PEG-scutellarin prodrugs: Synthesis, water solubility and protective effect on cerebral ischemia/reperfusion injury

Article abstract of DOI:10.1016/j.ejmech.2010.01.006

Fifteen PEG-scutellarin prodrugs were synthesized by modifying carboxyl and phenolic hydroxyl groups of scutellarin with mPEG of different molecular weight (400-3000). The water solubility of prodrugs increased remarkably and reached the maximum value of

Full text of DOI:10.1016/j.ejmech.2010.01.006

European Journal of Medicinal Chemistry 45 (2010) 1731–1738
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
PEG-scutellarin prodrugs: Synthesis, water solubility and protective effect on
cerebral ischemia/reperfusion injury
a
a
,
b
c
a
a
a,
*
*
Juan Lu , Changmei Cheng , Xinge Zhao , Qingfei Liu , Ping Yang , Yiming Wang , Guoan Luo
a
Department of Chemistry, Tsinghua University, Beijing 100084, PR China
Jiangsu Simcere Pharmaceutical Research Institute, Nanjing 210042, PR China
School of Medicine, Tsinghua University, Beijing 100084, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 October 2009
Received in revised form
Fifteen PEG-scutellarin prodrugs were synthesized by modifying carboxyl and phenolic hydroxyl groups
of scutellarin with mPEG of different molecular weight (400–3000). The water solubility of prodrugs
increased remarkably and reached the maximum value of 783.88 mg/mL (scutellarin, 0.02 mg/mL). The
anti-infarct effects of four PEG prodrugs with high water solubility were evaluated by Cerebral Ischemia/
Reperfusion in the Middle Cerebral Artery Occlusion (MCAO) model. The results showed that the prodrug
31 December 2009
Accepted 5 January 2010
Available online 14 January 2010
7
e could significantly reduce the infarct area from 27.2% to 12.2% (33.3% for the control) and decrease the
neurological deficit score from 2.77 to 1.32 (2.85 for the control). The half-life (18.62 min) of the prodrug
e was significantly longer than that of scutellarin (3.03 min).
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Scutellarin
PEG-scutellarin prodrugs
Cerebral ischemia/reperfusion injury
Middle cerebral artery occlusion model
Half-life
7
1
. Introduction
ROS and subsequently prevent the loss of GS activity, exerting
protective effect on cerebral I/R injury.
0
Scutellarin (4 ,5,6-trihydroxyflavone-7-glucuronide) (Chart 1) is
Although scutellarin has been clinically used for a long time, its
drawbacks are also very obvious. First, it has low solubility in both
water (0.056 mg/mL) and lipid (logP ¼ ꢀ2.56 in PBS at pH 4.2) [8].
Scutellarin needs to be dissolved in water for intravenous injection
during the treatment of acute cerebral infarction. In the current
study, in order to improve the water solubility, scutellarin was
dissolved in aqueous solution of disodium ethylenediamine tet-
raacetate (EDTA–2Na), which is a metal ion chelating agent and
could mediate cation (including calcium) equilibrium in vivo. On
the other hand, the low lipid solubility may account for the low
permeability of the blood–brain barrier (BBB). Secondly, probably
due to the low lipid solubility and BBB permeability, scutellarin
shows low activity against cerebral infarction in rat MCAO model.
Although scutellarin has been proved to be effective for the treat-
ment of acute cerebral infarction and paralysis in clinical trials, only
limited effects were observed in MCAO rats subjected to cerebral I/
R injury following scutellarin pretreatment (i.g.) for 7 days [9]. In
addition, if scutellarin was used after I/R injury in rat MCAO model,
very few protective effects could be observed. As such, scutellarin is
always regarded as a protective drug but not a therapeutical drug
an active ingredient extracted from traditional Chinese medicine
Erigeron breviscapus (vant) Hand–Mazz [1,2], which has been clin-
ically used to treat acute cerebral infarction and paralysis induced
by cerebrovascular diseases such as hypertension, cerebral throm-
bosis, cerebral haemorrhage in China since 1984 [3,4]. Clinical
studies have shown that the total effective rate for acute cerebral
infarction treatment is as high as 85% in 180 cases [5] and the
effective rate for paralysis treatment is even as high as 89.3% in 469
cases [6]. The protective activity of scutellarin against cerebral
Ischemia/Reperfusion (I/R) injury in the rat MCAO model has been
well established, which is mainly attributed to the scavenging effect
on reactive oxygen species (ROS). Excessive ROS production during
cerebral I/R may lead to the oxidative inactivation of glutamine
synthetase (GS), which may be a critical factor in the neurotoxicity
produced after cerebral I/R injury [7]. Scutellarin could scavenge
Abbreviations: I/R, Ischemia/Reperfusion; MCAO, Middle cerebral artery occlu-
sion; ROS, Reactive oxygen species; BBB, Blood–brain barrier; ECA, External carotid
artery; ICA, Internal carotid artery.
[9]. Third, the half-life of scutellarin is very short in vivo. Pharma-
*
Corresponding authors. Tel./fax: þ86 10 62784642. (C.C.); Tel./fax: þ86 10
cokinetic studies showed that the elimination half-life of scutellarin
in dog plasma was 1.0 min, which might be attributed to the
cleavage of the glycoside bond [10]. The short half-life might be
6
2781688 (G.L.).
