Synthesis and biological evaluation of methylated scutellarein analogs based on metabolic mechanism of scutellarin in vivo

Article abstract of DOI:10.1016/j.ejmech.2015.10.039

Scutellarin (1) could be hydrolyzed into scutellarein (2) in vivo and then converted into methylated, sulfated and glucuronidated forms. In order to investigate the biological activities of these methylated metabolites, eight methylated analogs of scutellarein (2) were synthesized via semi-synthetic methods. The antithrombotic activities of these compounds were evaluated through the analyzation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB). Their antioxidant activities were assessed by measuring their scavenging capacities toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and the ability to protect PC12 cells against H₂O₂-induced cytotoxicity. Furthermore, the physicochemical properties of these compounds including aqueous solubility and lipophilicity were also investigated. The results showed that 6-O-methylscutellarein (5) demonstrated potent antithrombotic activity, stronger antioxidant activity and balanced solubility and permeability compared with scutellarin (1), which warrants further development of 5 as a promising lead for the treatment of ischemic cerebrovascular disease.

Full text of DOI:10.1016/j.ejmech.2015.10.039

Accepted Manuscript
Synthesis and biological evaluation of methylated scutellarein analogs based on
metabolic mechanism of scutellarin in vivo
Zhi-Hao Shi, Nian-Guang Li, Zhen-Jiang Wang, Yu-Ping Tang, Ze-Xi Dong, Wei
Zhang, Peng-Xuan Zhang, Ting Gu, Wen-Yu Wu, Jian-Ping Yang, Jin-Ao Duan
PII:
S0223-5234(15)30321-4
10.1016/j.ejmech.2015.10.039
EJMECH 8174
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 July 2015
Revised Date: 10 October 2015
Accepted Date: 22 October 2015
Please cite this article as: Z.-H. Shi, N.-G. Li, Z.-J. Wang, Y.-P. Tang, Z.-X. Dong, W. Zhang, P.-X.
Zhang, T. Gu, W.-Y. Wu, J.-P. Yang, J.-A. Duan, Synthesis and biological evaluation of methylated
scutellarein analogs based on metabolic mechanism of scutellarin in vivo, European Journal of Medicinal
Chemistry (2015), doi: 10.1016/j.ejmech.2015.10.039.
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of methylated scutellarein analogs based on
metabolic mechanism of scutellarin in vivo
Zhi-Hao Shia,b, Nian-Guang Li*a, Zhen-Jiang Wanga, Yu-Ping Tang*a, Ze-Xi Donga, Wei Zhanga,
Peng-Xuan Zhanga, Ting Gua, Wen-Yu Wua, Jian-Ping Yanga and Jin-Ao Duan*a
aJiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,
Jiangsu Key Laboratory for High Technology Research of TCM Formulae, National and Local
Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and
Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu
210023, P.R. China
bDepartment of Organic Chemistry, China Pharmaceutical University, Nanjing, Jiangsu 211198,
P.R. China
_________________
* Corresponding author. Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources
Industrialization, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, P.R. China.
** Corresponding authors. Tel./fax: +86 25 85811916
E-mail addresses: linianguang@njutcm.edu.cn (N.-G. Li), yupingtang@njutcm.edu.cn (Y.-P. Tang),
dja@njutcm.edu.cn (J.-A. Duan).
ABSTRACT: 6-O-methyl-scutellarein (5) demonstrated potent antithrombotic
activity, stronger antioxidant activity and balanced solubility and permeability
compared with scutellarin (1), which warrants further development for the treatment
of ischemic cerebrovascular disease.
OH
OH
OH
OH
O
HO
O
O
O
In vivo
metabolite
Methylation and
biological evaluation
HO
HO
O
O
O
HO
HO
O
O
H3CO
OH
OH
HO
6-O-methyl-scutellarein (5)
OH
OH
scutellarin (1)
scutellarein (2)
potent antithrombotic activity
stronger antioxidant activity
balanced solubility and permeability

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of methylated scutellarein analogs based on
metabolic mechanism of scutellarin in vivo
Zhi-Hao Shia,b, Nian-Guang Li*a, Zhen-Jiang Wanga, Yu-Ping Tang*a, Ze-Xi Donga, Wei Zhanga,
Peng-Xuan Zhanga, Ting Gua, Wen-Yu Wua, Jian-Ping Yanga and Jin-Ao Duan*a
aJiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,
Jiangsu Key Laboratory for High Technology Research of TCM Formulae, National and Local
Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and
Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu
210023, P.R. China
bDepartment of Organic Chemistry, China Pharmaceutical University, Nanjing, Jiangsu 211198,
P.R. China
_________________
* Corresponding author. Jiangsu Collaborative Innovation Center of Chinese Medicinal
Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023,
P.R. China.
** Corresponding authors. Tel./fax: +86 25 85811916
E-mail addresses: linianguang@njutcm.edu.cn (N.-G. Li), yupingtang@njutcm.edu.cn (Y.-P.
Tang), dja@njutcm.edu.cn (J.-A. Duan).
ABSTRACT: Scutellarin (1) could be hydrolyzed into scutellarein (2) in vivo and then
converted into methylated, sulfated and glucuronidated forms. In order to investigate
the biological activities of these methylated metabolites, eight methylated analogs of
scutellarein (2) were synthesized via semi-synthetic methods. The antithrombotic
activities of these compounds were evaluated through the analyzation of prothrombin
time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and
fibrinogen (FIB). Their antioxidant activities were assessed by measuring their
scavenging capacities toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and the
ability to protect PC12 cells against H2O2-induced cytotoxicity. Furthermore, the
physicochemical properties of these compounds including aqueous solubility and
lipophilicity were also investigated. The results showed that 6-O-methylscutellarein
(5) demonstrated potent antithrombotic activity, stronger antioxidant activity and
balanced solubility and permeability compared with scutellarin (1), which warrants
further development of 5 as a promising lead for the treatment of ischemic
cerebrovascular disease.
Keywords: Scutellarin, Scutellarein, Metabolism, Methylation, Thrombin,
Antioxidant
1

