Pyridothiadiazine derivatives as aldose reductase inhibitors having antioxidant activity

Article abstract of DOI:10.1080/14756366.2016.1178638

A series of aldose reductase (ALR2) inhibitors based on pyridothiadiazine were prepared and evaluated for their activities in ALR2 inhibition, DPPH scavenging, and MDA inhibition. Comparison studies were carried out between analogs having either hydroxyl

Full text of DOI:10.1080/14756366.2016.1178638

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Pyridothiadiazine derivatives as aldose reductase
inhibitors having antioxidant activity
Shaojuan Zhu, Shuzhen Zhang, Xin Hao, Xiangyu Qin, Shagufta Parveen,
Shaoqi Yang, Bing Ma & Changjin Zhu
To cite this article: Shaojuan Zhu, Shuzhen Zhang, Xin Hao, Xiangyu Qin, Shagufta Parveen,
Shaoqi Yang, Bing Ma & Changjin Zhu (2016): Pyridothiadiazine derivatives as aldose reductase
inhibitors having antioxidant activity, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.1080/14756366.2016.1178638
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–5
2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1178638
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ORIGINAL ARTICLE
Pyridothiadiazine derivatives as aldose reductase inhibitors having
antioxidant activity
Shaojuan Zhu, Shuzhen Zhang, Xin Hao, Xiangyu Qin, Shagufta Parveen, Shaoqi Yang, Bing Ma, and Changjin Zhu
Department of Applied Chemistry, Beijing Institute of Technology, Beijing, China
Abstract
Keywords
A series of aldose reductase (ALR2) inhibitors based on pyridothiadiazine were prepared and
evaluated for their activities in ALR2 inhibition, DPPH scavenging, and MDA inhibition.
Comparison studies were carried out between analogs having either hydroxyl or methoxy
groups substituted on the N2-benzyl side chains of the compounds. Most of the hydroxy-
substituted compounds were found to be more potent compared to their methoxy-substituted
analogs with respect to DPPH inhibition (493%) and MDA inhibition (473%). However, ALR2
inhibitory activity was found to be affected by the electron-withdrawing substituent at the C7
position in addition to the effect of the N2-substituted benzyl group. These results provide an
array of multifunctional ALR2 inhibitors possessing capacities both for ALR2 inhibition and as
antioxidants.
Aldose reductase inhibitors, antioxidant
activity, pyridothiadiazine derivatives,
structure-activity relationships
History
Received 30 January 2016
Revised 12 March 2016
Accepted 14 March 2016
Published online 3 May 2016
Introduction
prevention and treatment of diabetic complications. To this
purpose, a large number of ALR2 inhibitors (ARIs) have been
developed in the past few decades (Figure 2)6–9. Unfortunately,
few of these have advanced to the later stages of clinical trials. At
present, epalrestat is only the drug available on the market for
treatment of diabetic complications. Therefore, the development
of more ARIs suitable for use to prevent DM complications is still
needed.
Diabetes mellitus (DM), a life-threatening disease distributed
worldwide, is associated with various complications such as
retinopathy, nephropathy, cataract, neuropathy and stroke1,2
.
Diabetic complications triggered by hyperglycemia have been
proposed to involve three major pathways: protein kinase C
(PKC) isoform activation, increased glucose flux via the aldose
reductase pathway and higher production of glucose-derived
advanced glycation end products (AGE). As the starting event of
DM development is hyperglycemia, mechanisms such as glucose-
stimulated free radical production and hyperglycemia-induced
overproduction of mitochondrial superoxide have also been
studied3–5. Under hyperglycemic conditions, the polyol metabolic
pathway is activated (Figure 1) and leads to the accumulation of
sorbitol. Aldose reductase (E.C.1.1.1.21, ALR2), as the first and
rate-determining enzyme, catalyzes the reduction of excess
glucose to sorbitol in the presence of co-factor NADPH. In
turn, sorbitol dehydrogenase converts sorbitol to fructose through
an NAD+-dependent reaction. Sorbitol accumulation causes
osmotic stress, which results in tissue damage. Furthermore, the
increase in the NADH/NAD+ ratio promotes the activation of
PKC isoform that are believed to act as mediators for biochemical
It has been suggested that the discovery of ARIs possessing
antioxidant activity is very desirable when designing drug
candidates that are effective against diabetic complications,
since increased oxidative stress is a widely accepted participant
in the development and progression of DM and its complica-
tions3,10–13. A phenolic group is commonly involved in many
natural and synthetic products exhibiting antioxidant activity14–16
.
Previously, we have designed a series of ARIs based on the
scaffold of pyrido[2,3-e][1,2,4]thiadiazine-1,1-dioxide17,18. The
present study is focused on the introduction of phenolic groups
into the framework in order to obtain ARIs with antioxidant
functionality.
Results and discussion
and functional alterations induced by hyperglycemia. Moreover, The key intermediate compounds 1 in the synthesis were obtained
the activity of the NADPH-requiring enzyme glutathione (GSH) by starting from 2-amino-pyridines, and the procedure has been
reductase decreases with the depletion of NADPH cofactor. The previously reported17.
decrease in GSH level is associated with a concomitant rise in
toxic lipid peroxidation products like malondialdehyde (MDA)
and trans-4-hydroxy-2-nonenal (HNE). Therefore, the inhibition
of ALR2 has received considerable attention as a means for the
NADP+
Sorbitol
NAD+
NADH
NADPH
Glucose
Fructose
aldose
reductase
sorbitol
dehydrogenase
Address for correspondence: Dr Changjin Zhu, Department of Applied
Chemistry, Beijing Institute of Technology, No. 5, Zhongguancun South
Street, Beijing 100081, China. E-mail: zcj@bit.edu.cn
Figure 1. The polyol pathway of glucose metabolism.