E-mail addresses: chengcm@mail.tsinghua.edu.cn (C. Cheng), luoga@mail.
tsinghua.edu.cn (G. Luo).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.006

1732
J. Lu et al. / European Journal of Medicinal Chemistry 45 (2010) 1731–1738
OH
O
O
OH
O
O
m PEG O
O
HO
O
HO
O
HO
O
mPEG
O
O
HO
HO
HO
HO
HOBT,DCC
OH
OH
OH
2
2
2
2
2
a:mPEG=400
b:mPEG=600
c:mPEG=1000
d:mPEG=2000
e:mPEG=3000
OH
(2)
(1)
Scheme 1. Esterification of carboxyl group of scutellarin with mPEG.
associated with its low activity. Finally, scutellarin had a poor
bioavailability. The absolute bioavailability of scutellarin for oral
administration in Beagle dogs was only 0.4%, which indicated that
scutellarin was almost not absorbed after oral administration [11].
The absorption studies of breviscapine (containing 95% scutellarin)
in small intestine of the rat revealed that the mechanism was
mainly passive transport and the bioavailability was determined by
the concentration of scutellarin in solution passing through the
intestine [12]. In other words, the poor oral bioavailability might be
ascribed to the low water solubility of scutellarin.
To overcome the above disadvantages of scutellarin, many
approaches, including liposome-based drug delivery [13], sustained
and controlled release preparations [14,15], formation of cyclo-
dextrin inclusion compound [16] and prodrug synthesis [4,8,17],
have been reported. Among which, prodrug approach has become
a widely used method to overcome the disadvantages of scutellarin.
A prodrug is a biologically inactive derivative of a parent drug
molecule that usually requires hydrolysis or enzymatic trans-
formation within the body to release the active drug [18],
composed of parent drug and carrier. In recent years, macromole-
cule prodrug has attracted great attentions, in which macromole-
cule was used as a carrier. The favorable macromolecules include
water soluble cellulose, hydrogel, plasma protein, PEG, etc. In this
paper, PEG was chosen as a carrier to modify scutellarin because
PEG conjugate can both increase water and lipid solubility of a drug.
The improved water solubility may enhance the oral bioavailability
of scutellarin as the concentration in intestine can be increased and
improved lipid solubility may facilitate the penetration of scu-
tellarin through the BBB. In addition, PEG as a carrier may extend
the half-life of scutellarin. This can be attributed, in part, to the
increased molecular weight of the conjugate, which is beyond the
limit of molecular size for renal filtration. Reduced enzymolysis of
glycosidase caused by PEG encapsulation may be also involved [19].
Furthermore, PEG has been approved for intravenous (i.v.), oral, and
dermal applications in human by FDA [20].
Zhou et al. [4] described the synthesis of a series of ester pro-
drugs of scutellarin using PEG (200–1000) as a carrier. The water
solubility of the prodrugs was increased to 18.77–62.34 mg/mL,
which was still not achieved the practical level for i.v. and no
activity data on cerebral I/R injury has been reported.
The objective of the present study was to systematically
synthesis the PEG-scutellarin prodrug by linking the difference
molecular weight of mPEGs to difference reactive sites of scu-
tellarin and to elucidate the effect of PEGylation of scutellarin on
solubility, activity and half-life.
2. Chemistry
00
2.1. The synthesis of scutellarin-6 -PEG esters (2a–e) (Scheme 1)
In the literature [4], the esterification of scutellarin carboxyl
group with mPEG employed 4-dimethylaminopyridine (DMAP) and
DCC as the coupling system, and DMF as the solvent. The molar
ratio of scutellarin to mPEG (400–1000) was 1:100. In the begin-
ning, the target product was not obtained. Thus, many different
approaches have been tried to achieve improvements. Firstly, the
moisture is not easy to be completely removed from PEG, and the
large excess of PEG might increase the total number of water
molecules in the coupling system. Therefore, the molar ratio of
scutellarin to mPEG was reduced to 1:1.2, which also greatly
reduced the difficulty in purification. Secondly, coupling agents of
DMAP and DCC were not suitable for this reaction system. Various
other coupling systems, such as DMAP/EDCI, HOBT/DCC, and HOBT/
EDCI were tested. HOBT and DCC were finally selected as the
O
NK
O
NH
3 2
CH CH OH
O
O
O
(
3)
2 4 2
N H ·H O
N mPEG
mPEG-NH2
(6)
O
O
S
Cl
S OmPEG
O
O
mPEG
H C
O
(5)
3
3
H C
(4)
OH
O
O
H
N
HO
O
4
a,5a,6a,7a:mPEG=400
m PEG
Scutellarin
HOBT,DCC
O
4b,5b,6b,7b:mPEG=600
4c,5c,6c,7c:mPEG=1000
HO
HO
O
4
4
d,5d,6d,7d:mPEG=1300
e,5e,6e,7e:mPEG=2000
OH
OH
(7)
Scheme 2. Amidation of carboxyl group of scutellarin with mPEG.

J. Lu et al. / European Journal of Medicinal Chemistry 45 (2010) 1731–1738
1733
O
O
O
O
mPEG
O
C
2 2
CH CH COOH
mPEG
(
8)
HO
O
OH
O
O
OH
O
O
HO
O
MeO
O
HO
O
O
HO
HO
O
HO
HO
OH
OH
OH
OH
(9)
OH
O
O
HO
MeO
O
O
O
HO
HO
O
O
OH
O
mPEG 8a, 10a:mPEG=400
8
8
8
8
b, 10b:mPEG=600
c, 10c:mPEG=1000
d, 10d:mPEG=2000
e, 10e:mPEG=3000
O
(10)
Scheme 3. Esterification of phenolic hydroxyl group with mPEG-succinate.
coupling reagents due to a good yield of 2a (76.3%). Thirdly, four
kinds of solvents including DMF/CH Cl [21], pyridine, DMSO and
synthesized by HOBt/DCC coupling procedure, with the same
condition as esterification.
2
2
DMF were examined and DMF was proven to be the best solvent for
this reaction.