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1. Introduction
Nowadays, ischemic cerebrovascular disease is
a
common and
frequently-occurring disease that seriously endangers human health, and it has been
one of the leading causes of disability and death worldwide [1]. Some evidences
suggest that thrombin plays a critical role in ischemic cerebrovascular disease [2], it
is generated in response to vascular injury. As a multifunctional serine protease,
thrombin can catalyze the proteolytic cleavage of the soluble plasma-protein
fibrinogen to form insoluble fibrin, which leads to clot formation. In addition,
thrombin also serves as a potent platelet agonist and amplifies its own generation by
feedback activation of several steps in the coagulation cascade [3]. Furthermore,
oxidative stresses are major causes of ischemic cerebrovascular disease [4,5], some
−•
reactive oxygen species (ROS) including superoxide anion radical (O2 ), hydroxyl
radical (•OH), hydrogen peroxide (H2O2), singlet oxygen (1O2) and nitric oxide (NO•)
are constantly generated in the human body [6]. However, the excessive production
of ROS may lead to increased level of low-density lipoprotein (LDL), and the
oxidative modification of LDL can impair the endothelial derived relaxing factor
(EDRF, nitric oxide, NO)-mediated bioactions [7].
On the above basis, some researchers have proposed and searched some natural
products with antithrombotic capacity and antioxidant activity for the treatment of
cerebrovascular disease [8]. Traditional Chinese medicines 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 constituent (>85%) of
breviscapine, a natural drug consisting of total flavonoids of Erigeron breviscapus
(Vant.) Hand-Mazz. (Compositae), has been used for the treatment of cerebral
infarction, angina pectoris and coronary heart disease with a large market share in
China [9]. Nowadays, the research on scutellarin (1) has become a hot topic in China
due to its distinguished efficacy in the clinical therapy. Pharmacological studies
found that scutellarin (1) exhibited antithrombotic and antioxidant activities to
attenuate neuronal damage, thus had a wide range of benefits to brain injury caused
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by cerebral ischemia/reperfusion [10-12].
(Fig. 1)
Interestingly, some researches showed that scutellarin (1) is readily hydrolyzed
into scutellarein (2) (Fig. 1) before absorption in vivo, and scutellarein (2) is absorbed
into the blood and can metabolite into methylated, sulfated or glucuronidated forms
[13]. 6-O-Methylscutellarein (5) (Fig. 1), which is one of the major in vivo
metabolites, might be responsible for the therapeutic effects of scutellarin (1) [13]. In
order to study the biological activities of 6-O-methylscutellarein (5) and other
methylated scutellarein analogs, in this paper, we report here on the synthesis of a
series of monomethylated scutellarein, dimethylated scutellarein and trimethylated
scutellarein derivatives via semi-synthetic methods. Then the antithrombotic
activities of these compounds were evaluated through the analyzation of prothrombin
time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and
fibrinogen (FIB). Their antioxidant activities were assessed by measuring their
scavenging capacities toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and the
ability to protect PC12 cells against H2O2-induced cytotoxicity using MTT assay
method. Furthermore, the physicochemical properties including aqueous solubility
and lipophilicity were also investigated in this report.
2. Results and discussion
2.1. Synthesis
2.1.1. Synthesis of monomethylated scutellarein analogs
The 4ʹ-O-methylscutellarein (3) is synthesized as summarized in Scheme 1.
Scutellarein (2) was obtained from scutellarin (1) by hydrolysis with 6N HCl in 90%
ethanol under reflux [14,15]. Treatment of
2
with 1.5 equiv. of
dichlorodiphenylmethane in diphenyl ether at 175 °C [16] led to the formation of
desired product 11 in 85% yield after the reaction mixture had proceeded 30 min.
Then the reaction of 11 with iodomethane (1.5 equiv.) afforded 12 in 91% yield with
a methyl group at the C-4ʹ phenol hydroxyl position. Under hydrogenolysis
3

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conditions, the diphenylmethylene ketal could be cleaved and the best results were
obtained with 10% palladium on carbon as a catalyst in EtOH, this procedure allowed
deprotection to afford 3 in 92% yield and no side product was detected by TLC
analysis.
(Scheme 1)
The 5-O-methylscutellarein (4) is synthesized as summarized in Scheme 2.
Reaction of 2 with an excess of chloromethyl methyl ether (4.5 equiv.) led to the
formation of 13 in 72% yield, whose phenolic functions except the hydroxyl group at
C-5 position were protected [17]. Because the phenol hydroxyl group at C-5 position
could form a hydrogen bond with the carbonyl group at C-4 position, the reaction of
13 with iodomethane and K2CO3 did not work. Fortunately, the treatment of 13 with
1.5 equiv. of iodomethane and 2.0 equiv. of NaH in the solution of DMF led to 14 in
78% yield with a methyl group at the C-5 phenol hydroxyl position. At last, the
hydrolysis of the MOM ether groups by hydrochloric acid provided the
5-O-methylscutellarein (4) in 90% yield.
(Scheme 2)
The synthesis of 6-O-methylscutellarein (5) is shown in Scheme 3. The reaction
of 11 with 1.5 equiv. of benzyl bromide in the presence of K2CO3 afforded 15 in 93%
yield. From compound 15, we focused on the deprotection of diphenylmethylene
ketal, two different methods were available for the cleavage of this protecting group:
hydrogenolysis or hydrolysis. Fortunately, under hydrolysis conditions the
diphenylmethylene ketal was deprotected by using the HOAc solution. This method
gave 16 in 95% yield and no side product was detected by TLC analysis. Benzylation
of 16 with benzyl bromide and K2CO3 in DMF led to the di-benzyl ether product 17
in 93% yield. A cross-peak observed in the ROESY spectrum of 17 between 5.03
(–OCH2Ph) with 6.87 (C8–H) indicated that one of the two benzyl group was at C-7
phenol hydroxyl position, other cross-peaks between 5.23 (–OCH2Ph) with 7.18
(C3′,5′–H) indicated that the other benzyl group was at C-4' phenol hydroxyl position.
Treatment of 17 with 1.2 equiv. of iodomethane led selectively to 18 with the desired
4

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methyl group was at the C-6 phenol hydroxyl position in 94% yield. Then the
deprotection of di-benzyl groups under hydrogenation conditions using 10%
palladium on carbon as the catalyst in THF/EtOH afforded 5 in 96% yield.
(Scheme 3)
Preparation of 7-O-methylscutellarein (6) is shown in Scheme 4. Scutellarein (2)
was converted into 19 (90% yield) after it was reacted with acetic anhydride under
the catalyst of 4-N,N-dimethylaminopyridine (DMAP) in pyridine. Interestingly, the
reaction of 19 with benzyl bromide issued the substitution of acetic group by benzyl
group to afford 20 in 70% yield. The position of benzyl group in 20 was confirmed
by ROESY spectrum, a cross-peak between 5.36 (–OCH2) with 6.87 (C8–H)
indicated that the benzyl group was at the C-7 phenol hydroxyl position. This
preferential reaction [18,19] might be that the electronically deficient C-4 carbonyl
group in 19 was para to the C-7 acetoxy unit, thus the C-4 carbonyl group could
accept electron density from the C-7 oxygen, which contributed to a weakening of
the ester bond and facilitated its rupture, thus leading to the benzyl substituted
intermediate 20. Deprotection of the benzyl group in 20 was accomplished under
hydrogenation conditions with 10% palladium on carbon as the catalyst in
EtOH/CH2Cl2 gave 21 in 95% yield, then the treatment of 21 with iodomethane (1.5
equiv.) led to 22. Finally, the hydrolysis of acetyl groups in 22 with a solution of
sodium hydroxide afforded 6 in 81% yield.
(Scheme 4)
2.1.2. Synthesis of dimethylated and trimethylated scutellarein analogs
The synthesis of 6,7-dimethylscutellarein (7) is shown in Scheme 5. Treatment
of 16 with 3.0 equiv. of iodomethane led to 23 in 86% yield, then the cleavage of the
benzyl group by hydrogenolysis gave 7 in 91% yield. Because the reactivity of two
phenol hydroxyl groups at C-4' and C-7 positions in 6-O-methylscutellarein (5) were
very active, so the reaction of 5 with 1.5 equiv. of iodomethane produced 7 and 8 in
38% and 49% respectively (Scheme 5). Furthermore, the methylation of the free
phenol hydroxyl function at C-6 and C-7 positions in 3 with iodomethane (3.5 equiv.)
5

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afforded 9 and 10 in 32% and 54% respectively (Scheme 5).
(Scheme 5)
2.2. Biological investigations
2.2.1. Antithrombotic activity
Because the antithrombotic activity can be assessed by measuring the
prolongation of the plasma clotting time of TT, APTT, INR increasement of PT, and
reduction of FIB content according to our previous studies [20], so the thrombin time
of different compounds was investigated for TT, PT, APTT and FIB, and the results
were shown in Table 1.
From the results we could see that the antithrombotic activity remained when
the glucuronyl group in scutellarin (1) was hydrolyzed to produce scutellarein (2),
although the PT decreased and the FIB content increased in scutellarein (2), the TT
and APTT in scutellarein (2) increased compared to those in scutellarin (1), this result
indicated that the glucuronyl group was not important for the antithrombotic activity.
In the series of monomethylated scutellareins, the most active compounds were
6-O-methylscutellarein (5) and 7-O-methylscutellarein (6), these two compounds
prolonged TT and PT, decreased FIB content compared to scutellarein (2). The less
active compound was 4'-O-methylscutellarein (3), it decreased TT, APTT, and
increased FIB content compared to 5. Furthermore, three plasma coagulation
parameters of 3 including TT, PT and APTT were decreased in the comparation to 6.
Interestingly, when the phenol hydroxyl group at C-5 position was etherized by
methyl group, the antithrombotic activity decreased significantly, this result could be
seen that the TT and APTT of 5-O-methylscutellarein (4) decreased, and the FIB
content increased compared to those of 4'-O-methylscutellarein (3). So the phenol
hydroxyl group at C-5 position was not methylated in the subsequent multiple-methly
derivatives of scutellarein (2). These results showed that the methyl group etherized
at the C-6 phenol hydroxyl position was very important to keep the antithrombotic
activity.
6

