Then, the
mixture was purified via silica gel chromatography to give the
desired product 8.
1H), 4.67 ppm (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 169.21,
154.31, 146.72, 146.01, 139.21, 133.25, 131.85, 128.63, 124.57, 121.15,
121.10, 119.55, 118.07, 116.27, 113.76, 108.11, 43.45 ppm; HRMS (ESI)
m/z calcd for [M ꢃ H]ꢃ326.0670, found 326.0680.
4.1.6. General procedure for synthesis of methyl 2-(7-(4-
methoxyphenethyl)-2-oxobenzo[d]oxazol-3(2H)-yl)acetate
derivatives (6)
A mixture of the appropriate 4 (0.7 mmol) and 10% palladium on
carbon (0.33 g) in methanol (10 mL) was stirred at room temper-
ature for 6 h under a hydrogen atmosphere and then filtered
through Celite pad. The filtrate was concentrated and purified via
column chromatography to obtain the product 6.
4.1.8.1. Methyl 2-(2-oxo-7-phenylbenzo[d]oxazol-3(2H)-yl)acetate
(8a). Yield: 172 mg (61%); white solid; 1H NMR (400 MHz,
DMSO‑d6)
d
7.77 ppm (d, J ¼ 7.7 Hz, 2H), 7.54 ppm (t, J ¼ 7.6 Hz, 2H),
7.49e7.37 ppm (m, 2H), 7.41e7.28 ppm (m, 2H), 4.84 ppm (s, 2H),
3.74 ppm (s, 3H).
4.1.8.2. Methyl 2-(2-oxo-7-(4-(trifluoromethyl)phenyl)benzo[d]oxa-
zol-3(2H)-yl)acetate (8b). Yield: 161 mg (47%); white solid; 1H NMR
(400 MHz, DMSO‑d6)
d
8.01 ppm (d, J ¼ 8.1 Hz, 2H), 7.91 ppm (d,
4.1.6.1. Methyl
3(2H)-yl)acetate (6a). Yield: 142 mg (41%); white solid; 1H NMR
(400 MHz, DMSO‑d6)
2-(7-(4-methoxyphenethyl)-2-oxobenzo[d]oxazol-
J ¼ 8.0 Hz, 2H), 7.49 ppm (dd, J ¼ 7.1, 2.3 Hz, 1H), 7.44e7.33 ppm (m,
2H), 4.86 ppm (s, 2H), 3.75 ppm (s, 3H).
d
7.13 ppm (t, J ¼ 4.0 Hz, 5H), 7.01 ppm (dd,
J ¼ 6.0, 3.3 Hz, 1H), 6.83 ppm (d, J ¼ 8.0 Hz, 2H), 4.77 ppm (s, 2H),
3.72 ppm (d, J ¼ 2.7 Hz, 6H), 2.96 ppm (t, J ¼ 7.9 Hz, 2H),
2.92e2.83 ppm (m, 2H).
4.1.8.3. Methyl
3(2H)-yl)acetate (8c). Yield: 166 mg (53%); white solid; 1H NMR
(400 MHz, DMSO‑d6) 7.76e7.68 ppm (m, 2H), 7.40e7.22 ppm (m,
2-(7-(4-methoxyphenyl)-2-oxobenzo[d]oxazol-
d
3H), 7.14e7.06 ppm (m, 2H), 4.85 ppm (s, 2H), 3.82 ppm (s, 3H),
4.1.6.2. Methyl 2-(7-(4-hydroxy-3-methoxyphenethyl)-2-oxobenzo
3.74 ppm (s, 3H).
[d]oxazol-3(2H)-yl)acetate (6b). Yield: 167 mg (47%); white solid;
1H NMR (400 MHz, DMSO‑d6)
d
8.68 ppm (s, 1H), 7.16e7.07 ppm (m,
4.1.8.4. Methyl
3(2H)-yl)acetate (8d). Yield: 131 mg (38%); white solid; 1H NMR
(400 MHz, DMSO‑d6) 7.45e7.37 ppm (m, 1H), 7.38e7.25 ppm (m,
3H), 7.30e7.22 ppm (m, 1H), 7.11 ppm (dd, J ¼ 8.2, 2.3 Hz, 1H),
4.83 ppm (s, 2H), 3.84 ppm (s, 3H), 3.82 ppm (s, 3H), 3.74 ppm (s,
3H).