During the esterification and amidation processes of scutellarin,
a by-product of glucuronic acid g-lactone was formed via carboxyl
0
0
with its 4 -hydroxyl group.
2.2. The synthesis of scutellarin-PEG amide (7a–e) (Scheme 2)
0
2
.3. The synthesis of scutellarin-4 -PEG-succinate esters (10a–e)
It is well-known that hydrogen bond formed between amide NH
(Scheme 3)
2
and H O can effectively increase the water solubility of a drug. Thus
an amide group in PEG-scutellarin prodrug instead of an ester
group would be better to increase water solubility. As shown by
Scheme 2, a series of mPEG-scutellarin amide prodrugs (7a–e) were
In the early stage, ss-mPEG was chosen as activated mPEG to
react with scutellarin. However the product yield was low due to
poor stability of ss-mPEG. In addition, the unprotected carboxylic
Table 1
The 1H and 13C NMR data of scutellarin, Scutellarin-Methyl ester (9) and prodrug of 10c (
d in dimethyl sulfoxide-d ).
6
No
Carbon signals
Scutellarin
Proton signals
Scutellarin
9
10c
9
10c
2
3
4
5
6
7
8
9
1
1
2
3
4
5
6
1
2
3
4
5
6
7
164.2
102.6
182.4
146.9
130.5
151.0
93.7
149.1
105.9
121.4
128.5
116.1
161.3
116.1
128.5
100.0
72.8
164.1
102.6
182.3
146.9
130.5
151.0
93.5
149.0
105.9
121.3
128.4
116.0
161.2
116.0
128.4
99.9
162.8
104.8
182.5
146.8
130.7
151.2
93.5
149.2
104.8
122.4
127.9
122.5
153.2
122.5
127.9
99.9
6.82(H, s)
6.80(H, s)
7.01(H, s)
6.99(H, s)
7.00(H, s)
7.05(H, s)
0
0
0
0
0
0
0
7.94(H,d, J ¼ 8.6 Hz)
6.95(H, d, J ¼ 8.9 Hz)
7.94(H, d, J ¼ 8.9 Hz)
6.96(H, d, J ¼ 8.6 Hz)
8.15(H, d, J ¼ 8.6 Hz)
7.37(H, d, J ¼ 8.6 Hz)
6.95(H, d, J ¼ 8.9 Hz)
7.94(H, d, J ¼ 8.6 Hz)
5.23
6.96(H, d, J ¼ 8.6 Hz)
7.94(H, d, J ¼ 8.9 Hz)
5.28
7.37(H, d, J ¼ 8.6 Hz)
8.15(H, d, J ¼ 8.6 Hz)
5.29
0
0
0
0
0
0
0
0
0
0
0
0
0
0
72.8
75.3
71.4
75.1
169.2
52.0
72.8
75.3
71.3
75.0
169.2
52.0
75.6
71.4
75.3
170.1
4.06(H, d, J ¼ 9.3 Hz)
4.22(H, d, J ¼ 9.3 Hz)
4.22(H, d, J ¼ 9.3 Hz)
3.67(H, s)
3.67(H, s)

1734
J. Lu et al. / European Journal of Medicinal Chemistry 45 (2010) 1731–1738
acid made the reaction complicated. Therefore, in the current study,
mPEG-succinate was coupled with methyl ester of scutellarin in the
presence of HOBt and DCC to produce prodrug 10. Cao et al. [8]
reported the synthesis of ethyl ester of scutellarin by the reaction of
ethyl bromide with scutellarin sodium salt, but the yield was only
PEG chain compared with the A ring of flavones, Such orientation of
flexible chain would greatly disturb the intermolecular stack.
3. Pharmacology
19.8%. We quantitatively prepared the methyl ester of scutellarin by
3.1. Protective effects against cerebral I/R injury
the reaction of scutellarin with excess methanol in DMF under the
catalysis of hydrogen chloride.
The protective effects of those prodrugs on cerebral I/R injury
There are six hydroxyl groups in scutellarin molecule including
three phenolic and three sugar hydroxyl groups. The reaction
selectivity of the hydroxyl group is a critical problem. Fortunately,
were investigated using a rat MCAO model. In the model, nimodi-
pine was selected as a positive control drug, and the protective
effect of nimodipine against cerebral I/R injury in the rat MCAO
model has been well established [23,24]. And then, four prodrugs
including of 10b, 10d, 10e and 7e were selected for activity inves-
tigation according to the results of water solubility and preliminary
test on protective effects against cerebral I/R injury of the prodrugs.
13
C NMR and 1H NMR revealed that esterification with mPEG-
0
13
succinate only occurred at 4 -hydroxyl group. Table 1 shows C and
1
1
H NMR chemical shift values and H NMR coupling constants of
the parent drug Scutellarin [22], methyl ester of Scutellarin (9) and
prodrug 10c (10a, 10b, 10d and 10e are almost the same as 10c).
Comparison of the chemical shift values of the aromatic protons of
prodrug 10c with the 1H NMR spectrum of 9 revealed that the
3.1.1. Effects of 10b, 10d, 10e and 7e on the infarct area compared
with scutellarin
signal C
–H and C –H also had a downfield shift of 0.21 ppm and 0.11 ppm,
3 , 5 2 ,
–H showed an obvious downfield shift of 0.41 ppm, C
0 0 0
The evaluation of the infarct area is shown in Fig. 1a. The infarct
area was slightly reduced in the groups of I/Rþ10b, I/Rþ10d, I/
Rþ10e, I/R þ Scu and I/R þ Nim, but there was no significant
difference (p > 0.05, vs vehicle). The prodrug of 7e could signifi-
cantly reduce the infarct area (p < 0.05, vs vehicle), indicating that
0
6
3
respectively, while the other protons remained unchanged. Very
importantly, 13C NMR spectrum of prodrug 10c indicated that C-4
0
had an 8.0 ppm downfield shift compared with the methyl ester of
0
scutellarin. The less steric hindrance of 4 -hydroxyl group was also
an important factor of this selectivity.