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In the series of dimethylated scutellarein analogs, the most active compound
was 6,7-O-dimethylscutellarein (7), it prolonged TT and APTT, increased PT and
decreased FIB content significantly compared to scutellarein (2). The less active
compounds were 4',6-O-dimethylscutellarein (8) and 4',7-O-dimethylscutellarein (9),
compound 8 only increased TT compared to 7, compound 9 showed no stronger
antithrombotic activity compared to 7 in the above four indicators. For compound
4',6,7-O-dimethylscutellarein (10), where the three phenol hydroxyl groups were all
etherized by the methyl groups, the antithrombotic activity decreased, this
phenomenon could be seen that 10 only increased TT compared to 7. These
pharmacological activity results indicated that the methyl group both etherized at the
C-6 and C-7 phenol hydroxyl positions could improve the anticoagulant activity.
Interestingly, most of the synthesized analogs except 7 and 9 showed decreased
APTT compared to 2 in the four indicators, these results indicated that the other three
indicators including TT, PT and FIB might contribute to the antithrombic activity in
these methylated scutellarein analogs.
(Table 1)
2.2.2. Antioxidant activity
The in vitro antioxidant activities [21] of these scutellarein derivatives were
assessed by measuring their scavenging capacities toward DPPH assay and the ability
to protect PC12 cells against H2O2-induced cytotoxicity.
2.2.2.1. DPPH radical-scavenging activity
DPPH assay [22] measured the hydrogen-donating ability of antioxidants to
convert the stable DPPH free radical into 1,1-diphenyl-2-picrylhydrazine [23]. The
reaction was accompanied by a change in color from deep-yellow to light-yellow and
was monitored spectrophotometrically.
The data obtained in the DPPH assay was depicted in Table 2. From the results
we can infer that, the monomethylated scutellarein derivatives showed potent
antioxidant activities. 6-O-methylscutellarein (5) (IC50 = 23.67 ꢀM), in which only
the C-6 phenol hydroxy was etherized by the methyl group, showed stronger
7

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antioxidant activity in this DPPH assay compared to scutellarin (1) (IC50 = 27.93 ꢀM).
The less active compound was 4'-O-methylscutellarein (3), in which only the C-4'
phenol hydroxy was etherized by the methyl group, it showed the IC50 of 25.29 ꢀM in
the DPPH assay. 5-O-methylscutellarein (4), in which the C-5 phenol hydroxyl
function was etherized by methyl group, showed less antioxidant activity compared
to 3 with its IC50 was 28.85 ꢀM. 7-O-methylscutellarein (6), showed the least
antioxidant activity in the series of monomethylated scutellarein analogs, with its
IC50 was 30.17 ꢀM. Furthermore, if there were two methyl groups etherized at the
phenol hydroxyl functions in scutellarein (2), the antioxidant activity decreased, these
results could be seen that one methyl group etherized at the C-7 phenol hydroxyl
position and C-4' phenol hydroxyl position in 6-O-methyl-scutellarein (5) to produce
6,7-O-dimethylscutellarein (7) and 4',6-O-dimethylscutellarein (8), their IC50s
changed into 189.88 ꢀM and 169.82 ꢀM respectively. Although
4'-O-methylscutellarein (3) and 7-O-methylscutellarein (6) showed strong antioxidant
activity in this DPPH assay, when the methyl groups both etherized at the C-4' phenol
hydroxyl and C-7 phenol hydroxyl positions to produce 4',7-O-dimethylscutellarein
(9), the antioxidant activity was decreased with its IC50 was 158.67 ꢀM. At last, when
the three phenol hydroxyl functions at C-4', C-6 and C-7 positions were all etherized
by the methyl groups, the obtained product 10 lost its antioxidant activity with its
IC50 was 462.79 ꢀM.
2.2.2.2. Inhibitory Effect on PC12 Cells Induced by Oxidative Stress
PC12 cells can adopt a neuronal phenotype and have been used extensively as a
model for catecholamine-secreting neuronal cells [24]. Active mitochondria of living
cells can cleave MTT to produce formazan, the amount of which directly related to
the number of living cells. So the neuroprotective effects of the synthesized methyl
scutellarein derivatives were evaluated by the ability to protect PC12 cells against
H2O2-induced cytotoxicity using MTT assay method [25].
The results of the inhibition rate against cell damage were summarized in Table
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2. Comparison of the data reported in Table 2 indicated all the monomethylated
scutellarein derivatives showed higher inhibitory effect than scutellarin (1) against
H2O2-induced cytotoxicity in PC12 cells, especially for 4'-O-methylscutellarein (3)
and 6-O-methyl-scutellarein (5). In a concentration of 50 ꢀM, 3 and 5 showed
89.38% and 88.05% inhibition rate against cell damage, and in a concentration of 25
ꢀM, 3 and 5 showed 63.27% and 62.39% inhibition rate against cell damage
respectively, while the inhibiting rates of the parent compound 1 were 79.20% and
53.10% at 50 and 25 µM respectively. The less active compounds were
7-O-methylscutellarein (6) and 5-O-methylscutellarein (4), these two compounds
exhibited 82.30% and 81.42% inhibition rate against cell damage in a concentration
of 50 ꢀM respectively. All the dimethylated scutellarein derivatives were less
effective at protecting these neuronal cells against H2O2-induced oxidative injury
than scutellarin (1), for example, 6,7-O-dimethylscutellarein (7) and
4',7-O-dimethylscutellarein (9) showed 76.11% and 74.34% inhibition rate against
cell damage in a concentration of 50 ꢀM respectively. Unfortunately, the
trimethylated scutellarein derivative 10 lost its protective activity against cell damage
with its inhibition rate was 20.35% in a concentration of 50 ꢀM. In addition, all the
synthesized analogs 3-10 showed a lower inhibitory effect than 2, these results
indicated that the presence of more phenol hydroxyl groups would be more important
to protect the PC12 cells against H2O2-induced oxidative injury.
(Table 2)
2.2.3. Physicochemical property
Physicochemical profile plays a major role in modern drug discovery [26],
aqueous solubility [27] and lipophilicity, usually expressed as n-octanol/water
distribution coefficient, logP [28,29], are among the most important properties that
can provide a guideline for predicting a drug’s solubility characteristics [30]. These
properties influence the distribution of drugs within the body since drugs must
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possess some lipophilic properties to cross biological membranes, and hydrophilic
properties to be taken up in systemic circulation.
2.2.3.1. Water solubility determination
The aqueous solubility of the synthesized methylated scutellarein derivatives
was determined using UV spectrophotometer [31-33]. The results were shown in
Table 3. Although the introduction of methyl group in the scutellarein could increase
the lipid solubility, these methylated scutellarein derivatives still maintained some
water solubility. Among the monomethylated scutellarein derivatives,
4'-O-methylscutellarein (3) and 5-O-methylscutellarein (4) showed better water
solubility than the others with their values were 6.31 µg/ml and 5.72 µg/ml
respectively. 7-O-methylscutellarein (6) and 6-O-methylscutellarein (5) exhibited
good solubility in water with their values were 4.56 µg/ml and 4.27 µg/ml
respectively. Introduction of more methyl groups decreased the water solubility of
the scutellarein derivatives, this phenomenon could be seen that
4',7-O-dimethylscutellarein
(9),
4',6-O-dimethylscutellarein
(8)
and
4',6,7-trimethylscutellarein (10) exhibited remarkable decreases in solubility with
their values were 3.20 µg/ml, 1.36 µg/ml and 0.31 µg/ml respectively..
2.2.3.2. Partition coefficient
The partition coefficient between n-octanol and buffer for the methylated
scutellarein derivatives compared to the parent compounds scutellarin (1) and
scutellarein (2) was determined using traditional shake-flask method [34-36], the
buffer included three different solutions which were 0.1mol/L HCl, 0.15mol/L NaCl
and 0.15mol/L NaHCO3, and the results were summerized in Table 3. The logP
values of scutellarin (1) were found to be 0.5924, 0.3647 and -0.0513 in the three
different solutions respectively. Introduction of methyl groups in scutellarein
significantly altered the partition coefficient pattern in a range from 0.7846 to 2.6358
(Table 3). Interestingly, the 6-O-methylscutellarein (5) showed the best logP among
the monomethylated scutellarein derivatives, with its values were 1.7651, 1.6805 and
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1.0175 respectively. The logP values of 4',6,7-trimethylscutellarein (10) increased to
2.6358, 2.5492 and 2.2707 respectively when the three methyl groups etherized at the
C-4', C-6 and C-7 phenol hydroxyl positions. These results showed that the
introduction of the methyl group had a promising effect on the increasing of logP of
the synthesized compounds compared to scutellarin (1), thus increased the
lipophilicity of these compounds that may effect cell penetration.
(Table 3)
3. CONCLUSION
In summary, eight novel methylated scutellarein derivatives have been designed
and synthesized based on the metabolic mechanism of scutellarin (1) in vivo. The
antithrombotic activities of these compounds were evaluated through the analyzation
of PT, APTT, TT and FIB, the results showed that one methyl group etherized at the
C-6 phenol hydroxyl position was very important to keep the antithrombotic activity.
Their in vitro antioxidant activity was evaluated by DPPH radical-scavenging activity
assays and the protective effects on H2O2-induced cytotoxicity in PC12 cells using
MTT assay method, the presence of the phenol hydroxyl group was very important to
antioxidant activity. The results of aqueous solubility and lipophilicity showed that
the introduction of the methyl group had a promising effect on the increasing of the
lipophilicity of these compounds that may effect cell penetration.
6-O-methyl-scutellarein (5), which had only one methyl group etherized at the C-6
phenol hydroxyl position, demonstrated potent antithrombotic activity, stronger
antioxidant activity and balanced solubility and permeability compared with
scutellarin (1), warranted further development of this agent as a promising lead for
the treatment of ischemic cerebrovascular disease.
4. Experimental
4.1. Chemical synthesis
Reagents and solvents were purchased from commercial sources and used
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without further purification unless otherwise specified. Air- and moisture-sensitive
liquids and solutions were transferred via syringe or stainless steel cannula. Organic
solutions were concentrated by rotary evaporation below 45 ºC at approximately 20
mm Hg. All non-aqueous reactions were carried out under anhydrous conditions
using flame-dried glassware within nitrogen atmosphere in dry, freshly distilled
solvents, unless otherwise noted. Yields referred to chromatographically
homogeneous materials, unless otherwise stated. Reactions were monitored by
thin-layer chromatography (TLC) carried out on 0.15–0.20 mm Yantai silica gel
plates (RSGF 254) using UV light as the visualizing agent. Chromatography was
performed on Qingdao silica gel (160–200 mesh) using petroleum ether (60–90) and
ethyl acetate as the eluanting solvent. 1H NMR spectra were obtained using a Bruker
AV-300 (300 MHz) or Bruker AV-500 (500 MHz). Chemical shifts were recorded in
ppm downfield from tetramethylsilane. J values were given in Hz. Abbreviations
used were s (singlet), d (doublet), t (triplet), q (quartet), b (broad), and m (multiplet).
ESI-MS spectra were recorded on a Waters Synapt HDMS spectrometer.
4.1.1. 5,6,7-Trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (2)
Water (10 ml) was added to a stirring mixture of scutellarin (1) (10.0 g, 21.6 mmol)
and concentrated hydrochloric acid (120 ml) in ethanol (120 ml), then the reaction
mixture was refluxed under an N2 atmosphere for 36 h. After being cooled down to
the room temperature, the mixture was poured into water, the solid obtained was
filtered, and purified by column chromatography (50% ethyl acetate in petroleum
1
ether) to afford 2 (1.05 g, 17.0% yield) as a yellow solid. H NMR (DMSO-d6, 300
MHz) δ 6.57 (s, 1H, 8-H), 6.74 (s, 1H, 3-H), 6.91 (d, J = 8.8 Hz, 2H, 3',5'-H), 7.90 (d,
J = 8.8 Hz, 2H, 2',6'-H), 8.71 (s, 1H, 6-OH), 10.29 (s, 1H, 7-OH), 10.44 (s, 1H,
4'-OH), 12.78 (s, 1H, 5-OH); ESI-MS: m/z 287 [M+H]+.
4.1.2.
9-Hydroxy-6-(4-hydroxyphenyl)-2,2-diphenyl-8H-[1,3]dioxolo[4,5-g]chromen-8-
12