2
S. Zhu et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
F
Figure 2. Previously reported aldose reduc-
tase inhibitors.
F
N
S
CO2H
O
O
CO2H
N
O
Br
Cl
N
F
N
S
N
O
Br
CO2H
Epalrestat
Zenarestat
Lidorestat
OH
O
OH
O
N
CO2H
F
OH
Br
NH
O
O
N
N
N
HN
S
O
O
F
OH
OH
N
H2N
O
O
F
Acetic acid derivative
of oxadiazole
Sorbinil
PPP
Benzenesulfonamide
The introduction of a benzyl group as a side chain at the N2- N2-benzyl ring as shown in Table 1. All the compounds could be
position of esters 1a–c was achieved by an alkylation reaction categorized according to substituents at the N2-benzyl ring in two
with benzyl bromide as appropriate to achieve the corresponding types: the first type included 5a–c and 6b–c, which contained
methyl esters 2a–c, 4a–c and 7, which were then treated with both an electron-donating group and fluoro, an electron-with-
different methods to form the final carboxylic acids18–20. The drawing group, at the N2-benzyl ring. The second type included
procedures are depicted in Schemes 1–3, respectively.
Compounds 2a–c were demethylated to form the final acids namely compounds 13a–c, 8, 11 and 12. Of all the compounds,
3a–c by treatment with BBr3. 5a–c and 6b–c in the first type showed a more potent inhibitory
those with only the electron-donating group at the N2-benzyl ring,
The preparation of compounds 6a–c proceeded from 4a–c, activity against ALR2 with IC50 values ranging from 1.3 to
which involved two steps as they were first converted into 5a–c by 2.86 mM. The compounds in the second type bearing only
hydrolysis with aqueous sodium hydroxide (Scheme 2). electron-donating substituents at the N2-benzyl ring, namely
Compounds 11 and 12 were obtained by demethylation with 13b–c, 3a–c, 8, 11 and 12, displayed less inhibitory activity
AlCl3 (Scheme 3).
against ALR2. These results suggest that electron-donating
The synthesized compounds were evaluated for their ALR2 substituents at the N2-benzyl ring retained ALR2 inhibition
inhibition, scavenging effects on the DPPH free radical, and MDA activity, and the electron-withdrawing substituents at the N2-
inhibition. The results are summarized in Table 1. Previously benzyl ring improved the activity of ARIs based on the
reported compounds17 were also included and named as 13a–c in pyrido[2,3-e]-[1,2,4]-thiadiazine 1,1-dioxide scaffold. This is
Table 1 for comparison.
consistent with a previous study17. In addition, in comparing
the series 3a–c with the parent compounds 13a–c, an interesting
inhibition profile was observed. The C7-halogen, N2-4-hydroxyl
derivatives 3a and 3b showed a higher activity than 13a and 13b,
respectively. In contrast, the C7- methyl, N2-4-hydroxyl deriva-
tives 3c appeared to be less active than 13c. These findings
ALR2 inhibitory activity
The synthetic work led to a series of compounds containing
diversified groups located on the C7-position of the core and the
O
O
O
O
OH
O
N
N
OCH3
iii
N
N
S
OH
N
N
S
i
N
O
N
O
NH
O
R
S
R
R
OCH3
O
O
O
1
2
3
BrH2C
1a, 2a, 3a
1b, 2b, 3b
: R = Br
: R = Cl
1c, 2c, 3c: R = CH3
Scheme 1. Reagents and conditions: (i) K2CO3, (ii) CH3CN, 70 ꢀC; (iii) BBr3, CH2Cl2, 0 ꢀC.
O
O
O
O
O
F
OH
F
O
OH
F
N
N
S
N
N
S
N
N
S
N
N
S
ii
i
iii
N
O
N
N
NH
O
R
OCH3
R
OCH3
F
R
OH
R
O
O
O
O
O
O
1
4
5
BrH2C
OCH3
6
1a, 4a, 5a: R = Br
1b, 4b, 5b: R = Cl
1c 4c 5c
: R = CH3
11b: R = Cl
11c: R = CH3
,
,
Scheme 2. Reagents and conditions: (i) K2CO3, CH3CN, 70 ꢀC; (ii) NaOHaq., THF, then HClaq. and (iii) K2CO3, thiophenol, NMP, 150 ꢀC.