2.4. Solubility and partition coefficient
Aqueous solubility of the prodrugs and the parent drug was
determined using double-distilled water after 24 h equilibration.
Determination of partition coefficient was carried out in n-octanol/
ꢁ
phosphate buffer (pH 7.4) at 37 C. The values of solubility and
partition coefficient are shown in Table 2. Both solubility and lip-
ophilicity were significantly enhanced by introducing PEG into
scutellarin. The solubility of three series of prodrugs, 2a–e, 7a–e
and 10a–e, was 1.09–15.32 mg/mL, 276.14–756.02 mg/mL, and
2
00.34–783.88 mg/mL, respectively. Prodrugs 7a–e and 10a–e
showed much better solubility than 2a–e. The solubility of pro-
drugs increased remarkably with increased molecular weight of
mPEG. Generally, there are two main forces to reduce the solubility.
One is the planar stack force and the other is the intermolecular
hydrogen bonds. For scutellarin, as a flavonoide compound, the
stack force might be the major factor for molecular aggregation. The
remarkable increase in solubility of 10a–e can be explained by
decreased stack force, since the direct attachment of PEG to
aromatic ring would interfere the stack between the interplanar
flavonoides by the flexible long chain. However, the mechanism for
the dramatic increase in the solubility of amide 7a–e compared
with ester 2a–e is not clear. The probable reason is that the NH
group in amide may form hydrogen bond with 6-OH through
a deca-membered ring, leading to the vertical orientation of the
Table 2
The water solubility and partition coefficient of PEG–scutellarin prodrugs.
Compounds
Solubilitya
logPa
Compounds
Solubility
(mg/ml)
logP
0.04
ꢀ0.08
ꢀ0.31
ꢀ0.54
ꢀ0.21
ꢀ1.58
ꢀ1.19
ꢀ0.55
(
mg/ml)
Scutellarin
0.02
1.09
5.21
6.86
9.35
15.32
ꢀ2.37
ꢀ0.13
0.64
7c
7d
7e
10a
10b
10c
10d
10e
554.52
557.45
756.08
200.34
220.42
268.57
554.71
783.88
2
2
2
2
2
7
7
a
b
c
d
e
a
b
0.13
ꢀ0.11
0.06
276.14
321.70
ꢀ0.05
0.16
Fig. 1. The effects of 10b, 10d, 10e and 7e on (a) ratio of infarct area and (b) neuro-
logical score (Data were expressed as means ꢂ SD. *p < 0.05, compared with sham;
a
#
Values are the mean of three determinations.
p < 0.05, compared with vehicle; &p < 0.05, compared with I/R þ Scu.).

J. Lu et al. / European Journal of Medicinal Chemistry 45 (2010) 1731–1738
1735
Table 3
Pharmacokinetic parameters of scutellarin and 7e after i.v. administration to rats
OH
O
6
``
O
5
HO
HO
(
n ¼ 6, Mean ꢂ SD).
3
5
``
O
Parameter
Scutellarin
7e
Scutellarin
HO
HO
2`
1
``
released from 7e
O
O
OH
8
1
3`
`
AUC 0–240
(mmol/L*min)
1162.34 ꢂ 99.98
667.32 ꢂ 58.46
402.59 ꢂ 37.26
6
`
4
5`
OH
MRT 0–240 (min)
t1/2 (min)
29.67 ꢂ 3.18
3.03 ꢂ 0.29
82.98 ꢂ 10.04
18.62 ꢂ 3.65
21.40 ꢂ 2.98
2.70 ꢂ 0.25
Chart 1. The structure of scutellarin.
7
e had significant protective effect on ischemic injury compared
evaluated and the pharmacokinetic parameters were calculated. As
shown in Table 3, the pharmacokinetic parameters between scu-
tellarin and 7e are significantly different such as AUC0-240, MRT0-
with scutellarin.
3.1.2. Effects of 10b, 10d, 10e and 7e on the neurological score
240 and half-life (p < 0.05), implying that 7e is eliminated much
compared with scutellarin
more slowly than scutellarin. The slow elimination promotes better
access of 7e to remote targets [26]. But the molar concentrations of
both 7e and scutellarin released were much lower than that of the
parent drug scutellarin as shown in Fig. 2. The phenomenon can be
explained as that 7e was phargocytosised easily by the macrophage
of various organs such as liver and spleen because of its macro size,
which may lead to the accumulation in these positions. Besides, the
prodrugs can penetrate the BBB much more easily compared with
the parent drug scutellarin because the PEGylation could effectively
decrease the polarity and improve the solubility of scutellarin due
to the flexible segment of mPEG and its amphiphilic properties, and
so 7e may have the tendency of brain targeting. Both factors
mentioned above may lead to the decrease of 7e concentration in
the blood. The validity of the explanation can be verified by our
future research on the tissue distribution of 7e.
As shown in Fig. 1b, 10b and 10e and 7e could greatly ameliorate
the neurological deficits compared with scutellarin (10b and 10e:
p < 0.05, compared with the vehicle- and sham-operated groups;
7
e: p < 0.05, compared with sham-operated group, vehicle-oper-
ated group and scutellarin-treated I/R group).
Studies have demonstrated that the prodrugs of 7e, 10b, 10d and
10e could reduce infarct area and ameliorate the neurological
deficit after cerebral I/R at different degrees, among which 7e
exhibited obvious effect. Therefore, the data indicated that the
prodrug of 7e has protective effect on ischemic injury induced by
cerebral I/R in rats compared with the parent drug scutellarin.