ACCEPTED MANUSCRIPT
one (11)
To a stirring mixture of scutellarein (2) (10 g, 35 mmol) in diphenyl ether (200 ml)
was added dichlorodiphenylmethane (18 g, 52.5 mmol, 1.5 equiv.), then the reaction
mixture was heated at 175 °C for 30 min. After being cooled down to room
temperature, petroleum ether (1000 ml) was added, the solid obtained was filtered,
and purified by column chromatography (25% ethyl acetate in petroleum ether) to
1
afford 11 (13.35 g, 85% yield) as a yellow solid. H NMR (300 MHz, DMSO-d6) δ
6.69 (s, 1H, 3-H), 6.82 (s, 1H, 8-H), 6.96 (d, 2H, J = 8.7 Hz, 3′,5′-H), 7.47-7.50 (m,
6H, -Ph), 7.58-7.61 (m, 4H, -Ph), 7.94 (d, 2H, J = 8.7 Hz, 2′,6′-H), 10.41 (s, 1H,
4′-OH), 12.99 (s, 1H, 5-OH); ESI-MS: m/z 449 [M–H]-.
4.1.3.
9-Hydroxy-6-(4-methoxyphenyl)-2,2-diphenyl-8H-[1,3]dioxolo[4,5-g]chromen-8-
one (12)
To a stirring solution of 11 (200 mg, 0.44 mmol) in DMF (10 ml) was added
K2CO3 (107 mg, 0.78 mmol, 1.75 equiv.) and iodomethane (0.041 ml, 0.66 mmol, 1.5
equiv.). After stirring at 0 °C for 2 h, the reaction mixture was allowed to warm to
room temperature and the stirring was maintained for 6 h. Then the reaction mixture
was partitioned between 100 ml ethyl acetate and 100 ml water, and the organic layer
was washed with brine (100 ml), dried over Na2SO4, filtered and concentrated, the
crude material was purified by column chromatography (33% ethyl acetate in
1
petroleum ether) to afford 12 (186 mg, 91% yield) as a yellow solid. H NMR (300
MHz, DMSO-d6) δ 3.88 (s, 3H, -OCH3), 6.96 (s, 1H, 3-H), 7.11 (s, 1H, 8-H), 7.15 (d,
2H, J = 8.6 Hz, 3′,5′-H), 7.46-7.49 (m, 6H, -Ph), 7.55-7.60 (m, 4H, -Ph), 8.08 (d, 2H,
J = 8.6 Hz, 2′,6′-H), 13.10 (s, 1H, 5-OH); ESI-MS: m/z 465 [M+H]+
4.1.4. 5,6,7-Trihydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (3)
To a solution of 12 (160 mg, 0.34 mmol) dissolved in ethanol (30 ml) was added
10% Pd/C (16 mg) with vigorous stirring. Then the reaction vessel was evacuated and
13

