Antihyperglycemic and neuroprotective effects of one novel Cu-Zn SOD mimetic

Article abstract of DOI:10.1016/j.bmcl.2011.05.051

Increasing evidence supports that OS plays important roles in diabetes mellitus and cerebral ischemia. This suggests that recovering an impaired endogenous superoxide dismutase (SOD) enzyme system induced by OS with a mimetic would be beneficial and protective for these diseases. In present study, one nonpeptidyl small molecular weight compound (D34) was synthesized. Its SOD mimetic activity and the potential therapeutic actions were also evaluated both in vivo and in vitro. The in vitro nitro blue tetrazolium (NBT) assay indicated that D34 presents an SOD mimetic activity. D34 (20 μmol/kg) exhibited significant antihyperglycemic activity in alloxan-diabetic mice. D34 could also ameliorate the cerebral neuronal death in hippocampus of global cerebral ischemia mice. Furthermore, the D34 treatment significantly decreased malondialdehyde (MDA) contents and increased SOD activities in brains or livers of diabetes mice or cerebral ischemic mice. In conclusion, these preliminary findings support that D34 exhibits SOD mimetic activity and possesses significant antihyperglycemic and neuroprotective effects.

Full text of DOI:10.1016/j.bmcl.2011.05.051

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4320–4324
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antihyperglycemic and neuroprotective effects of one novel Cu–Zn SOD mimetic
Che Wang a,b, Song Li c, , De-Jing Shang b, Xiao-Ling Wang a, Zhong-Lu You a, Hui-Bing Li a
⇑
a Department of Pharmacy, School of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian 116029, China
b Key Laboratory of Biotechnology and Drug Discovery, School of Life Science, Liaoning Normal University, Dalian 116029, China
c Institute of Neuroinformatics, Dalian University of Technology, Dalian 116024, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 February 2011
Revised 5 May 2011
Accepted 17 May 2011
Available online 24 May 2011
Increasing evidence supports that OS plays important roles in diabetes mellitus and cerebral ischemia.
This suggests that recovering an impaired endogenous superoxide dismutase (SOD) enzyme system
induced by OS with a mimetic would be beneficial and protective for these diseases. In present study,
one nonpeptidyl small molecular weight compound (D34) was synthesized. Its SOD mimetic activity
and the potential therapeutic actions were also evaluated both in vivo and in vitro. The in vitro nitro blue
tetrazolium (NBT) assay indicated that D34 presents an SOD mimetic activity. D34 (20 lmol/kg) exhib-
Keywords:
SOD mimetic
Diabetes
Antihyperglycemic
Cerebral ischemia
Neuroprotective
ited significant antihyperglycemic activity in alloxan-diabetic mice. D34 could also ameliorate the cere-
bral neuronal death in hippocampus of global cerebral ischemia mice. Furthermore, the D34 treatment
significantly decreased malondialdehyde (MDA) contents and increased SOD activities in brains or livers
of diabetes mice or cerebral ischemic mice. In conclusion, these preliminary findings support that D34
exhibits SOD mimetic activity and possesses significant antihyperglycemic and neuroprotective effects.
Ó 2011 Elsevier Ltd. All rights reserved.
Oxidative stress (OS) is characterized by over-produced cellular
oxidants (e.g., superoxide and hydrogen peroxide) and/or de-
creased antioxidants and antioxidant enzymes [e.g., vitamin E
and superoxide dismutase, (SOD)]. Excessive OS can directly lead
to DNA and protein modification and lipid peroxidation,1–3 de-
creases insulin mRNA, cytosolic ATP, calcium influx into cytosol
and mitochondria, and causes apoptosis.4,5
Increasing evidence in studies of both rodents and human has
suggested that OS is associated with numerous pathological condi-
tions, including diabetes mellitus and cerebral ischemia. In diabe-
tes mellitus, OS participates not only in b-cell dysfunction and
insulin resistance but also in the genesis of other late complica-
tions of diabetes.6–8 Cerebral ischemia is another one pathological
condition correlated closely with OS. Temporary or permanent
hypoperfusion could induce depletion of SOD, and then production
of OS,9–11 ultimately leading to neuronal death.12
Over expression of SOD or supplements of antioxidants includ-
ing SOD mimetics, targeted to overcome OS, reduce reactive oxy-
gen species and increase antioxidant enzymes, has been shown
to prevent diabetes mellitus13–17 and reduce neuronal damage in
cerebral ischemia.18–23 Recently, the nonpeptidyl low molecular
weight compounds have been reported, which possess significant
antioxidant activity to that of the native SOD enzymes.24–29 The
use of these compounds suggests to provide a better therapeutic
approach in diseases mediated by OS.