According to the design of prodrugs reported by Jarkko Rautio
[25], the protective effect obtained may be attributed to the
following reasons. Because of the strong polar and non liposoluble
property, scutellarin can hardly penetrate the BBB. The modifica-
tion of scutellarin with mPEG could effectively decrease the polarity
and improve the solubility due to the flexible segment of mPEG and
its amphiphilic properties. As a result, the prodrugs can penetrate
the BBB much more easily than the parent drug of scutellarin. After
that the prodrugs were enzymatically hydrolyzed and/or chemi-
cally transformed to release scutellarin and exert the desired
pharmacological effect.
4. Conclusion
In this paper, fifteen PEG-scutellarin conjugates were designed
and synthesized. The water solubility of the prodrugs was increased
remarkably. The anti-infarct effects of four PEG prodrugs with high
water solubility were evaluated by Cerebral I/R in the MCAO model.
The results showed that the prodrug 7e could significantly reduce
the infarct area from 27.2% to 12.2% (33.3% for the control) and
decrease the neurological deficit score from 2.77 to 1.32 (2.85 for
the control). The half-life (18.62 min) of the prodrug 7e was
significantly longer than that of scutellarin (3.03 min). Therefore,
prodrug 7e increased the water solubility, improved the protective
effect on cerebral I/R injury and prolonged half-life compared to the
parent drug scutellarin.
3.2. Pharmacokinetic study of scutellarin and its prodrug 7e
After injecting scutellarin or 7e, the concentrations of scu-
tellarin, prodrug 7e and scutellarin released from 7e in blood were
5
5
5
. Experimental protocols
.1. Chemistry
.1.1. Reagents and chemicals
Scutellarin were purchased from Fengshanjian medicine
research Ltd. (Kunming, China) and further purified. The purity of
scutellarin obtained was above 98%. Methoxy PEG (mPEG) was
dried in toluene by azeotropic distillation followed by drying at
ꢁ
1
30 C in vacuum. The silica gel was obtained from Qingdao
Haiyang Chemical&Special Silica Gel CO, Ltd. Merck LC grade
acetonitrile, analytical grade formic acid were used as received.
Deionized water was prepared with a Milli-Q system (Millipore,
France).
5
.1.2. General methods
1H NMR and 13C NMR spectra were recorded using JOEL JNM-
ECA300 NMR instrument in the given solvent. In all the spectra TMS
was used as internal reference. Mass spectra were carried out on
Fig. 2. Concentration profiles of scutellarin and 7e in plasma after i.v. of scutellarin and
7e respectively.

1736
J. Lu et al. / European Journal of Medicinal Chemistry 45 (2010) 1731–1738
ion trap MS (Agilent) for low molecular weight prodrugs
MW < 2000) and 4800 plus MALDI-TOF/TOF (ABI) for high
mixture was stirred overnight and pH was adjusted to 3–4 with 3 M
hydrochloride solution. Then the reaction mixture was extracted
with methylene dichloride (three times, 50 mL each time) and the
solvent was removed under vacuum. The crude product was
precipitated from this solution after 300 mL ether was added
dropwise with rapid stirring. The precipitate was filtered and
washed several times with ether. The product was crystallized from
ethanol, filtered, washed, and dried under vacuum. The yields of 4a,
4b, 4c, 4d and 4e were 97.6%, 88.5%, 80.8%, 78.9% and 70.2%,
respectively [27].
(
molecular weight prodrugs (MW ꢃ 2000).
The post-processing method of product: the reaction mixture
was filtered. The mixture of acetic acid and tetrahydrofuran (mass
ratio ¼ 1:9) were added into the filtrate with stirring for 2 h and
then ether was added into the mixture. The precipitate was filtered
and purified by silica gel column chromatography (CH
CH
OH ¼ 10:1). Finally, the product was crystallized three times
from ethanol and dried under vacuum.
2 2
Cl /
3
All final products had a purity of ꢃ98%. The purity of final
products was determined by HPLC (Aglient 1200)-UV-ELSD (Alltech
5.1.4.3. Synthesis of amino PEG (6a–e). 3 (0.205 mol) and 4
5
(
00).
4.6 mm ꢄ 250 mm, 5
with 0.1% TFA, 0–5 min, 80% B; 5–15 min, 80%/60% B; 15–25 min,
Method:
Phenomenex
Gemini
C18
column
(0.020 mol) were stirred in 100 mL DMF in a nitrogen atmo-
sphere at 120 C for 4 h. The precipitate was filtered off and
dissolved in 200 mL ethanol, and then 10 mL hydrazine hydrate
was added. The mixture was heated under reflux for 4 h. After
cooling down to room temperature, the product was precipitated
by adding dropwise ether to the solution. The precipitate was
ꢁ
mm); mobile phase: A ¼ acetonitrile, B ¼ H
2
O
6
0% B; 26–30 min, 80% B; flow rate ¼ 0.6 mL/min;
l
¼ 334 nm; run
ꢁ
time ¼ 30 min; ELSD: drift tube temperature: 110 C; gas flow:
3
.0 L/min.
Reversed-phase HPLC was performed with L-2130 pump and
2 2
filtered and re-dissolved in 30 mL CH Cl , and the insoluble
L-2400 UV detector system (HITACHI, Japan). A binary isocratic
mobile phase composed of a mixture of 0.3% of formic acid in water
and acetonitrile was used on a Phenomenex RP-18C column
impurities were removed by filtration. The filtrate was concen-
trated and ether was slowly added. The precipitate was filtered,
washed, and dried in vacuum to give 6. The yields of the products
6a, 6b, 6c, 6d, and 6e were 93.5%, 86.7%, 81.6%, 77.4% and 69.3%,
respectively [27].