2-(7-(3,4-dimethoxyphenyl)-2-oxobenzo[d]oxazol-
2H), 7.05e6.98 ppm (m, 1H), 6.76 ppm (s, 1H), 6.71e6.56 ppm (m,
2H), 4.77 ppm (s, 2H), 3.72 ppm (s, 6H), 2.97 ppm (d, J ¼ 7.9 Hz, 2H),
2.83 ppm (t, J ¼ 7.9 Hz, 2H).
d
4.1.7. General procedure for synthesis of 2-(7-(4-
methoxyphenethyl)-2-oxobenzo[d]oxazol-3(2H)-yl)acetic acid
derivatives (7)
4.1.8.5. Methyl
3(2H)-yl)acetate (8e). Yield: 172 mg (47%); white solid; 1H NMR
(400 MHz, DMSO‑d6)
2-(7-([1,10-biphenyl]-4-yl)-2-oxobenzo[d]oxazol-
A suspension of the appropriate 6 (1 mmol) and saturated aq
LiOH (5 mL) in THF (5 mL) was stirred at room temperature until
the starting material disappeared. Then, the alkaline suspension
was acidified with 0.1 N HCl to pH 3 and extracted with ethyl ac-
etate (3 ꢁ 15 mL). The organic layer was collected, dried, filtered,
and evaporated in vacuo. The residue was recrystallized from ethyl
acetate to get the desired final product 7.
d
7.87 ppm (dt, J ¼ 9.8, 7.1 Hz, 4H), 7.76 ppm
(d, J ¼ 7.6 Hz, 2H), 7.55e7.38 ppm (m, 4H), 7.41e7.31 ppm (m, 2H),
4.85 ppm (s, 2H), 3.75 ppm (s, 3H).
4.1.8.6. Methyl
3(2H)-yl)acetate (8f). Yield: 127 mg (37%); white solid; 1H NMR
(400 MHz, DMSO‑d6)
2-(7-(4-(tert-butyl)phenyl)-2-oxobenzo[d]oxazol-
d
7.69 ppm (dd, J ¼ 8.4, 2.1 Hz, 2H), 7.55 ppm
4.1.7.1. 2-(7-(4-methoxyphenethyl)-2-oxobenzo[d]oxazol-3(2H)-yl)
(dd, J ¼ 8.5, 2.1 Hz, 2H), 7.38 ppm (d, J ¼ 7.1 Hz, 1H), 7.36e7.26 ppm
acetic acid (7a). Yield: 114 mg (35%); white solid; mp: > 300 ꢀC;
(m, 2H), 4.83 ppm (s, 2H), 3.32 ppm (s, 3H), 1.33 ppm (s, 9H).
purity: 99%; 1H NMR (400 MHz, DMSO‑d6)
d 7.18e7.03 (m, 4H), 6.98
(d, J ¼ 7.5 Hz, 1H), 6.87e6.79 (m, 2H), 4.51 (s, 2H), 3.71 (s 3H), 2.95
4.1.8.7. Methyl
3(2H)-yl)acetate (8g). Yield: 103 mg (30%); white solid; 1H NMR
(400 MHz, DMSO‑d6) 7.30e7.18 ppm (m, 3H), 7.16e7.09 ppm (m,
2-(7-(2,4-dimethoxyphenyl)-2-oxobenzo[d]oxazol-
(d, J ¼ 7.8 Hz, 2H), 2.87 (d, J ¼ 8.0 Hz, 2H); 13C NMR (101 MHz,
DMSO‑d6)
d
169.41, 158.01, 154.45, 140.62, 133.35, 131.46, 129.69,
d
124.11,123.93, 123.32, 114.16,107.76, 55.42, 43.94, 34.67, 31.09 ppm;
1H), 6.74e6.61 ppm (m, 2H), 4.80 ppm (s, 2H), 3.83 ppm (s, 3H),
HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ326.1034, found 326.1032.
3.76 ppm (s, 3H), 3.74 ppm (s, 3H).