In present study, we investigated the SOD mimetic activity,
antihyperglycemic and neuroprotective effects of D34, a novel,
low molecular weight, nonpeptidyl compound. The compound
(Fig. 1, structure formula: C21H24N2O4Cl2CuZn; molecular weight:
568.23) was synthesized in the laboratory of the Department of
Pharmacy, College of Chemistry and Chemical Engineering,
Liaoning Normal University, China, and is currently pending for
Chinese and international patent.
D34 is a liposoluble binuclear schiff base Cu(II)–Zn(II) complex
with good solubility in DMSO or methanol D34 exerts good stabil-
ity in aqueous environment without releasing of Copper or Zinc
ions due to its poor water-solubility.
The SOD mimetic activity of D34 was measured by nitro blue
tetrazolium (NBT) method in vitro.29,30 Superoxide anions were
produced from the riboflavin/methionine system. The indicator
À
utilized in this case is NBT, which reacts with O2 to form blue
formazane. Reaction system containing 3.3 Â 10À6 M riboflavin,
0.01 M methionine, 4.6 Â 10À5 M NBT, and 0.05 M phosphate buf-
fer pH 7.4, and 0–1.5
l
g/ml D34 or 2 Â 10À9–2 Â 10À8 M native
Cu–Zn SOD, were illuminated under fluorescent lamps. The absor-
bance at 560 nm increased linearly with time of illumination. The
reduction of NBT was measured in terms of increased absorbance
at 560 nm on a Shimadzu UV-240 spectrophotometer. All photo-
induced reactions were performed at 25 °C. Each test was per-
formed in triplicate.
To further investigate potential therapeutic effects of D34 for
diabetes and cerebral ischemia in vivo, alloxan-diabetic model
and bilateral common carotid artery occlusion (BCCAO) model
⇑
Corresponding author. Tel.: +86 411 84706039; fax: +86 411 84706046.
E-mail address: lisong_spu2005@yahoo.com (S. Li).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.051

C. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4320–4324
4321
N
Cu
O
N
O
CHO
OH
b
a
N
N
+
H2N
NH2
OH
HO
60 min
30 min
Zn
Cl Cl
OEt
OEt
OEt
O
O
Figure 1. Synthesis of ligand and D34, a novel nonpeptidyl copper(II)–zinc(II) superoxide dismutase mimetic. Reagents and conditions: (a) 3-ethoxysalicylaldehyde,
1,3-diaminopropane, methanol, 92% yield; (b) Cu(CH3COO)2ÁH2O and ZnCl2, methanol 50% yield.
were established in mice. For evaluating the hypoglycemic effects
of D34 on healthy mice with normal blood glucose level, mice were
administrated intraperitoneally (ip 0.2 ml/day) for 3 days with D34
A Student’s t-test was used to determine significant differences of
individual responses between groups. p <0.05 was determined to
be significant.
(20
lmol/kg body weight) or vehicle following an overnight fast-
The results of NBT tests in vitro were summarized in Figure 2.