(
250 ꢄ 4.60 mm, 5
mm). The detection wavelength was 334 nm and
the flow rate was 0.8 mL/min. This HPLC method was used to
measure the plasma concentration of drugs in Pharmacokinetic
study.
5.1.4.4. Synthesis of scutellarin-PEG amide (7a–e). Scutellarin
(
0.01 mol) was dissolved in 60 mL of anhydrous DMF, and to this
0
0
5
.1.3. Synthesis of scutellarin-6 -PEG esters (2a–e)
solution were added 6 (0.012 mol), HOBT (0.015 mmol) and DCC
(0.015 mol). The resulting solution was stirred at 40 C for an
ꢁ
Scutellarin (0.010 mol) was dissolved in 60 mL of anhydrous
DMF, and to this solution were added mPEG (0.012 mol), HOBT
appropriate time under nitrogen. The progress of the reaction was
monitored by TLC. The post-processing method was mentioned as
‘‘2.2. General methods’’. The yields of 7a, 7b, 7c, 7d and 7e were
80.3%, 75.4%, 67.5%, 58.0% and 39.8%, respectively.
(
0.015 mol) and DCC (0.015 mol). The resulting solution was stirred
ꢁ
at 40 C for an appropriate time under nitrogen. The progress of the
reaction was monitored by TLC. The post-processing method was
mentioned as ‘‘2.2. General methods’’. The yields of 2a, 2b, 2c, 2d,
13
1
The C and H NMR of 7c (7a, 7b, 7d and 7e are almost the same
1
2
e were 76.3%, 65.4%, 58.7%, 53.2% and 47.4%, respectively.
as 7c) were as follows: H NMR (DMSO-d
(s, 1H, C –OH), 8.58 (s, 1H, C –OH), 7.96, 7.94 (d, 2H, C
(s, H, C –H), 6.95–6.92 (d, 2H, C –H), 6.83 (s, 1H, C –H), 5.08–5.06
(s,1H, C ), 3.51–
00–H), 3.98–3.95 (d, 1H, C 00–H), 3.73 (t, 2H, –COOCH
3.17 (–OCH CH ), 3.24 (s, 3H, –OCH ). C NMR (75 MHz, DMSO-d
58.09, 68.08, 69.63–69.82, 71.33, 72.79, 75.62, 75.30, 93.88,
6
, 300MHz):
d
H
10.37
0 0
–H), 6.98
2 , 6
13
1
The C and H NMR chemical shift values of 2a (2b, 2c, 2d and
4
0
5
1
2
e are almost the same as 2a) were as follows: H NMR (DMSO-d
6
,
3
3
0
,5
0
8
3
00 MHz):
d
H
10.36 (s, 1H, C
4
0
–OH), 8.60 (s, 1H, C
–H), 6.96–6.93 (d, 2H, C
00–H), 4.22–4.19 (d,1H, C
), 3.51–3.24 (–OCH CH ), 3.17 (s, 3H, –OCH
): 53.82, 68.55, 69.27–70.02, 71.21, 73.18,
6.20, 76.42, 93.92, 100.78, 101.57, 102.48, 116.03,116.03, 121.45,
28.69, 130.76, 146.92, 149.09, 151.05, 161.18, 164.15, 169.21, 182.42.
a: 287.2, 749.4, 793.4, 837.4, 881.4, 925.4, 969.4,1013.4,1057.3; The
mass spectrum data: 2b: 961.7,1005.7,1049.6,1093.5,1137.5,1181.4,
225.4, 1269.3; 2c: 1399.7, 1443.7, 1487.7, 1531.7, 1575.8, 1619.8,
663.8, 1707.8, 1751.8; 2d: 2280.0, 2324.0, 2368.0, 2412.1, 2456.0,
5
–OH), 7.95, 7.92
–H), 6.82
00–H), 3.68
1
5
2
13
(d, 2H, C
(s, 1H, C
(s, 2H, –COOCH
2
0
, 6
0
–H), 7.00 (s, H, C
3
3
0
,5
0
2
2
3
6
)
8
–H), 5.30–5.26 (s, 1H, C
1
5
d
13
2
2
2
3
).
C
100.65, 102.55, 105.99, 116.06, 116.06, 121.31, 128.52, 128.52, 130.58,
146.85, 149.06, 151.07, 161.32, 164.16, 168.14, 182.42. The mass
spectrum data: 7a: 768.6, 812.7, 856.6, 900.3, 944.2, 988.1, 1031.9,
1075.8,1119.8; 7b: 826.3, 870.4, 914.4, 958.4, 1002.4, 1046.5, 1090.5,
1134.5; 7c: 1252.5, 1296.5, 1340.5, 1384.5, 1428.6, 1472.6, 1516.7,
1560.7; 7d: 1618.8, 1662.8, 1706.8, 1750.9, 1794.9, 1838.9, 1882.9,
1926.9, 19709.9; 7e: 2391.3, 2435.4, 2479.4, 2523.4, 2567.5, 2611.5,
2655.5, 2699.5, 2743.6. The highest peak of the normal distribu-
tions of 7a, 7b, 7c, 7d and 7e were 944.2, 1002.4, 1428.6, 1794.9 and
2567.5, respectively.
NMR (75 MHz, DMSO-d
7
1
2
6
d
1
1
2
3
500.0, 2544.0; 2e: 3100.0, 3144.2, 3188.2, 3232.4, 3276.5, 3320.7,
364.6, 3408.5, 3452.7. The highest peak of the normal distribu-
tions of 2a, 2b, 2c, 2d and 2e were 881.4, 1093.5, 1575.8, 2412.1 and
0
3
276.5, respectively.