ACCEPTED MANUSCRIPT
the atmosphere replaced with hydrogen. After 12 h, the reaction mixture was filtered,
and the filtrate was concentrated, the crude material was purified by column
chromatography (20% ethyl acetate in petroleum ether) to afford 3 (94mg, 92% yield)
as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 3.91 (s, 3H, -OCH3), 6.59 (s, 1H,
3-H), 6.78 (s, 1H, 8-H), 6.92 (d, 2H, J = 8.6 Hz, 3′,5′-H), 7.92 (d, 2H, J = 8.6 Hz,
2′,6′-H), 8.75 (s, 1H, 6-OH), 10.77 (s, 1H, 7-OH), 13.16 (s, 1H, 5-OH); ESI-MS: m/z
299 [M–H]-.
4.1.5.
5-Hydroxy-6,7-bis(methoxymethoxy)-2-(4-(methoxymethoxy)phenyl)-4H-chrom
en-4-one (13)
Chloromethyl ether (2.39 ml, 31.5 mmol, 4.5 equiv.) was added to a stirring
mixture of 2 (2 g, 7 mmol) and K2CO3 (5.80 g, 42 mmol, 6.0 equiv.) in dry acetone
(100 ml) at room temperature. The reaction mixture was refluxed gently for 6 h. After
being cooled down to room temperature, the reaction mixture was filtered, and the
filtrate was concentrated, the crude material was purified by column chromatography
(20% ethyl acetate in petroleum ether) to afford 13 (2.11 g, 72% yield) as a yellow
1
solid. H NMR 300 MHz, DMSO-d6) δ 3.66 (s, 3H, -OCH3), 3.76(s, 3H, -OCH3),
3.87 (s, 3H, -OCH3), 5.35 (s, 2H, -OCH2), 5.48 (s, 2H, -OCH2), 5.60 (s, 2H, -OCH2),
6.95 (s, 1H, 3-H), 7.09 (s, 1H, 8-H), 7.16 (d, 2H, J = 8.7 Hz, 3′,5′-H), 8.06 (d, 2H, J =
8.7 Hz, 2′,6′-H), 12.93 (s, 1H, 5-OH); ESI-MS: m/z 419 [M+H]+.
4.1.6.
5-Methoxy-6,7-bis(methoxymethoxy)-2-(4-(methoxymethoxy)phenyl)-4H-chrom
en-4-one (14)
To a stirring solution of 13 (200 mg, 0.48 mmol) in DMF (10ml) at 0 ºC was added
NaH (23 mg, 0.96 mmol, 2.0 equiv.), 30 min later, the iodomethane (0.04 ml, 0.66
mmol, 1.5 equiv.) was added, then the reaction mixture was allowed to warm to room
temperature. After 6 h, the reaction mixture was partitioned between 100 ml ethyl
14

ACCEPTED MANUSCRIPT
acetate and 100 ml water, and the organic layer was washed with brine (100 ml),
dried over Na2SO4, filtered and concentrated, the crude material was purified by
column chromatography (20% ethyl acetate in petroleum ether) to afford 14 (162 mg,
78% yield) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 3.46 (s, 3H, -OCH3),
3.47 (s, 3H, -OCH3), 3.48 (s, 3H, -OCH3), 3.49 (s, 3H, -OCH3), 5.32 (s, 2H, -OCH2),
5.33 (s, 2H, -OCH2), 5.34 (s, 2H, -OCH2), 6.43 (s, 1H, 3-H), 6.75 (s, 1H, 8-H), 7.23
(d, 2H, J = 8.7 Hz, 3′,5′-H), 8.06 (d, 2H, J = 8.7 Hz, 2′,6′-H); ESI-MS: m/z 433
[M+H]+.
4.1.7. 6,7-Dihydroxy-2-(4-hydroxyphenyl)-5-methoxy-4H-chromen-4-one (4)
To a stirring solution of 14 (100 mg, 0.23 mmol) in ether (5ml) and
dichloromethane (5ml) was added concentrated hydrochloric acid (1ml) at 0 ºC, then
the reaction mixture was allowed to warm to room temperature. After 6 h, the
resulting mixture was partitioned between 100 ml ethyl acetate and 100 ml water, and
the organic layer was washed with brine (100 ml), dried over Na2SO4, filtered and
concentrated, the crude material was purified by column chromatography (30% ethyl
acetate in petroleum ether) to afford 4 (62 mg, 90% yield) as a yellow solid. 1H NMR
(300 MHz, DMSO-d6) δ 4.23 (s, 3H, -OCH3), 6.67 (s, 1H, 3-H), 6.92 (d, 2H, J = 8.7
Hz, 3′,5′-H), 6.95 (s, 1H, 8-H), 7.92 (d, 2H, J = 8.7 Hz, 2′,6′-H), 8.73 (s, 1H, 6-OH),
10.30 (s, 1H, 4′-OH), 10.73 (s, 1H, 7-OH); ESI-MS: m/z 299 [M–H]-.
4.1.8.
6-(4-(Benzyloxy)phenyl)-9-hydroxy-2,2-diphenyl-8H-[1,3]dioxolo[4,5-g]chromen
-8-one (15)
To a stirring solution of 11 (200 mg, 0.44 mmol) in DMF (10 ml) was added
K2CO3 (107 mg, 0.78 mmol, 1.75 equiv.) and benzyl bromide (0.078 ml, 0.66 mmol,
1.5 equiv.). After stirring at 0 °C for 2 h, the reaction mixture was allowed to warm to
room temperature and the stirring was maintained for 12 h. Then the reaction mixture
15

ACCEPTED MANUSCRIPT
was partitioned between 100 ml ethyl acetate and 100 ml water, and the organic layer
was washed with brine (100 ml), dried over Na2SO4, filtered and concentrated, the
crude material was purified by column chromatography (33% ethyl acetate in
1
petroleum ether) to afford 15 (221 mg, 93% yield) as a yellow solid. H NMR (300
MHz, DMSO-d6) δ 5.22 (s, 2H, -OCH2), 6.95 (s, 1H, 3-H), 7.08 (s, 1H, 8-H), 7.22 (d,
2H, J = 8.7 Hz, 3′,5′-H), 7.32-7.48 (m, 11H, -Ph), 7.55-7.61(m, 4H, -Ph), 8.04 (d, 2H,
J = 8.7 Hz, 2′,6′-H), 10.40 (s, 1H, 4′-OH), 13.10 (s, 1H, 5-OH); ESI-MS: m/z 541
[M+H]+.
4.1.9. 2-(4-(Benzyloxy)phenyl)-5,6,7-trihydroxy-4H-chromen-4-one (16)
Compound 15 (150 mg, 0.28 mmol) was dissolved in 20 ml of HOAc/H2O (4:1)
solution and the reaction mixture was refluxed under a N2 atmosphere for 1.5 h. After
being cooled down to the room temperature, the mixture was poured into 100 ml
water, extracted with ethyl acetate (50 ml×3), then the organic layer was washed with
brine (100 ml), dried over Na2SO4, filtered and concentrated, the crude material was
purified by column chromatography (25% ethyl acetate in petroleum ether) to afford
16 (100 mg, 95% yield) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 5.28 (s,
2H, -OCH2), 6.81 (s, 1H, 3-H), 6.92 (d, 2H, J = 8.7 Hz, 3′,5′-H), 7.00 (s, 1H, 8-H),
7.35-7.45 (m, 3H, -Ph), 7.51-7.54 (m, 2H, -Ph), 7.94 (d, 2H, J = 8.7 Hz, 2′,6′-H), 8.74
(s, 1H, 6-OH), 10.35 (s, 1H, 7-OH), 12.69 (s, 1H, 5-OH); ESI-MS: m/z 375 [M–H]-.
4.1.10. 7-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-5,6-dihydroxy-4H-chromen-4-one
(17)
To a stirring solution of 16 (200 mg, 0.53 mmol) in dry DMF (20 ml) was added
K2CO3 (109 mg, 0.80 mmol, 1.5 equiv.) and benzyl bromide (0.08 ml, 0.68 mmol,
1.3 equiv.) at 0 °C, then the mixture was warmed to room temperature. After 12 h, the
reaction mixture was partitioned between 100 ml ethyl acetate and 100 ml water, then
the ethyl acetate layer was washed with brine (100 ml), dried over Na2SO4, filtered
and concentrated, the crude material was purified by column chromatography (25%
16

