Design, synthesis and biological evaluation of glucose-containing scutellarein derivatives as neuroprotective agents based on metabolic mechanism of scutellarin in vivo

Article abstract of DOI:10.1016/j.bmcl.2012.11.002

Based on metabolic mechanism of scutellarin in vivo that scutellarin could be hydrolyzed into scutellarein by β-glucuronide enzyme, some glucose-containing scutellarein derivatives were designed and synthesized through the introduction of glucose moiety at C-7 position of scutellarein via a glucosidic bond. Biological activity evaluation showed that these glucose-containing scutellarein derivatives exhibited potent DPPH radical scavenging activities. Furthermore, the improvement of physicochemical properties such as anticoagulant and neuroprotective activities alongside with the water solubility was achieved by introducing glucose. These findings suggest that the introduction of the glucose moiety to scutellarein wattants further development of this kind of compounds as neuroprotective agents.

Full text of DOI:10.1016/j.bmcl.2012.11.002

Bioorganic & Medicinal Chemistry Letters 23 (2013) 102–106
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of glucose-containing
scutellarein derivatives as neuroprotective agents based on metabolic
mechanism of scutellarin in vivo
Nian-Guang Li a,b, Min-Zhe Shen a, Zhen-Jiang Wang a, Yu-Ping Tang a, , Zhi-Hao Shi a,c, Yi-Fan Fu a,
⇑
Qian-Ping Shi a,b, Hao Tang a, Jian-Ao Duan a,
⇑
a Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210046, China
b Department of Medicinal Chemistry, Nanjing University of Chinese Medicine, Nanjing 210046, China
c Department of Organic Chemistry, China Pharmaceutical University, Nanjing 211198, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on metabolic mechanism of scutellarin in vivo that scutellarin could be hydrolyzed into scutella-
rein by b-glucuronide enzyme, some glucose-containing scutellarein derivatives were designed and syn-
thesized through the introduction of glucose moiety at C-7 position of scutellarein via a glucosidic bond.
Biological activity evaluation showed that these glucose-containing scutellarein derivatives exhibited
potent DPPH radical scavenging activities. Furthermore, the improvement of physicochemical properties
such as anticoagulant and neuroprotective activities alongside with the water solubility was achieved by
introducing glucose. These findings suggest that the introduction of the glucose moiety to scutellarein
wattants further development of this kind of compounds as neuroprotective agents.
Received 1 September 2012
Revised 31 October 2012
Accepted 2 November 2012
Available online 10 November 2012
Keywords:
Scutellarin
Scutellarein
Glucose
Ó 2012 Elsevier Ltd. All rights reserved.
Antioxidant
Anticoagulation
Neuroprotection
Solubility
Traditional Chinese medicines (TCMs) have been used clinically
for many years and can be regarded as potential sources for drug
discovery. Scutellarin (1) (Fig. 1), which is the main effective con-
stituent of breviscapine (>85%), a clinic natural drug consisting of
total flavonoids of Erigeron breviscapus (Vant.) Hand-Mazz. (Com-
positae), has been used for the treatment of cerebral infarction,
coronary heart disease, and angina pectoris in China.1 Due to the
distinguished efficacy of scutellarin in the clinical therapy, the re-
search of scutellarin has become a hot topic in China in recent
years. Pharmacological studies have demonstrated that scutellarin
is associated with a wide range of benefits to brain injury caused
by cerebral ischemia/reperfusion, these benefits are due to its
antioxidant and anticoagulant activities to attenuate neuronal
damage.2–4
OH
O
HO
HO
OH
O
OH
OH
B
O
O
C
HO
HO
O
O
A
HO
OH
1: scutellarin
O
OH
2: scutellarein
Figure 1. Chemical structures of scutellarin (1) and scutellarein (2).
been a major impediment to its overall effectiveness as an oral
drug. The other reason was that scutellarin is readily converted
into scutellarein (2) (Fig. 2) before absorption, the latter is
relatively easily absorbed into the blood and can metabolite into
glucuronidated, sulfated or methylated forms.12 Due to the low
bioavailability after oral administration, direct administration of
scutellarin by injection is the most common route of administra-
tion in clinical settings. Nonetheless, therapeutic effects elicited
by breviscapine require repeated injection daily for a long time,
this is highly inconvenient and results in low patient compliance.
Pharmacokinetic studies on scutellarin have been investigated
in rats,5–7 dogs8 and humans9 after oral administration, and the re-
sults showed that the oral bioavailability of scutellarin was quite
poor.10 One reason was its poor aqueous solubility and low lipo-
philicity,11 its poor ability to penetrate cell membranes has long
⇑
Corresponding authors. Tel./fax: +86 25 85811916.
E-mail addresses: yupingtang@njutcm.edu.cn (Y.-P. Tang), dja@njutcm.edu.cn
(J.-A. Duan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.002