5.1.5. Synthesis of scutellarin-4 -PEG-succinate esters (10a–e)
5.1.5.1. Synthesis of mPEG-succinate (8a–e). mPEG (100 mmol) was
5
5
.1.4. Synthesis of scutellarin-PEG amide (7a–e)
.1.4.1. Synthesis of potassium phthalimide (3). To a stirred solution
dissolved in 250 mL chloroform and to this resulting solution was
added succinic anhydride (125 mmol) and 10 mL pyridine. Then the
reaction mixture was refluxed for 48 h with stirring. After evapo-
ration of the reaction mixture to dryness, the residue was dissolved
in saturated sodium bicarbonate solution, filtered and then
extracted with ethyl acetate (twice, 200 mL each). Aqueous phase
of phthalimide (100 g, 0.68 mol) in 600 mL ethanol, was added
dropwise 1200 mL of a potassium hydroxide solution in methanol
(
80 g/L), and the stirring was continued for another 4 h at room
temperature. The reaction mixture was filtered. The filter cake was
washed three times with ethanol, and then evaporated under
reduced pressure to yield a white solid (122.02 g, 97.0%).
ꢁ
was cooled to 0 C and the pH was adjusted to 2.0 with 2 M
hydrochloric acid, and then extracted with methylene chloride
(
3 ꢄ 200 mL). The methylene chloride solution was dried over
5
.1.4.2. Synthesis of PEG-tosylate (4a–e). 0.01 mol mPEG was dis-
solved in methylene dichloride, followed by the addition of
.40 mol p-Toluenesulfonyl chloride and 5 mL pyridine. The
magnesium sulfate and then evaporated to dryness. The yields of
8a, 8b, 8c, 8d and 8e were 98.5%, 90.8%, 86.0%, 80.4% and 75.9%,
respectively.
0

J. Lu et al. / European Journal of Medicinal Chemistry 45 (2010) 1731–1738
1737
5
.1.5.2. Synthesis of scutellarin-methyl ester (9). Hydrochloride
synthesized in our lab. Rats were divided into 8 groups. The vehicle-
treated I/R groups and the sham-operated groups were treated with
5% i.v. glucose solution with the same volume as other groups. The
nimodipine-treated I/R groups as the positive control were i.v.
treated with 1.2 mg/kg nimodipine, which was divided into three
times. The scutellarin-treated I/R groups were treated with 60 mg/
kg scutellarin by i.v., which was divided into three times. The
administration dosage of groups I/R þ 7e, I/R þ 10b, I/R þ 10d and I/
R þ 10e were 318.3, 168.3, 342.5 and 462.9 mg/kg, respectively.
solution of methanol was prepared by adding dropwise 10 mL
sulfinyl chloride to 50 mL of anhydrous methanol at a temperature
ꢁ
of ꢀ10 C. The mixture was stirred for 10 min at the same
temperature to produce hydrochloride solution of methanol.
ꢁ
Hydrochloride solution of methanol prepared under 0 C was
added dropwise slowly to
a stirred solution of Scutellarin
(
0.010 mol) in 60 mL of anhydrous DMF. The reaction mixture was
stirred for another 15 min, and then the ice-bath was removed and
the mixture was heated to 40 C. The progress of the reaction was
ꢁ
monitored by HPLC. Then the pH was adjusted to 7 with saturated
sodium bicarbonate solution when the progress of the reaction
achieved 99%. After the addition of some water to the mixture
solution, the precipitate was filtered, washed several times with
water and dried under vacuum to yield the product (4.67 g, 98.1%).
5.2.1.2. Cerebral I/R procedure. Brain I/R injury was induced by
a middle cerebral artery occlusion (MCAO). Rats were anesthetized
with 10% chloral hydrate (3.5 mL/kg, i.v.). Briefly, the right common
carotid artery, external carotid artery (ECA) and internal carotid
artery (ICA) were isolated via a jugular midline incision after skin
disinfection. The vagus was stripped carefully and ECA was ligated
and snipped. Then pterygopalatine artery was ligated forward along
0
5
.1.5.3. Synthesis of scutellarin-4 -PEG-succinate esters (10a–
e). DCC (0.015 mol) and HOBT (0.015 mol) were added to a solution
of 9 (0.010 mol) and 8 (0.012 mol) in 60 mL DMF. The mixture was
stirred at 40 C for 12 h and filtered. The post-processing method
with the ICA. Monofilament nylon suture (40.285 mm), with its tip
rounded by heating near a flame, was introduced into the ECA lumen
and advanced into the ICA for a distance of about 20 mm in order to
block the origin of the MCA. After 1.5 h of ischemia, the nylon suture
was withdrawn to establish reperfusion. Then the scalp was sutured
and disinfected. After arousal from anesthesia, the rats were returned
to the cages. Sham-operated animals were not exposed to I/R. The
ꢁ
was mentioned as ‘‘2.2. General methods’’. The yields of 10a, 10b,
10c, 10d and 10e were 68.0%, 60.2%, 54.7%, 47.3% and 35.2%,
respectively.