4.1.7.2. 2-(7-(4-hydroxy-3-methoxyphenethyl)-2-oxobenzo[d]oxa-
4.1.8.8. Methyl
3(2H)-yl)acetate (8h). Yield: 98 mg (31%); white solid; 1H NMR
(400 MHz, DMSO‑d6) 9.22 ppm (s, 1H), 9.18 ppm (s, 1H),
2-(7-(3,4-dihydroxyphenyl)-2-oxobenzo[d]oxazol-
zol-3(2H)-yl)acetic acid (7b). Yield: 101 mg (29%); white solid; mp:
198e200 ꢀC; purity: 99%; 1H NMR (400 MHz, DMSO‑d6)
d
13.35 (s,
d
1H), 8.66 (s, 1H), 7.12 (t, J ¼ 3.7 Hz, 2H), 7.00 (q, J ¼ 4.1 Hz, 1H), 6.76
(s, 1H), 6.70e6.57 (m, 2H), 4.63 (s, 2H), 3.71 (s, 3H), 2.91 (d,
J ¼ 8.0 Hz, 2H), 2.84 (d, J ¼ 8.0 Hz, 2H); 13C NMR (101 MHz,
7.32e7.17 ppm (m, 4H), 7.06 ppm (d, J ¼ 8.2 Hz, 1H), 6.87 ppm (d,
J ¼ 8.2 Hz, 1H), 4.81 ppm (s, 2H), 3.74 ppm (s, 3H).
DMSO‑d6)
d
169.21, 154.38, 147.82, 145.12, 140.64, 132.24, 131.24,
4.1.8.9. Methyl 2-(7-(naphthalen-1-yl)-2-oxobenzo[d]oxazol-3(2H)-
124.21, 123.59, 120.86, 115.76, 112.93, 107.70, 55.99, 43.43, 35.19,
31.06 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ342.0983, found
342.0982.
yl)acetate (8i). Yield: 165 mg (50%); white solid; 1H NMR (400 MHz,
DMSO‑d6)
d
8.06 ppm (d, J ¼ 8.3 Hz, 2H), 7.64 ppm (s, 1H), 7.62 ppm
(d, J ¼ 3.5 Hz, 1H), 7.61e7.48 ppm (m, 3H), 7.47e7.35 ppm (m, 2H),
7.25 ppm (d, J ¼ 7.5 Hz, 1H), 4.86 ppm (s, 2H), 3.76 ppm (s, 3H).
4.1.8. General procedure for synthesis of methyl 2-(2-oxo-7-
phenylbenzo[d]oxazol-3(2H)-yl)acetate derivatives (8)
A mixture of the appropriate 3 (1 mmol), (PPh3)4Pd (0.03 mmol),
the corresponding phenylboronic acid (1.1 mmol), K2CO3 (0.65 g,
4.1.8.10. Methyl 2-(7-(naphthalen-2-yl)-2-oxobenzo[d]oxazol-3(2H)-
yl)acetate (8j). Yield: 179 mg (54%); white solid; 1H NMR (400 MHz,
DMSO‑d6)
d 8.34 ppm (s, 1H), 8.10 ppm (s, 1H), 8.08 ppm (d,
14

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
J ¼ 2.2 Hz, 1H), 8.00 ppm (dt, J ¼ 6.4, 2.7 Hz, 1H), 7.97e7.90 ppm (m,
2H), 7.65e7.60 ppm (m, 1H), 7.58e7.53 ppm (m, 2H), 7.37 ppm (s,
1H), 4.84 ppm (s, 2H), 3.78 ppm (s, 3H).
1H), 7.34 ppm (d, J ¼ 6.5 Hz, 2H), 4.70 ppm (s, 2H); 13C NMR
(101 MHz, DMSO‑d6) d 169.21, 154.27, 140.40, 139.90, 139.45, 133.70,
132.29, 129.49, 129.02, 128.20, 127.62, 127.15, 124.97, 122.94, 122.11,
109.25, 43.56 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ344.0928,
found 344.0923.