ACCEPTED MANUSCRIPT
ethyl acetate in petroleum ether) to afford 17 (230 mg, 93% yield) as a yellow solid.
1H NMR (300 MHz, DMSO-d6) δ 5.03 (s, 2H, -OCH2), 5.23 (s, 2H, -OCH2), 6.62 (s,
1H, 3-H), 6.87 (s, 1H, 8-H), 7.18 (d, 2H, J = 8.6 Hz, 3′,5′-H), 7.31-7.53 (m, 10H,
-Ph), 8.04 (d, 2H, J = 8.6 Hz, 2′,6′-H), 10.82 (s, 1H, 6-OH), 13.11 (s, 1H, 5-OH);
ESI-MS: m/z 465 [M–H]-.
4.1.11.
7-(Benzyloxy)-2-(4-(benzyloxy)phenyl)-5-hydroxy-6-methoxy-4H-chromen-4-one
(18)
To a stirring solution of 17 (84 mg, 0.18 mmol) in dry DMF (20 ml) was added
K2CO3 (48 mg, 0.35 mmol, 1.4 equiv.) and iodomethane (0.014 ml, 0.22 mmol, 1.2
equiv.) at room temperature. After 12 h, the reaction mixture was then partitioned
between 100 ml ethyl acetate and 100 ml water. The ethyl acetate layer was then
washed with brine (100 ml), dried over Na2SO4, filtered and concentrated, the crude
material was purified by column chromatography (25% ethyl acetate in petroleum
1
ether) to afford 18 (81 mg, 94% yield) as a yellow solid. H NMR (300 MHz,
DMSO-d6) δ 4.00 (s, 3H, -OCH3), 4.95 (s, 2H, -OCH2), 5.28 (s, 2H, -OCH2), 6.62 (s,
1H, 3-H), 6.94 (d, 2H, J = 8.6 Hz, 3′,5′-H), 7.00 (s, 1H, 8-H), 7.32-7.42 (m, 6H, -Ph),
7.44-7.54 (m, 4H, -Ph), 7.94 (d, 2H, J = 8.6 Hz, 2′,6′-H), 12.69 (s, 1H, 5-OH);
ESI-MS: m/z 481 [M+H]+.
4.1.12. 5,7-Dihydroxy-2-(4-hydroxyphenyl)-6-methoxy-4H-chromen-4-one (5)
To a solution of 18 (100 mg, 0.21 mmol) dissolved in ethanol (10 ml) and THF
(10 ml) was added 10% Pd/C (2 mg) with vigorous stirring. The reaction vessel was
then evacuated and the atmosphere replaced with hydrogen. After 8 h, the reaction
mixture was filtered and the filtrate was concentrated, the crude material was purified
by column chromatography (50% ethyl acetate in petroleum ether) to afford 5 (60 mg,
96% yield) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 3.72 (s, 3H, -OCH3),
6.58 (s, 1H, 3-H), 6.89 (d, 2H, J = 8.6 Hz, 3′,5′-H), 7.11 (s, 1H, 8-H), 7.87 (d, 2H, J =
17

ACCEPTED MANUSCRIPT
8.6 Hz, 2′,6′-H), 10.19 (s, 1H, 4′-OH), 10.72 (s, 1H, 7-OH), 12.76 (s, 1H, 5-OH);
ESI-MS: m/z 299 [M–H]-.
4.1.13. 5,6,7-Triacetoxy-2-(4-acetoxyphenyl)-4H-chromen-4-one (19)
Pyridine (5 ml) and 4-dimethylaminopyridine (DMAP) (12.2 mg, 0.1 mmol) were
added to a stirring solution of 2 (286 mg, 1.0 mmol) in acetic anhydride (5 ml) at
0 °C, then the mixture was warmed to room temperature. After 4 h, the reaction
mixture was poured into 100 ml water, and the solid obtained was filtered and
purified by column chromatography (25% ethyl acetate in petroleum ether) to afford
19 (408 mg, 90% yield) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 2.32 (s,
3H, –COCH3), 2.35 (s, 3H, –COCH3), 2.37 (s, 3H, –COCH3), 2.38 (s, 3H, –COCH3),
6.95 (s, 1H, 8-H), 7.36 (d, J = 8.7 Hz, 2H, 3',5'-H), 7.84 (s, 1H, 3-H), 8.14 (d, J = 8.7
Hz, 2H, 2',6'-H); ESI-MS: m/z 455 [M+H]+.
4.1.14. 7-(Benzyloxy)-5,6-diacetoxy-2-(4-acetoxyphenyl)-4H-chromen-4-one (20)
To a stirring solution of 19 (227 mg, 0.50 mmol) in dry acetone (20 ml) was added
K2CO3 (484 mg, 3.50 mmol, 7.0 equiv.) and benzyl bromide (0.07 ml, 0.60 mmol,
1.2 equiv.), after the reaction was refluxed for 6 h, the mixture was partitioned
between 100 ml ethyl acetate and 100 ml water. The ethyl acetate layer was then
washed with brine (100 ml), dried over Na2SO4, filtered, and concentrated, the crude
material was purified by column chromatography (25% ethyl acetate in petroleum
1
ether) to afford 20 (186 mg, 74% yield) as a yellow solid. H NMR (300 MHz,
DMSO-d6) δ 2.31 (s, 3H, –COCH3), 2.32 (s, 3H, –COCH3), 2.34 (s, 3H, –COCH3),
5.36 (s, 2H, CH2), 6.87 (s, 1H, 8-H), 7.36–7.45 (m, 7H, 3',5'-H and ArH), 7.60 (s, 1H,
3-H), 8.13 (d, J = 8.8 Hz, 2H, 2',6'-H); ESI-MS: m/z 503 [M+H]+.
4.1.15. 7-Hydroxy-5,6-diacetoxy-2-(4'-acetoxyphenyl) -4H-1-benzopyran-4-one
(21)
To a stirring mixture of 20 (426 mg, 0.85 mmol) in dichloromethane (50 ml) and
18