13
1
The C and H NMR of 10a (10b, 10c, 10d and 10e are almost the
1
ꢁ
same as 10a) were as follows: H NMR (DMSO-d
.15–8.12 (d, 2H, C –H), 7.37–7.34 (2H, C –H), 7.05 (s, 1H, C
H), 7.01 (s, 1H, C –H), 5.30–5.26 (s, 1H, C
), 3.61 (s, 3H, –COOCH ), 3.49–3.34 (–OCH
.23 (s, 3H, –OCH ), 3.91–2.87 (t, 2H, –OCOCH ), 2.74–2.70 (t, 2H,
). C NMR (75 MHz, DMSO-d 28.53, 28.88, 51.97, 58.03,
3.63, 68.23, 70.55–69.78, 71.28, 72.75, 75.02, 75.28, 93.5, 99.91,
04.79, 106.1, 122.38, 122.49, 122.49, 127.92, 127.92, 130.66, 146.84,
49.15, 151.24, 153.17, 162.81, 169.15, 170.59, 171.68, 182.48. The
mass spectrum data: 10a: 834.1, 878.1, 921.9, 965.9, 1009.7, 1053.4,
097.4, 1121.4, 1165.4; 10b: 1119.4, 1163.4, 1207.4, 1251.4, 1295.4,
339.4, 1383.4, 1427.4, 1471.4; 10c: 1339.4, 1383.4, 1427.4, 1471.4,
515.5, 1559.5, 1603.5, 1647.5, 1691.5; 10d: 2318.3, 2362.3, 2406.4,
6
, 300MHz):
d
H
body temperature of the rats was maintained at 37.0 ꢂ 0.5 C during
8
2
0
, 6
0
3
0
,5
0
3
–
the surgical procedure with heating plate and a desk lamp of 60 W.
00–H), 4.19–4.16 (m, 3H,
8
1
C
3
–
6
5
00–H, –COOCH
2
3
2
CH ),
2
5.2.1.3. Behavioral tests and measurement of infarct area.-
Neurological deficit of each rat after 48 h reperfusion was
expressed as a total score obtained using improved Bederson 5
point’s system (Table 4) [31] by a single experimenter, who was
blinded to the experimental design.
3
2
1
3
OCOCH
2
6
) d
1
1
Then the rats were anesthetized with 10% chloral hydrate
(3.5 mL/kg, i.v.) and subsequently decapitated. The brains without
olfactory bulb cerebellar and lower brainstem were collected. The
brains obtained were washed with normal saline, and wiped with
tissue paper to remove the adhering liquid. The brain samples were
1
1
1
ꢁ
2
3
450.4, 2494.4, 2538.5, 2582.5, 2626.5, 2670.6; 10e: 3211.8, 3255.8,
299.8, 3343.8, 3387.9, 3431.9, 3475.9, 3519.9, 3564.0. The highest
kept at ꢀ80 C for 7 min. Then a series of thin sections were
selected from the thick slices at 2 mm intervals to determine the
infarct areas. The slices were immersed in 20 g/L triphenylte-
trazolium chloride, which was prepared freshly with PBS (0.2 mol/L
peak of the normal distributions of 10a, 10b, 10c, 10d and 10e were
009.7, 1295.4, 1515.5, 2494.4 and 3387.9, respectively.
1
ꢁ
pH 7.4–7.8), and incubated at 37 C for 90 min in a water bath.
5
.1.6. Determination of solubility and apparent partition coefficient
Solubility determinations were carried out in deionized water as
During this period, the infarct tissue remained unstained, whereas
the normal part was stained red. The slices were then washed with
normal saline and queued in a row quickly. Photos were taken for
each slice for later investigation. The infarct area and the total area
of the right side were summed up by a computerized image anal-
ysis system. The ratio of the infarct area was calculated as follows:
the method described by Dias et al. [28]. Parallel experiments were
performed to determine the solubility of scutellarin in the same
media. Partitioning studies were performed with n-octanol and
phosphate buffer (pH 7.4) following standard procedures [28].
Parallel experiments were performed to determine the apparent
partition coefficient of scutellarin.
The infarct areas % ¼ 100 ꢄ ðThe infarct areaÞ=ðThe total areaÞ
5.2. Pharmacology
Table 4
Neurologic examination grading system.
Sprague–Dawley rats (Grade SPF) were obtained from the
Neurological Neurological deficit
score
Experimental Animal Center of the Chinese Academy of Medical
Sciences weighing 250–300 g and housed at a constant tempera-
0
Both forelimbs were extended toward the floor and that had no
other neurological deficit
ꢁ
ture of 22–25 C under a 12 h light–dark cycle with free access to
food and drinking water. The protocol was duly approved by the
Institutional Animal Care and Use Committee of Tsinghua Univer-
sity (Beijing, China)
1
Rats with infarction consistently flexed the forelimb contra-
lateral to the injured hemisphere; posture varied from mild
wrist flexion and shoulder adduction with extension at the
elbow to severe posturing with full flexion of wrist, elbow, and
adduction with internal rotation of the shoulder
Rats had consistently reduced resistance to lateral push toward
the paretic side
5
5
.2.1. Protective effect against cerebral I/R injury [9,29,30]
.2.1.1. Drugs and animal treatment. Scutellarin was dissolved in
2
3
4
Rats circled toward the paretic side consistently
Limbs of flaccid paralysis and no spontaneous activity
glucose solution before use. Nimodipine was ready for i.v. with
specification of 10 mg/50 mL. The prodrugs of scutellarin were

1
738
J. Lu et al. / European Journal of Medicinal Chemistry 45 (2010) 1731–1738
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5
.3. Statistical analysis
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.3.1. Pharmacokinetic study of scutellarin and its prodrug 7e
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[
fasted for 12 h prior to the experiment of intravenous administra-
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3
[
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plasma samples were collected. The plasma concentrations of
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[
(
Acknowledgment
1
[
The authors would like to thank the efficient analysis provided
by Dongya Zhu, Haiyin Wu, etc., Pharmacy College of China Phar-
maceutical University. We also greatly appreciate the financial
support from Jiangsu Simcere Pharmaceutical Research Institute.
3
[
[
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