4.1.8.11. Methyl 2-(7-(4-(hydroxymethyl)phenyl)-2-oxobenzo[d]oxa-
zol-3(2H)-yl)acetate (8k). Yield: 139 mg (44%); white solid; 1H NMR
(400 MHz, DMSO‑d6)
d
7.73 ppm (d, J ¼ 7.8 Hz, 2H), 7.47 ppm (d,
4.1.9.6. 2-(7-(4-(tert-butyl)phenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)
J ¼ 7.9 Hz, 2H), 7.40 ppm (d, J ¼ 7.2 Hz, 1H), 7.32 ppm (d, J ¼ 6.8 Hz,
2H), 5.27 ppm (t, J ¼ 5.9 Hz, 1H), 4.84 ppm (s, 2H), 4.57 ppm (d,
J ¼ 5.7 Hz, 2H), 3.74 ppm (s, 3H).
acetic acid (9f). Yield: 88 mg (27%); white solid; mp: > 300 ꢀC;
purity: 98%; 1H NMR (400 MHz, DMSO‑d6)
d 13.49 ppm (s, 1H),
7.69 ppm (d, J ¼ 7.9 Hz, 2H), 7.55 ppm (d, J ¼ 8.0 Hz, 2H),
7.40e7.24 ppm (m, 3H), 4.67 ppm (s, 2H),1.33 ppm (s, 9H); 13C NMR
4.1.9. General procedure for synthesis of 2-(2-oxo-7-phenylbenzo
[d]oxazol-3(2H)-yl)acetic acid derivatives (9)
(101 MHz, DMSO‑d6) d 169.23, 154.30, 151.28, 139.38, 132.22, 131.83,
128.22, 126.18, 124.88, 123.39, 122.10, 108.89, 43.57, 34.87,
31.52 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ324.1241, found
324.1253.
A mixture of the appropriate 8 (1 mmol) and saturated aq LiOH
(5 mL) in THF (5 mL) was stirred at room temperature until the
starting material disappeared. Then, the alkaline suspension was
acidified with 0.1 N HCl to pH 3 and extracted with ethyl acetate
(3 ꢁ 15 mL). The organic layer was collected, dried, filtered, and
evaporated in vacuo. The residue was recrystallized from ethyl
acetate to get the desired final product 9.
4.1.9.7. 2-(7-(2,4-dimethoxyphenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)
acetic acid (9g). Yield: 49 mg (15%); yellow solid; mp: > 300 ꢀC;
purity: 95%; 1H NMR (400 MHz, DMSO‑d6)
d
7.24 ppm (d, J ¼ 8.4 Hz,
1H), 7.16 ppm (d, J ¼ 7.9 Hz, 1H), 7.08e6.97 ppm (m, 2H), 6.71 ppm
(s, 1H), 6.65 ppm (d, J ¼ 8.5 Hz, 1H), 4.25 ppm (s, 2H), 3.83 ppm (s,
4.1.9.1. 2-(2-oxo-7-phenylbenzo[d]oxazol-3(2H)-yl)acetic acid (9a).
3H), 3.76 ppm (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 161.28,
Yield: 108 mg (40%); white solid; mp: > 300 ꢀC; purity: 99%; 1H
158.03, 154.66, 140.08, 132.25, 131.85, 123.91, 123.62, 121.04, 116.54,
108.43, 105.71, 99.32, 56.05, 55.82, 45.42 ppm; HRMS (ESI) m/z
calcd for [M ꢃ H]ꢃ328.0827, found 328.0834.
NMR (400 MHz, DMSO‑d6)
J ¼ 7.6 Hz, 2H), 7.51e7.42 ppm (m, 2H), 7.32 ppm (q, J ¼ 2.3 Hz, 2H),
4.70 ppm (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
169.20, 154.27,
d 7.80e7.73 ppm (m, 2H), 7.54 ppm (t,
d
139.40,134.68, 132.21,129.38,128.77,128.52,124.94, 123.48,122.30,
109.19, 43.49 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ268.0615,
found 268.0610.
4.1.9.8. 2-(7-(3,4-dihydroxyphenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)
acetic acid (9h). Yield: 44 mg (15%); red solid; mp: > 300 ꢀC; purity:
98%; 1H NMR (400 MHz, DMSO‑d6)
d 8.86 ppm (s, 1H), 7.99 ppm (s,
1H), 6.90 ppm (s, 1H), 6.74 ppm (q, J ¼ 10.1, 9.4 Hz, 3H), 6.39 ppm
4.1.9.2. 2-(2-oxo-7-(4-(trifluoromethyl)phenyl)benzo[d]oxazol-
3(2H)-yl)acetic acid (9b). Yield: 92 mg (37%); white solid; mp: >
(dd, J ¼ 34.5, 7.8 Hz, 2H), 3.84 ppm (s, 2H); 13C NMR (101 MHz,
DMSO‑d6)
d 169.24, 154.38, 146.30, 145.89, 138.98, 132.13, 125.67,
300 ꢀC; purity: 97%; 1H NMR (400 MHz, DMSO‑d6)
d
13.20 ppm (s,
124.78, 123.82, 121.63, 119.70, 116.45, 115.81, 108.00, 43.43 ppm;
1H), 8.01 ppm (d, J ¼ 8.0 Hz, 2H), 7.91 ppm (d, J ¼ 8.1 Hz, 2H),
HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ300.0514, found 300.0517.