DOI: 10.1080/14756366.2016.1178638
Antioxidant activity in pyridothiadiazine derivatives
3
O
O
O
OH OCH3
O
OCH3
O
N
N
S
N
N
S
N
N
S
i
iii
N
O
N
O
Br
O
OCH3
Br
OCH3
NH
O
BrH2C
OCH3
O
O
O
8
7
1
ii
BrH2C
OCH3
O
N
O
OH
O
OCH3
N
N
S
N
N
S
+
N
O
Br
OH
Br
OH
O
O
O
9
10
iii
iii
O
N
O
N
OH OH
OH OCH3
N
N
S
N
N
S
Br
OH
Br
OH
O
O
O
O
11
12
Scheme 3. Reagents and conditions: (i) K2CO3, CH3CN, 70 ꢀC; (ii) AlCl3, CH2Cl2, 40 ꢀC and (iii) NaOHaq., THF, then HCaq.
Table 1. Biological evaluation of pyridothiadiazine derivatives.
O
OH
N
N
S
R2
N
O
R1
O
Product
R2
DPPH
inhibition, n (%)y
MDA
(nmol/mgpro)z
MDA
inhibition, n (%)ô
Comp. no.
R1
ALR2 IC50 (mM)*
MDA IC50 (mM)x
13a
13b
13c
3a
3b
3c
5a
5b
5c
6b
Br
Cl
CH3
Br
Cl
CH3
Br
Cl
CH3
Cl
CH3
Br
Br
Br
4-OCH3
4-OCH3
4-OCH3
4-OH
4-OH
4-OH
2-F,5-OCH3
2-F,5-OCH3
2-F,5-OCH3
2-F,5-OH
2-F,5-OH
3-OCH3,5-OCH3
3-OH,5- OCH3
3-OH,5-OH
18.2 (0.52–20.76)
7.54 (4.32–8.01)
7.2 (4.79–7.98)
10.1 (7.85–13.92)
6.4 (3.21–10.13)
12.4 (4.85–16.54)
1.3 (1.03–1.24)
1.74 (0.79–1.75)
1.82 (0.71–2.12)
2.86 (1.31–3.16)
2.86 (1.58–4.31)
14.2 (4.10–17.91)
11.7 (6.22–14.38)
13.1 (5.77–12.07)
0.12 (0.08–0.13)
6.2
5.9
3.1
17.0
93.2
5.2
8.7
8.6
6.4
17.2
9.8
11.5
21.3
49.8
0
1.115 0.008
1.125 0.018
1.056 0.017
0.838 0.001
0.392 0.156
1.152 0.034
1.115 0.004
1.012 0.022
1.143 0.021
1.036 0.009
1.138 0.053
1.109 0.010
0.934 007
2.6
1.6
8.4
29.9
73.9
0
2.3
12.7
0
10.4
0.3
3.2
20.4
38.7
0
–
–
–
–
124.4 (74.7–153.5)
–
–
–
–
–
–
–
–
–
–
–
6c
8
11
12
0.799 0.005
1.148 0.032
0.052 0.002
0.127 0.002
1.141 0.056
Epalrestat
Trolox
Blank
Control
95.0
98.1
*IC50 (95% CL) values represent the concentration of the tested compounds required to decrease enzymatic activity by 50%.
yInhibitory effects were evaluated at a concentration of 100 mM.
zBlank: brain homogenate; control: brain homogenate + the oxidant system (FeCl3 20 mM and ascorbic acid 100 mM); the inhibitory effect was
estimated at a concentration of 100 mM.
ôThe MDA inhibitory activity % was calculated using the following equation: Inhibitory effect % ¼ [(control ꢁ test)/(control ꢁ blank)] ꢂ 100%. Blank:
brain homogenate; control: brain homogenate + the oxidant system (FeCl3 20 mM and ascorbic acid 100 mM); test: brain homogenate + the oxidant
system (FeCl3 20 mM and ascorbic acid 100 mM) + compound.
xIC50 (95% CL) values represent the concentration of the tested compounds required to inhibit the production of MDA by 50%.