N.-G. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 102–106
103
OH
O
HO
glucose
OH
O
OH
OH
HO
Hydrolysis
Hydrolysis
O
O
O
O
O
β-glucuronide
β-glucuronide
enzyme
enzyme
HO
HO
OH
O
OH
3: glucose-containing
1: scutellarin
scutellarein derivatives
Figures 2. Design of glucose-containing scutellarein derivatives.
It has been reported that scutellarin (1) is readily hydrolyzed
into scutellarein (2) by b-glucuronide enzyme and the microbial
population in the intestine prior to absorption.12 Since the glucose
connected to scutellarein via a glucosidic bond is unlikely to be
cleaved by b-glucuronide enzyme and the microbial population
in the intestine, in the present study, the glucose moiety was
introduced to scutellarein at C-7 position via a glucosidic bond
(3) (Fig. 2). We assessed the anti-oxidation and anticoagulant
activities, as well as the neuroprotective activities of 3a–3c to
investigate whether the biological activities of parent scutellarin
1 was retained in these glucose-containing scutellareins. Further-
more, the solubility of these glucose-containing scutellarein
derivatives was also evaluated.
OH OH
Ac2O, HClO4
OAc OAc
OAc OAc
HO
HO
AcO
AcO
AcO
AcO
Red P, Br2
O
O
O
OH
10
OAc
11
Br
8
Scheme 2. Reagents and conditions: (a) Ac2O (10.0 equiv, HClO4 (0.05 equiv); (b)
red phosphorus (0.2 equiv), Br2 (10 equiv), H2O, 25 °C, 40% over two steps.
O-Acetyl-
(10) in two steps as shown in Scheme 2, firstly, penta-O-acetyl-
glucose (11) was synthesized after -glucose (10) was reacted with
D-glucosyl bromide 8 was synthesized from D-glucose
D
-
D
acetic anhydride under the catalyst of perchloric acid, then the
reaction mixture was reacted with red phosphorus and liquid bro-
mine directly without separation, and the target O-acetyl-D-gluco-
syl bromide 8 was obtained in 40% yield over these two steps.
The formation of 3b and 3c were obtained through the intro-
duction of O-acetyl-D-manmosyl bromide and O-acetyl-D-galacto-
The regioselective D-glucose-containing scutellarein derivative
(3a) was achieved using the Mitsunobu method,13 which involved
the coupling of protected scutellarein (7) with protected glucose
(8) (Scheme 1). Scutellarin (1) was firstly hydrolyzed by refluxing
with 6 N HCl in ethanol under a N2 atmosphere to generate scutel-
larein (4) in 17.0% yield. Compound 4 was then converted into 5
(78.9% yield) after it was reacted with acetic anhydride and cata-
lytic 4-N,N-dimethylaminopyridine (DMAP) in pyridine. Interest-
ingly, when compound 5 reacted with BnBr using K2CO3 and
catalytic KI in dry acetone, the desired intermediate 6 was obtained
in 70% yield. Deprotection of the benzyl group in 6 was accom-
plished under hydrogenation conditions with 10% palladium on
carbon as the catalyst in EtOH/CH2Cl2 gave 7 in 95% yield. Glycosyl-
ation of hydroxyl group took place completely at the position 7
using 2.5 equiv of K2CO3 and 2.0 equiv of AgO as bases in quinoline
that led to 9.14 Finally, the hydrolysis of acetyl groups in 9 with a
solution of sodium hydroxide afforded 3a in 41% yield.
pyranosyl bromide, respectively, using a procedure similar to
that described above.
The antioxidant activities of the synthesized glucose-containing
scutellarein derivatives 3a–3c were evaluated by examining DPPH
radical scavenging according to our previous procedure.15 For
comparison purposes, the antioxidant activity of scutellarin (1)
was included as positive controls. As shown in Table 1, the synthe-
sized compounds showed DPPH radical scavenging activities
(IC50 = 25.31–34.86
lM) that were similar to those of the scutella-
rin (1) (IC50 = 26.78
lM), which indicated that the introduction of
the glucose moiety had little effect on the change of DPPH radical
OH
O
HO
HO
OH
OAc
c
OAc
O
OH
OH
b
AcO
AcO
O
BnO
AcO
O
O
O
O
HO
HO
O
O
a
HO
OAc O
OAc O
OH
OH
5
6
4
1
OAc OAc
OH OH
OAc OAc
O
AcO
AcO
HO
HO
AcO
AcO
O
O
OAc
OAc
O
OH
O
HO
O
O
O
O
Br
f
d
8
AcO
AcO
HO
e
OAc O
9
OAc O
OH
3a
7
Scheme 1. Reagents and conditions: (a) 6 N Concentrated hydrochloric acid, EtOH, N2, 120 °C, 17.0%; (b) Ac2O (10.0 equiv), pyridine (10.0 equiv), DMAP (0.1 equiv), 25 °C,
12 h, 78.9%; (c) PhCH2Br (3.0 equiv), K2CO3 (7.0 equiv), KI (1.0 equiv), acetone, reflux, 6 h, 70%; (d) Pd/C (10 wt %), H2 (1 atm), CH2Cl2/Et2OH, 25 °C, 24 h, 95%; (e) 8 (4.0 equiv),
CuSO4 (2.5 equiv), AgO (2.0 equiv), quinoline, 25 °C, 12 h, 40%; (f) NaOH (2.5 M), N2, CHCl3, 0 °C, 41%.