7.47 ppm (d, J ¼ 7.4 Hz, 1H), 7.43e7.33 ppm (m, 2H), 4.72 ppm (s,
2H); 13C NMR (101 MHz, DMSO‑d6)
d
169.15, 154.11, 139.58, 138.76,
4.1.9.9. 2-(7-(naphthalen-1-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)acetic
132.33, 129.31, 126.28, 126.25, 126.01, 125.08 (d, J ¼ 179), 122.43,
121.84, 110.19, 49.06 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ
336.0489, found 336.0482.
acid (9i). Yield: 129 mg (40%); white solid; mp: > 300 ꢀC; purity:
98%; 1H NMR (400 MHz, DMSO‑d6)
d 13.47 ppm (s,1H), 8.06 ppm (d,
J ¼ 8.2 Hz, 2H), 7.68e7.60 ppm (m, 2H), 7.60e7.55 ppm (m, 2H),
7.52 ppm (t, J ¼ 7.7 Hz, 1H), 7.47e7.35 ppm (m, 2H), 7.23 ppm (d,
J ¼ 7.5 Hz, 1H), 4.71 ppm (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
4.1.9.3. 2-(7-(4-methoxyphenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)ace-
tic acid (9c). Yield: 75 mg (25%); white solid; mp: 279e281 ꢀC;
d 169.24,154.19, 140.24,133.73,132.86,131.80,131.11,129.18, 128.90,
purity: 98%; 1H NMR (400 MHz, DMSO‑d6)
d
13.48 ppm (s, 1H),
128.27, 127.14, 126.65, 126.03, 125.60, 124.80, 124.62, 122.75, 109.48,
43.61 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ318.0772, found
318.0769.
7.72 ppm (d, J ¼ 8.2 Hz, 2H), 7.35 ppm (d, J ¼ 7.7 Hz, 1H),
7.33e7.20 ppm (m, 2H), 7.10 ppm (d, J ¼ 8.3 Hz, 2H), 4.65 ppm (s,
2H), 3.82 ppm (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 169.24,
159.75, 154.33, 139.13, 132.25, 129.73, 126.92, 124.83, 123.18, 121.78,
114.84, 108.45, 55.69, 43.63 ppm; HRMS (ESI) m/z calcd for [M ꢃ
H]ꢃ298.0721, found 298.0730.
4.1.9.10. 2-(7-(naphthalen-2-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)ace-
tic acid (9j). Yield: 111 mg (35%); white solid; mp: 235e237 ꢀC;
purity: 96%; 1H NMR (400 MHz, DMSO‑d6)
d 8.33 ppm (s, 1H),
8.06 ppm (t, J ¼ 10.5 Hz, 2H), 8.02e7.95 ppm (m, 1H), 7.92 ppm (d,
J ¼ 8.6 Hz, 1H), 7.65e7.56 ppm (m, 2H), 7.54 ppm (d, J ¼ 7.2 Hz, 1H),
7.37 ppm (d, J ¼ 7.7 Hz, 2H), 4.72 (s, 2H); 13C NMR (101 MHz,
4.1.9.4. 2-(7-(3,4-dimethoxyphenyl)-2-oxobenzo[d]oxazol-3(2H)-yl)
acetic acid (9d). Yield: 54 mg (16%); yellow solid; mp: > 300 ꢀC;
purity: 95%; 1H NMR (400 MHz, DMSO‑d6)
d
7.40 ppm (d, J ¼ 7.6 Hz,
DMSO‑d6) d 169.21, 154.33, 139.62, 133.47, 132.95, 132.33, 132.19,
1H), 7.36e7.24 ppm (m, 4H), 7.11 ppm (d, J ¼ 8.2 Hz, 1H), 4.69 ppm
128.90, 128.77, 128.05, 127.66, 127.16, 127.09, 126.29, 125.01, 123.39,
122.61, 109.30, 43.60 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ
318.0772, found 318.0785.