4
S. Zhu et al.
J Enzyme Inhib Med Chem, Early Online: 1–5
indicated that the introduction of a phenolic group does not containing products 11, 12 formed from 9, and 10, respectively
necessarily increase inhibitory activity against ALR2. This is showed a much greater free radical scavenging activity (21.3 and
further evidenced by the fact that compound 11 is more active 49.8%, respectively) compared with their parent compound 8
than compound 8 but less active than compound 12, resulting in (11.5%), and compound 12 containing two hydroxyl groups
the rank order 1141248. In the case of compounds 13b and 3b, showed more than twice the activity of compound 11 containing
the introduction of a hydroxyl group increased the activity, which one hydroxyl group. Of all the hydroxyl contained compounds,
is likely due to the enhanced binding behavior between 3b and with respect to the effect of a C7-substituent at the core only, the
ALR2.
C7-chloro substitution undoubtedly had a more remarkable
influence on activity than that of C7-bromo and C7-methyl,
when comparing 3b with 3a and 3c, and 6b with 6c.
DPPH radical scavenging activity
The free radical scavenging potential of all the compounds were
assessed in vitro using a model reaction with the stable free
Inhibition of lipid peroxidation
radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Results are sum- To further confirm the antioxidant potential of the newly
marized in Table 1. While their free radical scavenging activity synthesized compounds described above, the inhibitory effect of
was less than that of Trolox, the positive control for antioxidant the compounds on hydroxyl radical dependent lipoperoxidation
potency, compounds 3a,b, 6b, 8, 11 and 12 showed good to induced in rat brain homogenate by the oxidant system Fe(III)/
excellent DPPH radical scavenging activity, and compound 3b ascorbic acid was also examined. The level of MDA is an
was the most active achieving 93.2% activity at a concentration of indicator of lipid peroxidation in cellular membranes. In mem-
100 mM, which approached that of Trolox (95%).
branes, relative antioxidant reactivity is probably different from
Structure-activity relationship studies were based on three that in a homogeneous system, because of the role played by
comparisons. Among all the compounds in Table 1, the additional factors such as the subcellular location of the antioxi-
compounds containing a phenolic hydroxyl group showed dants and radicals, and to some extent, the differing activities
higher free radical scavenging activity compared to their parent between aqueous and lipophilic compartments. The antioxidation
analogs, revealing that the phenolic subunit is a structural results from the DPPH radical scavenging experiment were
determinant of DPPH free radical scavenging activity in the perfectly replicated in this assessment demonstrating suppression
present study. This is further confirmed by the effect of the of lipid peroxidation with the compounds. As shown in Table 1,
number of the hydroxyl group in compounds 8, 11, 12 on the the tested compounds inhibited the production of MDA and the
DPPH radical scavenging activity in which the hydroxyl group- thiobarbituric acid reactive substances (TBARS), and reduced the
Figure 3. Molecular docking of compound 13b (A & B) and 3b (C & D). (A & C) Docking of compounds 13b or 3b into the ALR2 active site. The
protein structure is shown in ribbon and tube representation with selected residues labeled and shown in line representation; ligand 13b (or 3b) and
NADPH are shown as stick models. The docked pose of 13b and 3b are shown in yellow. Hydrogen bonds are shown as green dashed lines. (B & D)
Surface representation of protein active site residues in the docking of 13b or 3b into the active site of ALR2.

DOI: 10.1080/14756366.2016.1178638
Antioxidant activity in pyridothiadiazine derivatives
5
index of lipid peroxidation, with varying degrees of efficacy. References
Compounds 3a,b, 10b, 11b, 11 and 12 showed appreciable
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compounds. In particular, the inhibitory activity of the most active
compound 3b was 73.9% with an IC50 value of 124.4 mM, further
demonstrating the importance of the hydroxyl substituents in the
derivatives for their antioxidant activity. This is consistent with
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Molecular docking
To understand the mechanistic details in the inhibition of ALR2
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molecular docking of compounds 13b and 3b, a pair of structural
analogs with 3b possessing good activities both in ALR2
inhibition and antioxidant activity, were performed on a human
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Conclusions
The developed pyridothiadiazine derivatives having the electron
withdrawing group at the C7 position and a hydroxyl group on the
N2-benzyl ring were active in ALR2 inhibition. The hydroxyl
group on the N2-benzyl ring was more effective than a methoxy
group for ARI activity. Furthermore, compounds 3a–b and 10
showed appreciable antioxidant properties in the tests both of
DPPH scavenging and MDA inhibition. In addition, the increase
in the number of the hydroxyl group on the N2-benzyl ring
significantly promoted the DPPH scavenging activity and MDA
inhibition of compounds.
Declaration of interest
This work was supported by the National Natural Science
Foundation of China (Grant No. 21272025), the Research
Fund for the Doctoral Program of Higher Education of
China (Grant No. 20111101110042) and the Science and
Technology Commission of Beijing, China (Grant No.
Z131100004013003).
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Supplementary material available online