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N.-G. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 102–106
Table 1
However, when the PC12 cells were co-incubated with scutellarin
In vitro antioxidatant activity in DPPH assay (IC50 in
glucose-containing scutellarein derivatives
lM) and water solubility of
(1) and glucose-containing scutellarein derivatives 3a–3c, H2O2-
induced cell toxicity was significantly attenuated, and the
protective effect of these compounds was resulted in concentra-
tion-dependent increases in cell survival (Table 2). Of the
compounds tested, although no clear structure–activity relation-
ship was found, 3a and 3c were more effective at protecting these
neuronal cells against H2O2-induced oxidative injury than scutell-
arin (1). 3c showed the most potency in the inhibition of oxidative
injury, with inhibiting rates were 83.33% and 60.98% at 50 and
Compd Structure
OH
IC50 (lM) Solubility (lM)
O
HO
OH
O
OH
OH
OH
OH
HO
1
26.78
25.31
34.86
28.42
7.53
O
O
O
HO
25
lM, respectively (Table 2), the inhibiting rates of the parent
OH
OH OH
compounds 1 were 78.79% and 52.65% at 50 and 25
lM, respec-
tively. Based on these findings, we conclude that glucose addition
to the parent structures had some positive effect on the protective
actions of the parent compounds against H2O2-induced oxidative
neuronal damage.
HO
HO
O
O
3a
3b
3c
7.85
8.19
8.93
O
O
Because the anticoagulant activities can be assessed through
the inhibition of thrombin activities, which can be evaluated
through the analyzation of the prolongation of the plasma clotting
time of thrombin time (TT), activated partial thromboplastin time
(APTT), prothrombin time (PT), and reduction of fibrinogen (FIB)
content according to our previous studies,18 so the anticoagulant
activities of these glucose-containing scutellarein derivatives were
investigated for TT, PT, APTT and FIB and the results were shown in
Table 3. The most active compound was 3c, it significantly pro-
longed TT (29.17 s) and APTT (39.62 s), increased PT (9.93 s) and
decreased FIB content (20.81 g/L) compared to scutellarin (1). In
addition, 3a had stronger anticoagulant activity than scutellarin
(1), although it decreased TT (18.13 s), however, it prolonged APTT
(43.06 s) and PT (7.81 s), decreased FIB content (32.43 g/L) com-
pared to scutellarin (1). 3b showed no more active effect on plasma
coagulation parameters than 1, despite it prolonged APTT (38.20 s),
decreased FIB content (26.68 s), but TT (19.54 s) and PT (6.57 s) de-
creased compared to 1. These results indicated that 7-hydroxyl po-
sition can be modified by glucosyl group without reducing the
anticoagulant activity.
HO
OH
OH OH
HO
HO
O
O
O
O
HO
OH
OH OH
HO
HO
O
O
O
O
HO
OH
In the thrombin inhibition activity tests, 3c showed the stron-
gest inhibitory activity on thrombin, so scutellarin (1) and 3c
was selected for the subsequent molecular docking experiment
with thrombin (2R2M) (Fig. 3). There were three pockets (S1, S2,
S3) in the thrombin as suggested by molecular modeling augured
well for anticipating in vitro activity as well according to our pre-
vious studies.18 In the binding mode of the scutellarin (1) with
thrombin (2R2M), as shown in the up part of Figure 3, the B ring
in scutellarin (1) mainly interacted with S1 pocket, the C ring
mainly interacted with S2 pocket, and the glucuronic acid group
in A ring mainly interacted with the S2 pocket. In the binding mode
of the 3c with thrombin (2R2M), as shown in the down part of
Figure 3, the B ring in 3c mainly interacted with S1 pocket, the C
ring mainly interacted with S2 pocket, and the galactopyranosyl
group in A ring mainly interacted with the S2 pocket. As displayed
in the up part of Figure 4, scutellarin (1) formed five hydrogen
bonds with the active site residues of 2R2M in the binding mode,
and the active site residues were Tyr83, Asp229, Ala230 and
scavenging activity. Furthermore, the results showed that the
DPPH radical scavenging activities did not appear to be altered
significantly by the type of glucose linkage in these three
glucose-containing scutellarein derivatives.
PC12 cells can adopt a neuronal phenotype and have been used
extensively as
a model for catecholamine-secreting neuronal
cells.16 Active mitochondria of living cells can cleave MTT to pro-
duce formazan, the amount of which it directly related to the num-
ber of living cell. So the neuroprotective effects of the synthesized
glucose-containing scutellarein derivatives 3a–3c were evaluated
by protective effects on H2O2-induced cytotoxicity in PC12 cells
using MTT assay method.17 As shown in Table 2, PC12 cells viabil-
ity markedly decreased after PC12 cell were exposed to H2O2.
Table 2
Attenuation of H2O2-induced PC12 cell damage by glucose-containing scutellarein
derivatives (mean S.D., n = 4)
Compd (
l
M)
A517
Inhibiting rate (%)
Table 3
Normal
H2O2
1/50
0.728 0.081
0.464 0.033
0.672 0.022
0.603 0.051
0.675 0.026
0.637 0.035
0.613 0.073
0.526 0.046
0.684 0.043
0.625 0.066
—
—
The anticoagulant activities of glucose-containing scutellarein derivatives
(mean S.D., n = 4)
78.79
52.65
79.92
65.53
56.44
23.48
83.33
60.98
1/25
Compd (100
lM)
Plasma coagulation parameters
3a/50
3a/25
3b/50
3b/25
3c/50
3c/25
TT (s)
PT (s)
APTT (s)
FIB (g/L)
1
21.35 1.45
18.13 1.59
20.54 1.25
29.17 1.78
6.91 0.17
7.81 0.76
6.57 0.62
9.93 0.52
33.06 1.79
43.06 1.78
38.20 1.96
39.62 1.58
37.96 1.48
32.43 1.05
26.68 1.32
20.81 1.20
3a
3b
3c