(s, 2H), 3.84 ppm (s, 3H), 3.82 ppm (s, 3H); 13C NMR (101 MHz,
DMSO‑d6)
d 169.22, 154.31, 149.47, 149.36, 139.19, 132.16, 127.14,
124.79, 123.52, 122.08, 121.19, 112.50, 112.07, 108.48, 56.10, 56.05,
43.45 ppm; HRMS (ESI) m/z calcd for [M ꢃ H]ꢃ328.0827, found
328.0828.
4.1.9.11. 2-(7-(4-(hydroxymethyl)phenyl)-2-oxobenzo[d]oxazol-
3(2H)-yl)acetic acid (9k). Yield: 79 mg (26%); white solid; mp: >
300 ꢀC; purity: 98%; 1H NMR (400 MHz, DMSO‑d6)
d 13.40 ppm (s,
4.1.9.5. 2-(7-([1,10-biphenyl]-4-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)
1H), 7.73 ppm (d, J ¼ 7.8 Hz, 2H), 7.47 ppm (d, J ¼ 7.9 Hz, 2H),
7.39 ppm (d, J ¼ 7.1 Hz,1H), 7.31 ppm (d, J ¼ 6.4 Hz, 2H), 5.27 ppm (s,
1H), 4.69 ppm (s, 2H), 4.57 ppm (s, 2H); 13C NMR (101 MHz,
acetic acid (9e). Yield: 131 mg (38%); white solid; mp: > 300 ꢀC;
purity: 98%; 1H NMR (400 MHz, DMSO‑d6)
d 13.50 ppm (s, 1H),
7.86 ppm (q, J ¼ 8.2 Hz, 4H), 7.76 ppm (d, J ¼ 7.6 Hz, 2H), 7.51 ppm (t,
DMSO‑d6)
d 169.21, 154.29, 143.25, 139.37, 132.99, 132.21, 128.23,
J ¼ 7.7 Hz, 2H), 7.46 ppm (d, J ¼ 6.7 Hz, 1H), 7.42 ppm (d, J ¼ 7.4 Hz,
127.36,124.90,123.43,122.18,108.98, 63.05, 43.49 ppm; HRMS (ESI)
15

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
m/z calcd for [M ꢃ H]ꢃ298.0721, found 298.0718.
spectrophotometer, after vortexing thoroughly and leaving for
30 min at room temperature. The experiments were performed in
triplicate. The percentage of DPPH radical scavenging was deter-
mined by the following equation.
4.2. In vitro experiments
4.2.1. Enzyme assays
Enzyme activity was performed on a Shimadzu UV-1800 spec-
trophotometer by monitoring the decrease in absorption of NADPH
ꢀ
ꢁ
Asample ꢃ Ablank
percentage of scavenging ð%Þ ¼ 1 ꢃ
Acontrol
at
l 340 nm. Wistar rats (200e250 g) were used as ALR2 and ALR1
donor, supplied by SPF Biotechnology Co.,Ltd. ALR2 and ALR1 were
prepared in accordance with the method of Kinoshita [44] and
Xiangyu Qin [23] et al.
Acontrol ¼ DPPH þ methanol
Asanple ¼ tested compound þ DPPH þ methanol
Ablank ¼ tested compound þ methanol
ALR2 activity assay was performed at 30 ꢀC in a reaction mixture
containing sodium phosphate buffer (0.25 mL, 0.1 M, pH 6.2),
enzyme extract (0.1 mL), deionized water (0.15 mL), NADPH
(0.25 mL, 0.10 mM), and D, L-glyceraldehyde (0.25 mL, 10 mM) [23].