ACCEPTED MANUSCRIPT
ethanol (50 ml) was added 10% Pd/C (43 mg). The reaction vessel was then
evacuated and the atmosphere replaced with hydrogen. After 12 h, the reaction
mixture was filtered, concentrated and purified by column chromatography (25%
ethyl acetate in petroleum ether) to afford 21 (322 mg, 92% yield) as a yellow solid.
1H NMR (300 MHz, DMSO-d6) δ 2.31 (s, 3H, -OC-CH3), 2.34 (s, 3H, -OC-CH3),
2.35 (s, 3H, -OC-CH3), 6.78 (s, 1H, 3-H), 7.08 (s, 1H, 8-H), 7.38 (d, 2H, J = 8.8Hz,
3′,5′-H), 8.18 (d, 2H, J = 8.8Hz, 2′,6′-H), 11.52 (s, 1H, 7-OH); ESI-MS: m/z 411
[M–H]-.
4.1.16. 7-Methoxy-5,6-diacetoxy-2-(4'-acetoxyphenyl) -4H-1-benzopyran-4-one
(22)
To a stirring solution of 21 (200 mg, 0.49 mmol) in dry DMF (10 ml) was added
K2CO3 (117 mg, 0.85 mmol, 1.75 equiv.) and iodomethane (0.05 ml, 0.74 mmol, 1.5
equiv.) at room temperature. After 6 h, the reaction mixture was then partitioned
between 100 ml ethyl acetate and 100 ml water. The ethyl acetate layer was then
washed with brine (100 ml), dried over Na2SO4, filtered and concentrated, the crude
material was purified by column chromatography (25% ethyl acetate in petroleum
1
ether) to afford 22 (188 mg, 90% yield) as a yellow solid. H NMR (300 MHz,
DMSO-d6) δ 2.30 (s, 3H, -OC-CH3), 2.31 (s, 3H, -OC-CH3), 2.33 (s, 3H, -OC-CH3),
4.09 (s, 3H, -CH3), 6.88 (s, 1H, 3-H), 7.36 (d, 2H, J = 8.8Hz, 3′,5′-H), 7.38 (s, 1H,
8-H), 8.12 (d, 2H, J = 8.8Hz, 2′,6′-H); ESI-MS: m/z 427[M+H]+ꢀ
4.1.17. 5,6-Dihydroxy-2-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one (6)
A solution of sodium hydroxide (0.5 M, 1 ml) was added to a stirring mixture of
22 (85 mg, 0.20 mmol) in methanol (10 ml) at 0 °C under N2 atmosphere, after 1h,
the mixture was then partitioned between 100 ml ethyl acetate and 100 ml water. The
ethyl acetate layer was then washed with brine (100 ml), dried over Na2SO4, filtered,
and concentrated, the crude material was purified by column chromatography (25%
ethyl acetate in petroleum ether) to afford 6 (49 mg, 81% yield) as a yellow solid. 1H
19

ACCEPTED MANUSCRIPT
NMR (300 MHz, DMSO-d6) δ 4.49 (s, 3H, -CH3), 6.59 (s, 1H, 3-H), 6.78 (s, 1H,
8-H), 6.93 (d, 2H, J = 8.8Hz, 3′,5′-H), 7.92 (d, 2H, J = 8.8Hz, 2′,6′-H), 10.33 (s, 1H,
4′-OH), 13.16 (s, 1H, 5-OH); ESI-MS: m/z 299 [M–H]-.
4.1.18.
(23)
2-(4-(Benzyloxy)phenyl)-5-hydroxy-6,7-dimethoxy-4H-chromen-4-one
To a stirring solution of 16 (200 mg, 0.53 mmol) in dry DMF (10 ml) was added
K2CO3 (256 mg, 1.86 mmol, 3.5 equiv.) and iodomethane (0.11 ml, 1.59 mmol, 3.0
equiv.) at room temperature. After 6 h, the reaction mixture was then partitioned
between 100 ml ethyl acetate and 100 ml water. The ethyl acetate layer was then
washed with brine (100 ml), dried over Na2SO4, filtered and concentrated, the crude
material was purified by column chromatography (25% ethyl acetate in petroleum
1
ether) to afford 23 (184 mg, 86% yield) as a yellow solid. H NMR (300 MHz,
DMSO-d6) δ 3.76 (s, 3H, -CH3), 3.87 (s, 3H, -CH3), 5.29 (s, 2H, -CH2), 6.95 (s, 1H,
3-H), 7.08 (s, 1H, 8-H), 7.14 (d, 2H, J = 8.7 Hz, 3′,5′-H), 7.35-7.53 (m, 8H, -Ph),
7.65-7.74 (m, 2H, -Ph), 8.06 (d, 2H, J = 8.7 Hz, 2′,6′-H), 12.90 (s, 1H, 5-OH);
ESI-MS: m/z 405 [M+H]+.
4.1.19. 5-Hydroxy-2-(4-hydroxyphenyl)-6,7-dimethoxy-4H-chromen-4-one (7)
To a solution of 23 (100 mg, 0.25 mmol) dissolved in ethanol (30 ml) was added
10% Pd/C (10 mg) with vigorous stirring. The reaction vessel was then evacuated and
the atmosphere replaced with hydrogen. After 12 h, the reaction mixture was filtered
and the filtrate concentrated, the crude material was purified by column
chromatography (20% ethyl acetate in petroleum ether) to afford 7 (71 mg, 91% yield)
as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 3.92 (s, 3H, -CH3), 3.98 (s, 3H,
-CH3), 6.86 (s, 1H, 3-H), 7.52 (s, 1H, 8-H), 7.36 (d, 2H, J = 8.7 Hz, 3′,5′-H), 8.15 (d,
2H, J = 8.7 Hz, 2′,6′-H), 10.40 (s, 1H, 4′-OH), 13.05 (s, 1H, 5-OH); ESI-MS: m/z 313
[M–H]-.
20

ACCEPTED MANUSCRIPT
4.1.20. 5,7-Dihydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (8)
To a stirring solution of 5 (200 mg, 0.67 mmol) in dry DMF (10 ml) was added
K2CO3 (184 mg, 1.33 mmol, 2.0 equiv.) and iodomethane (0.07 ml, 1.00 mmol, 1.5
equiv.) at room temperature. After 6 h, the reaction mixture was then partitioned
between 100 ml ethyl acetate and 100 ml water. The ethyl acetate layer was then
washed with brine (100 ml), dried over Na2SO4, filtered and concentrated, the crude
material was purified by column chromatography (25% ethyl acetate in petroleum
ether) to afford 7 (80 mg, 38% yield) and 8 (103mg, 49% yield) as yellow solids. 8
1H NMR (300 MHz, DMSO-d6) δ 3.40 (s, 3H, -CH3), 3.51 (s, 3H, -CH3), 6.63 (s, 1H,
3-H), 6.88 (s, 1H, 8-H), 7.19 (d, 2H, J = 8.7 Hz, 3′,5′-H), 8.04 (d, 2H, J = 8.7 Hz,
2′,6′-H), 10.91 (s, 1H, 7-OH), 13.07 (s, 1H, 5-OH); ESI-MS: m/z 313 [M–H]-.
4.1.21. Synthesis of 9 and 10
To a stirring solution of 3 (200 mg, 0.67 mmol) in dry DMF (10 ml) was added
K2CO3 (368 mg, 2.67 mmol, 4.0 equiv.) and iodomethane (0.16 ml, 2.33 mmol, 3.5
equiv.) at room temperature. After 8 h, the reaction mixture was then partitioned
between 100 ml ethyl acetate and 100 ml water. The ethyl acetate layer was washed
with brine (100 ml), dried over Na2SO4, filtered and concentrated, the crude material
was purified by column chromatography (25% ethyl acetate in petroleum ether) to
afford 9 (67 mg, 32% yield) and 10 (118 mg, 54% yield) as yellow solids
4.1.21.1 5,6-Dihydroxy-7-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (9)
1H NMR (300 MHz, DMSO-d6) δ 3.73 (s, 6H, -CH3), 3.91 (s, 6H, -CH3), 6.67 (s,
1H, 3-H), 7.14 (s, 1H, 8-H), 7.16 (d, 2H, J = 8.7 Hz, 3′,5′-H), 7.98 (d, 2H, J = 8.6 Hz,
2′,6′-H), 8.98 (s, 1H, 6-OH), 12.59 (s, 1H, 5-OH); ESI-MS: m/z 313 [M–H]-.
4.1.21.2 5-Hydroxy-6,7-dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (10)
1H NMR (300 MHz, DMSO-d6) δ 3.78 (s, 3H, -CH3), 3.91 (s, 3H, -CH3), 3.94 (s,
3H, -CH3), 6.71 (s, 1H, 3-H), 7.16 (d, 2H, J = 8.6 Hz, 3′,5′-H), 7.21 (s, 1H, 8-H), 8.06
(d, 2H, J = 8.7 Hz, 2′,6′-H), 12.95 (s, 1H, 5-OH); ESI-MS: m/z 329 [M+H]+ꢀ
4.2. Antithrombotic assay
21