The results showed a considerable antioxidant activity of D34.
ing. Blood samples were collected by tail nipping before the first
administration and 1 h after the last administration. Blood glucose
levels were determined using a commercially available quantifica-
tion kit based on glucose oxidase method. For evaluating the anti-
hyperglycemic effect, the alloxan-diabetic animal model was
established as described previously.31 Briefly, Mice were injected
with alloxan (150 mg/kg body weight, ip) after overnight fasting
for 12 h. Seventy-two hours after alloxan injection, mice with
blood glucose levels above 11.1 mmol/l were included in the study,
and then treated daily with D34 or saline for 2 days. Blood samples
were collected by tail nipping for the following assessment for
blood glucose. Finally, the mice were sacrificed, and the livers
and brains were removed and weighed. The tissues were rapidly
homogenized for bioassay or stored at À80 °C until required.
For global cerebral ischemia, mice were anesthetized with Chlo-
ral Hydrate (400 mg/kg, ip) and then subjected to transient cere-
bral hypoperfusion, as previously described with minor
modification.32 In brief, transient cerebral hypoperfusion was in-
duced by BCCAO with aneurysm clips for 20 min, and circulation
was restored by removing the clips. During the surgical procedure,
rectal temperature was maintained at 36 0.5 °C with heating pad.
After reperfusion, the animals were placed in a warm incubator
(32–33 °C). Hippocampal neuronal damage were evaluated 3 days
after BCCAO surgery since in our preliminary study significant neu-
ronal cell death was found in the CA1, CA3 and dentate gyrus (DG)
subregions of hippocampus at this time point. Briefly, mice were
anesthetized with Chloral Hydrate (400 mg/kg, ip). After decapita-
tion, brains were rapidly removed and homogenized for bioassay,
or frozen quickly on dry-ice powder and stored at À80 °C for the
histological examination. For the assessment of neural damage,
While the IC50 value of native Cu–Zn SOD is 0.015
lue of D34 is 1.31 M (0.76 g/ml, Table 1).
lM, the IC50 va-
l
l
The effect of the D34 on blood glucose was assessed in fasting
normal mice. The average levels of blood glucose in each group
are shown in Figure 3A. D34 treated mice did not exhibit any sig-
nificant alteration in their blood glucose levels after 3 days treat-
ment. The antihyperglycemic effect of the D34 on the blood
glucose levels of fasting diabetic mice is shown in Figure 3B. Intra-
peritoneal injection of alloxan monohydrate (150 mg/kg) led a sig-
nificant elevation of blood glucose level (p <0.01). Blood glucose
levels were 1.8-fold higher in diabetic mice than control group.
Treatment of the D34 for 2 days led to a significant fall in blood
glucose levels (p <0.05). The blood glucose level of D34 was about
26.7% lower than those of the diabetes group.
Ischemic neuronal damage in 20 min BCCAO mice was signifi-
cantly increased in hippocampus CA1, CA3, and dentate gyrus
(DG) subregions as compared with control mice (Fig. 4). Pre-treat-
ment with the D34 30 min before BCCAO significantly ameliorated
neuronal damage.
Figures 5 and 6 shows the MDA level and SOD activity in liver or
brain homogenates of normal and experimental animals. MDA was
significantly increased in livers of diabetic mice and in brains of
BCCAO mice whereas SOD activities were significantly decreased
0.16
0.14
0.12
A
1
2
3
4
0.01
5
0.08
0.06
0.04
0.02
0
6
7
coronal sections (10 lm) were prepared and then stained with
0.1% (w/v) cresyl violet. Morphologically abnormal neurons were
quantified at 400Â magnifications. Damaged neurons in each brain
subregion were semi-quantitatively scored as: (0) no ischemic
neurons; (1) 1–30% ischemic neurons; (2) 31–65% ischemic neu-
rons; (3) 66–100% ischemic neurons. The utility of this approach
has been demonstrated in a previous study with MF1 and C57Bl/
6J mice.33,34
Collected tissues were thawed, weighed and homogenized with
Tris–HCl (5 mmol/l containing 2 mmol/l EDTA, pH 7.4). Homoge-
nates were centrifuged (1000g, 15 min, 4 °C) and the supernatant
was used immediately for the assays of SOD activities and MDA
levels in liver or brain homogenates following the commercial kits
instructions.