The reaction mixture was incubated for 10 min except for D,L-
glyceraldehyde. The substrate was then added to start the reac-
4.2.3. Lipid peroxidation determination
tion, and the rate of decrease in absorption at
l 340 nm was
Fresh isolated rat brain was crushed with ice-cold normal saline
and homogenized. Then the homogenate was centrifuged and the
supernatant was used for biochemical analyses. A total protein
quantification kit based on the Coomassie Brilliant Blue method
was used to determine the protein concentration in the superna-
tant. A commercially available kit was used to determine the MDA
concentration in the homogenate according to the protocols pro-
vided by the manufacturer.
monitored for 4 min. Appropriate blanks were employed for cor-
rections, containing sodium phosphate buffer (0.25 mL, 0.1 M, pH
6.2), enzyme extract (0.1 mL), deionized water (0.15 mL), NADPH
(0.25 mL, 0.10 mM), and D, L-glyceraldehyde (0.25 mL, 10 mM) and
0.5% dimethyl sulfoxide as solvent.
ALR1 activity was performed at 37 ꢀC in a reaction mixture
containing sodium phosphate buffer (0.25 mL, 0.1 M, pH 7.2),
enzyme extract (0.1 mL), NADPH (0.25 mL, 0.12 mM), deionized
water (0.15 mL), and sodium
The reaction mixture was incubated for 10 min except for sodium
glucuronate. The substrate was then added to start the reaction,
and the rate of decrease in absorption at 340 nm was monitored
for 4 min. Appropriate blanks were employed for corrections,
containing sodium phosphate buffer (0.25 mL, 0.1 M, pH 7.2),
enzyme extract (0.1 mL), NADPH (0.25 mL, 0.12 mM), deionized
D-glucuronate (0.25 mL, 20 mM) [23].
D
-
4.2.4. MTT assay
293Tcells were seeded in 96 well plates at 1 ꢁ105 cells/well, and
treated in the absence or presence of compound 5i (25, 50, 100 and
200) for 24 h at 37 ꢀC. Cell viability was assessed using MTT assay
(Solarbio) based on the protocols provided by the manufacturer.
l
water (0.15 mL), and sodium D-glucuronate (0.25 mL, 20 mM) and
0.5% dimethyl sulfoxide as solvent.
All compounds were dissolved in dimethyl sulfoxide. The
compounds inhibitory activity against ALR2 and ALR1 was analyzed
4.3. Ethics
All experiments were conducted in compliance with all relevant
local ethical regulations.
by adding 5 mL of the inhibitor solution to the reaction mixture
described above. The NADPH oxidation rate in the presence of all
the reaction mixture components except the substrate was sub-
tracted from each experimental rate to correct for the nonenzy-
matic oxidation of NADPH. The IC50 values were calculated by least-
square analysis of the linear portion of the log (dose) versus
response curves (r2 > 0.95), which were generated using at least
three concentrations of compound with inhibitory activity between
20% and 80%. The experiments were performed in triplicate.
Kinetic studies were performed with two concentrations (10,
5 nM) of inhibitor 5i [45], containing varied concentrations of the
substrate DL-glyceraldehyde (7.5, 3.75, 1.88, 0.94 mM) while in-
hibitor and cofactor concentrations (0.025 mM) were held constant.
For NADPH studies, the concentrations of the cofactor NADPH were
4.4. Mice
Kunming mice (23e25 g) were allowed to acclimate for a week
and then were rendered diabetic by an intraperitoneal injection of
STZ (50 mg/kg) for 5 days, which had been freshly dissolved in
0.03 M citrate buffer, pH 4.5. One week later, tail blood glucose was
measured using a One Touch II blood glucose meter. Diabetic mice
whose blood glucose levels were more than 16 mmol/L were ran-
domized according to blood glucose levels into 6 groups with 12
mice in each group, namely 1) stz group; 2) metformin group; 3)
epalrestat group; 4) 5i-high-dose group; 5) 5i-low-dose group; 6)
5i-intervened group. For comparison, mice without injection of stz
were served as normal group. Then each group dosed daily for 8
weeks by gavage with dose of 1) 0.5% sodium carboxymethylcel-
lulose (CMC) solution (1 mL/kg); 2) metformin in 0.5% CMC solu-
tion, (80 mg/kg; 1 mL/kg); 3) epalrestat in 0.5% CMC solution,
(80 mg/kg; 1 mL/kg); 4) compound 5i in 0.5% CMC solution,
(160 mg/kg; 1 mL/kg); and 5) compound 5i in 0.5% CMC solution,
(80 mg/kg; 1 mL/kg) respectively. 5i-intervened group received
compound 5i in 0.5% CMC solution (80 mg/kg; 1 mL/kg) per day for
4 weeks when mice having been positive in total urine protein.