ACCEPTED MANUSCRIPT
4.2.1. Blood collection
Male New Zealand white rabbits, weighing 2-2.5 kg were obtained from the
experimental animal center of Nanjing University of Chinese Medicine and were
approved by Animal Ethics Committee of Nanjing University of Chinese Medicine.
They were kept in plastic cages at 22 ± 2°C with free access to pellet food and water
and on a 12 h light/dark cycle. Animal welfare and experimental procedures were
carried out in accordance with the guide for the care and use of laboratory animals
(National Research Council of USA, 1996) and related ethical regulations of Nanjing
University of Chinese Medicine. Rabbits were anesthetized with pentobarbital (50
mg/kg) and blood was drawn from the common carotid artery. Blood was collected
into plastic tubes with 3.8% sodium citrate (citrate/blood: 1/9, v/v) for plasma
anticoagulation. Platelet-poor plasma (PPP) was separated from blood by
centrifugation at 3000 rpm for 10 min.
4.2.2. Plasma antithrombotic assay
TT, PT, APTT and FIB were examined with commercial kits following the
manufacturer’s instructions by a coagulometer (Model LG-PABER-I, Steellex Co.,
China). All the compounds were dissolved in 80% ethanol, and the concentration was
100 ꢀM. TT was determined by incubating 40 ꢀl PPP solution for 3 min at 37 °C,
followed by addition of 40 ꢀl thrombin solution and 20 ꢀl sample for 3 min at 37 °C.
PT was determined by incubating 40 ꢀl PPP solution for 3 min at 37 °C, followed by
addition of 40 ꢀl thromboplastin agent and 20 ꢀl sample. APTT was determined by
incubating 10 ꢀl sample solution and 50 ꢀl PPP solution with 50 ꢀl APTT-activating
agent for 3 min at 37 °C, followed by addition of 50 ꢀl CaCl2. FIB was determined
by incubating 10 ꢀl PPP solution with 90 ꢀl imidazole buffer for 3 min at 37 °C,
followed by addition of 50 ꢀl FIB agent and 10 ꢀl sample solution. The
antithrombotic activity was assessed by assaying the prolongation of the plasma
clotting time of TT, APTT, INR increasement of PT, and reduction of FIB content
.
4.3. Antioxidant assay
4.3.1. DPPH radical scavenging assay
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The DPPH radical scavenging activity was measured according to previous
studies [14]. The solution of the sample (100 µL) in dimethylsulfoxide (DMSO) was
added to 100 µL of DPPH radical in ethanol (0.2 mM) in 96-well plate. The sample
solution refers to the tested compounds and the reference antioxidants at various
concentrations, as well as DMSO as a control. The solutions of the tested compounds
had concentrations ranging from 5 µM to 500 µM. The reaction leading to the
scavenging of DPPH radical was completed within 30 min at 25 ºC. The absorbance
of the mixture was then measured at 517 nm with a microplate reader. The inhibition
of DPPH radical was expressed as percentage [14]: Scavenging
rate % = [1 − (A1 − A2)/A0] × 100%, where A1 was the absorbance of the test
substance acted with DPPH, A2 was the absorbance of the test substance without
DPPH and A0 was the absorbance of the blank. The IC50 value was defined as the
concentration of sample that causes 50% loss of the DPPH radical.
4.3.2. PC12 cells antioxidant assay
Cell viability was estimated by MTT assay. Briefly, PC12 cells were seeded in
96-well plates at a density of 5×104 cells/ml (100 ꢀl per well), then they were
cultured at 37 °C in humidified air containing 5% CO2 for 24 h. The experiment was
divided into control group and H2O2 damage model group, each set five wells. In the
control group was serum-free DMEM medium. The H2O2 damage model group was
exposure to 400 ꢀM H2O2 for 1 h before the test drugs were added in. Drug
concentrations were 50 ꢀM and 25 ꢀM. The cells were incubated with 20 ꢀl of MTT
solution for 4 h at 37 °C. The formazan crystals formed in intact cells were dissolved
in 150 ꢀl of DMSO, and then vigorously shaking for 10 min. Finally, the absorbance
was assessed at 517 nm. The inhibiting rate of H2O2-induced cytotoxicity in PC12
cells was calculated as inhibiting rate (%) = [(A1 − A2)/(A0 − A2)] × 100%, where A1
was the absorbance in the presence of sample and H2O2, A2 was the absorbance in the
presence of H2O2 (model), A0 was the absorbance without sample and H2O2 (normal).
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4.4. Physicochemical properties
4.4.1. Solubility determination
The solubility of scutellarein derivatives in water was determined using the
known method [31-33] with minor modifications. The UV/UV device is composed of
a UV source (for UV photodegradation) and a UV absorption detector (for on-line
UV measurement). The UV source is a low pressure mercury lamp and the UV
detector is a UV-vis spectrophotometer Anthelie (Secomam) controlled by software
Dathelie, version 4.1f. The pathlength of the Suprasil quartz cell is 10 mm and the
scan speed is 2000 nm min–1. Under the influence of UV radiation, methylated
scutellarein derivatives were monitored by UV absorption spectrophotometry at the
wavelength of maximum absorbance (334 nm) from the whole spectrum using a
multicomponent exploitation method. Each tested compound (250 µg) was dissolved
in 25 ml CH3OH. The solutions of the tested compounds had concentrations ranging
from 0.1 µg/ml to 10 µg/ml. Different concentration solutions of each compound
were determined by UV scanning, and the absorbances were obtained. The results
showed a good linear relationship, then all the standard curves were completed. Each
tested compound (150 µg) was ultrasound dissolved in 10 ml pure water for 1 h at
room temperature. The solutions were stranded for 30 min and centrifuged at the
speed of 30000 r/min. The aqueous solution of each compound was determined by
UV scanning, and the absorbances were obtained. Then through the analyzation of
standard curve, all the compounds in the water solubility were obtained.
4.4.2. Determination of partition coefficient
The partition coefficient (logP) between n-octanol and the buffer was
determined by a slight modification of the previous method [36]. Briefly, 100 ꢀl of a
stock solution (10 ꢀg/ml) was diluted with 1.9 ml of appropriate buffer solution
(0.1mol/L HCl, 0.15mol/L NaCl and 0.15mol/L NaHCO3 respectively) and mixed
with 2 ml of n-octanol (the organic and aqueous phase was mutually saturated), the
vials were protected from light by wrapping in aluminum foil. The two phases were
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vortexed for 3 min and agitated for 5 h in a shaking water bath at 25 ± 0.1 ºC. After
equilibration, the n-octanol phase was removed with a Pasteur pipette and both
phases were assayed spectrophotometrically (λmax = 334 nm) to determine drug
concentration. The partition coefficient was calculated as the ratio between molar
concentration in n-octanol and aqueous phase. The total concentration in both phases
was measured by spectrophotometry and the experimental partition coefficients (logP)
was calculated by the following equation: logP = log[C] n-octanol/[C] aqueous.
Acknowledgments
This work was supported by National Natural Science Foundation of China (No.
81274058, 21302225), China Scholarship Council (No. 201407060046), Natural
Science Foundation of Jiangsu Province (BK20151563), the Program for New
Century Excellent Talents by the Ministry of Education (NCET-12-0741), 333
High-level Talents Training Project Funded by Jiangsu Province, Six Talents Project
Funded by Jiangsu Province (2013-YY-010), Technology Innovation Venture Fund
by Nanjing University of Chinese Medicine (CX201301), Program for Excellent
Talents in School of Pharmacy of Nanjing University of Chinese Medicine
(15ZYXET-1), Jiangsu Collaborative Innovation Center of Chinese Medicinal
Resources Industrialization (ZDXMHT-1-13), Project Funded by the Priority
Academic Program Development of Jiangsu Higher Education Institutions and
Project Funded by the Flagship Major Development of Jiangsu Higher Education
Institutions (PPZY2015A070).
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