0
0.5
1
1.5
2
2.5
3
3.5
T (min)
B
100
90
80
70
60
50
40
30
20
10
0
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
C (µg/mL)
All values are reported as mean SEM. Statistical analysis was
performed with SPSS software (for windows, version 13.0). Overall
significance within and between groups was determined using
analysis of variance (ANOVA). Dunnett’s post hoc test was applied
when significant differences were found using analysis of variance.
Figure 2. Antioxidant activity of D34 in vitro. (A) Absorbance values of D34 in NBT
assay in vitro [complex/M: (1) 0 (in absence of the D34); (2) 0.05 g/ml; (3) 0.15 g/
ml; (4) 0.25 g/ml; (5) 0.5 g/ml; (6) 1.0 g/ml and (7) 1.5 g/ml. (B) Inhibition
percentage of NBT reduction with an increase in the concentration of the complex.
l
l
l
l
l
l

4322
C. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4320–4324
Table 1
genetic manipulations have been found to limit the extent of path-
SOD-mimetic activities described by IC50
ological conditions. Thus, administration of native SOD or SOD mi-
metic could be a promising therapeutic approach,35–40 although
few of them have shown clinical potency.
a
Complex
IC50
(
l
M)
Reference
Native Cu–Zn SOD
D34
ZnCl2
0.015
1.31
127.5
5.12
6.15
3.16
2.25
3.9
This work
This work
This work
This work
29
In present study, the antioxidant and SOD mimetic activity of
D34, one nonpeptidyl low molecular weight compound, were eval-
uated in vitro by NBT method, a convenient and commonly used
bio-assay for screening of SOD mimetics.41 Antioxidant activity of
a test complex is usually defined as one unit SOD activity, which
is the concentration of a test complex or enzyme, which causes
50% inhibition reduction of NBT. The more efficient a test complex
is, the lower the concentration that corresponds to 50% inhibition
of NBT or reduction the IC50 value. Here, we reported that the
NBT reduction was effectively inhibited by D34, though the activity
is about 100 times less than that of the native Cu–Zn SOD. How-
ever, this complex is still a potent SOD mimic considering the very
low molecular weight when compared with that of the native SOD
enzyme (molecular weight, 32000 Da), which suggests that the
D34 possessed antioxidant activities for ameliorating the OS and
have the potential use in diabetes mellitus and cerebral ischemic
CuCl2
[Cu(C9H7NO3)(C12H8N2)]
[Cu(en)2](sal)2
[Cu(Pu-6-MePy)(H2O)](ClO4)2
SALCuCl2
C
22H24Cl2CuN2O4Zn
3.127
a
The reaction was conducted in anoxic conditions.
as compared with normal mice. The D34 treatment could reverse
these changes.
OS is defined in general as over-production and/or deficient re-
moval of highly reactive molecules, and indirectly initiates lipid
peroxidation. Both inhibition of production and enhanced detoxifi-
cation of reactive oxygen species with pharmacological agents or
A
B
12
20
Control
D34
**
10
8
16
#
12
8
6
4
4
2
0
0
Day 3
Day 0
Control Diabetic
D34
Figure 3. Antiglycemic activity of D34 in normal mice (A) and alloxan-diabetic mice (B). Blood samples were collected from the mice before the first administration and 1 h
after the last administration of the D34, and were processed for the analysis of glucose concentrations. ⁄⁄p <0.01 when compared with control group; #p <0.05 when compared
with diabetic group (n = 8–10).