Normal group received 0.5% CMC solution (1 mL/kg) per day for 8
weeks. Six hours after the final dose was administered, the mice
were sacrificed and kidney and liver were removed, stored
at ꢃ40 ꢀC.
varied (25, 12.5, 6.25, 3.125 mM) while inhibitor and substrate DL-
glyceraldehyde (2.5 mM) were held constant. The double-
reciprocal plots of enzyme velocity versus glyceraldehyde con-
centration were analyzed to determine the nature of inhibition
produced by each concentration of 5i.
4.2.2. DPPH assay
A method based on the scavenging of the stable free radical
DPPH was used to investigate the antiradical activity of tested
compounds. Add methanolic solution (100
compounds to DPPH methanolic solution (1 mL, 0.025 mg/mL) and
methanol (1.9 mL) to give final concentrations of 100, 50, and 10
for the tested compounds, respectively. The optical density was
measured at 517 nm using the Shimadzu UV-1800
mL) with various tested
m
M
l
16

X. Zhang, H. Chen, Y. Lei et al.
European Journal of Medicinal Chemistry 215 (2021) 113269
4.5. Glucose tolerance tests
4.14. Hematoxylin and eosin staining
After 8 weeks, glucose tolerance tests were performed in mice
fasted for 10 h as described previously. The mice were injected
intraperitoneally with glucose (1.5 g/kg body weight), and blood
glucose levels were measured.
Some of kidneys were dissected, washed in 0.9% ice-cold saline,
immediately fixed in buffered 10% formalin solution for 24 h, and
embedded in paraffin. Sections of 5
mM thickness were cut and
stained by both hematoxylin and eosin (HE) for histologic exami-
nation. Histologic observations were made under brightfield using
a Nikon Eclipse Ci (trinocular light microscope) by an independent
observer who was blind to the treatment groups.
4.6. Hypoglycemic in pharmacokinetic tests
Hypoglycemic in pharmacokinetic tests were performed in mice
fasted for 10 h as described previously. The blood glucose levels of
mice were measured from 1 h after gavage to 5 h after gavage.
4.15. Docking studies
Docking was performed using Molegro Virtual Docker (version
5.0) with the same protocol as described before [23a].
4.7. Tissue sorbitol analysis
Author contributions
The kidney sorbitol levels were measured by a standard enzy-
matic method that sorbitol dehydrogenase catalyzes the stoichio-
metric conversion of NADþ by sorbitol to NADH. The kidney sample
(30e60 mg) was placed into 6% perchloric acid and homogenized.
The homogenate was centrifuged at 1050g for 15 min at 4 ꢀC and
then the supernatant was neutralized with 2 M K2CO3. The reaction
mixture contained glycine buffer (1 mL, 0.1 mM, pH 9.4), NADþ
(0.5 mL, 1.3 mM), sorbitol dehydrogenase (0.5 mL, 1.3 U/mL), and
the tissue extract (1.0 mL). The mixture was allowed to stand for
60 min at 37 ꢀC. The fluorescence intensity was measured at
365 nm excitation wavelength and 454 nm emission wavelength
using a fluorospectrophotometer. The sorbitol concentration was
quantitated by comparison with standards of sorbitol and
expressed as nmol/wet weight.
Xin Zhang, Changjin Zhu and Bing Ma designed the experi-
ments, analyzed the data and wrote the manuscript. Xin Zhang,
Huan Chen, Yanqi Lei, Xiaonan Zhang, Long Xu, Wenchao Liu,
Zhenya Fan, Zequn Ma, Lingyun Li and Zhechang Yin collected data
or performed experiments for the study.
Declaration of competing interest
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.
Acknowledgments
4.8. Liver MDA measurement
This work was supported by the National Natural Science
Foundation of China (grant no. 21572021). Furthermore, the au-
thors would like to thank Analysis & Testing Center, Beijing Insti-
tute of Technology (China) for technical support for performing in
the structural analysis of synthetic compounds.
Liver MDA were measured using a kit based on the protocols
provided by the manufacturer.
4.9. Determination of intracellular superoxide dismutase (SOD)
activity
Appendix A. Supplementary data
Total SOD activities of the samples was determined using the
Total Superoxide Dismutase Assay Kit with WST-8 based on the
protocols provided by the manufacturer.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113269.
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