N.-G. Li et al. / Bioorg. Med. Chem. Lett. 23 (2013) 102–106
105
Figures 3. Scutellarin (1) (up) and 3d (down) docked into the pockets of thrombin
(2R2M).
Figure 4. Scutellarin (1) (up) and 3d (down) docked into residues of 2R2M,
hydrogen bonding interactions are shown as red dashed lines.
Gly258, respectively. 3c also formed five hydrogen bonds with the
active site residues Glu130, Asp229, Ala230 and Gly258 of 2R2M
(the down part of Fig. 4). These results confirmed our strategy for
designing glucose-containing scutellarein derivatives as throm-
bin-inhibitors.
The aqueous solubility of the synthesized glucose-containing
scutellarein derivatives has been determined using ultraviolet
(UV) spectrophotometer method.19,20 As presented in Table 1, the
water solubitily of the glucose-containing scutellarein derivatives
3a–3c was better compared to scutellarin (1). The best soluble
compound was compound 3c with its solubility in water was
Acknowledgments
This work was supported by National Natural Science Founda-
tion of China (No. 81001382, 81274058), the Program for New
Century Excellent Talents by the Ministry of Education (NCET-
09-0163), Research Fund for the Doctoral Program of Higher
Education of China (20093237120012), the Main Training Fund
of Nanjing University of Chinese Medicine (10XPY02), 333 High-
level Talents Training Project Funded by Jiangsu Province, Six Tal-
ents Project Funded by Jiangsu Province (2011-D-078), Program for
Outstanding Scientific and Technological Innovation Team of Jiang-
su Higher Education (2009), National Key Technology R&D Pro-
gram (2008BAI51B01), Key Research Project in Basic Science of
Jiangsu College and University (No. 07KJA36024, 10KJA360039),
Project Funded by the Priority Academic Program Development
of Jiangsu Higher Education Institutions (ysxk-2010), and Con-
struction Project for Jiangsu Engineering Center of Innovative Drug
from Blood-conditioning TCM Formulae.
8.93
pound 3a showed similar water solubility compared with scutella-
rin (1), with its value was 7.85 g/mL, and compound 3b exhibited
good water solubility with its value was 8.19 g/mL. These results
lg/mL, which was greater than that of scutellarein (2). Com-
l
l
indicated that the introduction of glucosyl group at 7-hydroxyl po-
sition could help to improve the water solubility.
In conclusion, we synthesized glucose-containing scutellarein
derivatives based on metabolic mechanism of scutellarin in vivo,
and evaluated their antioxidant, anticoagulant and neuroprotec-
tive activities as well as their water solubility. The results showed
these glucose-containing scutellarein derivatives exhibited potent
DPPH radical scavenging activities. Furthermore, the improvement
of physicochemical properties such as anticoagulant and neuropro-
tective activities alongside with the water solubility was achieved
by introducing glucose. Taken together, these findings suggest that
the introduction of the glucose moiety to scutellarein wattants fur-
ther development this kind of compounds as neuroprotective
agents.
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