CA1
CA3
DG
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
**
**
*
#
Control
Control
Control
BCCAO D34
BCCAO D34
BCCAO D34
Figure 4. Neuronal damage in CA1, CA3, and DG subregions of hippocampus in BCCAO ischemia mice. Coronal sections (10
neurons in each brain region were scored semi-quantitatively. ⁄p <0.01 when compared with control group.
lm) were stained with cresyl violet. Ischemic

C. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4320–4324
4323
In order to further evaluate the antihyperglycemic and neuro-
protective effects in vivo, the alloxan-diabetic model and BCCAO
model in mice were established. Diabetes mellitus is a group of
metabolic diseases characterized by abnormality of the blood glu-
cose metabolism resulting from altered insulin production or activ-
ity. The precise cellular and molecular mechanisms underlie the
etiology and progressions of diabetes are still not fully understood.
However, OS is thought to play a central role on the development
of diabetes and many diabetic complications. In line with this, an
increase in lipid peroxidation and deficits in the antioxidant de-
fense systems have been observed in a variety of experimental
models of diabetes.42–53 Among those animal models, alloxan-in-
duced diabetic model has been widely accepted. Alloxan induces
type I diabetes mellitus (insulin-dependent diabetes) by inducing
ROS formation and resulting in the selective necrosis of beta
cells.54 Thus, compounds that present antioxidant effects are of po-
tential therapeutic interest for the treatment of human and animal
diabetics. This postulates that D34 having high antioxidant poten-
tial may have a role to prevent the development of diabetes. The
present investigation showed that D34 could reverse the hypergly-
cemia in mice induced by alloxan treatment, without influence on
normal blood glucose level of heathy animals. The decreased SOD
activity and the enhanced MDA level in both brains and livers of
diabetic mice were reversed by D34, suggesting that the antidia-
betic activities of D34 might be due to its SOD mimetic property
and antioxidant activities.
The present investigation also revealed that D34 exhibited the
neuroprotective potential against BCCAO induced OS and neuronal
damage. It is observed that D34 attenuated the neuronal death in
CA1, CA3, and DG. The activity of D34 appears to work by restoring
the altered antioxidants enzymes as well as decrease the produc-
tion of MDA induced by BCCAO. There is a considerable evidence
supports that the role of OS in the pathogenesis of cerebral ische-
mia.55,56 Brain reperfusion after ischemia frequently leads to neu-
ronal death, which occurs preferentially in some brain regions,
such as hippocampus. Such neuronal death has long been associ-
ated with excessive production of reactive oxygen species during
OS, which reacts with cellular macromolecules and leads to oxida-
tive damage of the neurons. Therefore, the potent antioxidant
properties or LPO inhibiting ability of SOD mimetic compounds
protecting the neurons from OS may provide useful therapeutic
agent for cerebral ischemia.
A
30
25
20
15
10
5
Brain
Liver
#
*
#
*
0
Control
Control
Diabetic D34
Diabetic D34
Liver
B
4
3
2
1
0
Brain
**
*
# #
#
Control
Control
Diabetic D34
Diabetic D34
Figure 5. SOD activities and MDA levels in liver or brain of alloxan-diabetic mice. ⁄
p
<0.05 when compared with control group; #p <0.05 when compared with diabetic
group. ⁄⁄p <0.01 when compared with control group; ##p <0.01 when compared
with diabetic group (n = 8–10).
diseases. Moreover, IC50 value of D34 is higher than Cu2+ and Zn2+
indicating that the antioxidant activity of D34 might not be due to
released ions.
In summary, these preliminary experimental findings reveal
that D34 possesses SOD mimetic activity, and exhibits antihyper-
A
B
4
30
*
25
20
3
*
2
1
0
15
10
5
0
Control
Control
BCCAO D34
BCCAO D34
Figure 6. SOD activities and MDA levels in brain homogenates of BCCAO mice. ⁄p <0.05 when compared with control group (n = 8–10).

4324
C. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4320–4324
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ing the antioxidant defense system. These results support the effi-
cacy of D34 for diabetes and cerebral ischemia treatment.
However, further studies are still necessary for the evaluation of
the detailed mechanism of antidiabetic and neuroprotective activ-
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clinical treatment potential for human.
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This study was supported partly by the China Fundamental Re-
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.051.
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