Synthesis of scutellarein derivatives to increase biological activity and water solubility

Article abstract of DOI:10.1016/j.bmc.2015.09.047

In order to improve the biological activity and water solubility of scutellarin (1), some derivatives of its main metabolite (scutellarein) were designed and synthesized. All the compounds were tested for their thrombin inhibition activity through the analyzation of thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) 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, their water solubility were also assessed by ultraviolet (UV) spectrophotometer. The results showed that compound 8b demonstrated stronger anticoagulant and antioxidant activity, better water solubility compared with scutellarein (2), which warrants it as a promising agent for the treatment of ischemic cerebrovascular disease.

Full text of DOI:10.1016/j.bmc.2015.09.047

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of scutellarein derivatives to increase biological activity
and water solubility
a
a
a
a,
Zhi-Hao Shi a,b, Nian-Guang Li a, , Qian-Ping Shi , Wei Zhang , Ze-Xi Dong , Yu-Ping Tang
⇑
⇑
,
Peng-Xuan Zhang a, Ting Gu a, Wen-Yu Wu a, Fang Fang a, Xin-Xue a, He-Min Li a, Jian-Ping Yang a,
Jin-Ao Duan a,
⇑
a Jiangsu Collaborative Innovation Center of Chinese Medicine Resources Industrialization, Jiangsu Key Laboratory for High Technology Research of TCM Formulae, and National
and Local Collaborative Engineering Center of Chinese Medicine Resources, Nanjing University of Chinese Medicine, Nanjing 210023, China
b Department of Organic Chemistry, China Pharmaceutical University, Nanjing, Jiangsu 211198, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to improve the biological activity and water solubility of scutellarin (1), some derivatives of its
main metabolite (scutellarein) were designed and synthesized. All the compounds were tested for their
thrombin inhibition activity through the analyzation of thrombin time (TT), activated partial thrombo-
plastin time (APTT), prothrombin time (PT) 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, their water solubility
were also assessed by ultraviolet (UV) spectrophotometer. The results showed that compound 8b demon-
strated stronger anticoagulant and antioxidant activity, better water solubility compared with scutel-
larein (2), which warrants it as a promising agent for the treatment of ischemic cerebrovascular disease.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 13 August 2015
Revised 28 September 2015
Accepted 30 September 2015
Available online xxxx
Keywords:
Scutellarin
Scutellarein
Thrombin
Antioxidant
Solubility
1. Introduction
an impairment of endothelial derived relaxing factor (EDRF, nitric
oxide)-mediated bioactions.7
As one of the leading causes of disability and death worldwide,1
ischemic cerebrovascular disease has been a common and fre-
quently-occurring disease that seriously endangers human health
nowadays. Firstly, the increasing evidence2 suggests that thrombin
play a critical role in ischemic cerebrovascular disease, it acts as a
multifunctional serine protease and catalyzes the proteolytic
cleavage of the soluble plasma-protein fibrinogen to form insoluble
fibrin, thus leading 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
Secondly, oxidative stresses are major causes of ischemic cere-
brovascular disease,4,5 some reactive oxygen species (ROS) includ-
ing the superoxide anion radical (Oꢀ2 Å), hydroxyl radical (ÅOH), nitric
oxide (NOÅ), hydrogen peroxide (H2O2) and singlet oxygen (1O2) are
constantly generated in the human body by various physiological
functions.6 These excessive ROS may result in increased levels of
low-density lipoprotein (LDL), oxidative modification of LDL, and
On this basis, some researchers have recently proposed and
searched some natural products with antithrombotic capacity
and antioxidant activity for the treatment of ischemic cerebrovas-
cular disease.8 Traditional Chinese medicines have been used clin-
ically 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. (Com-
positae), 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 attenu-
ate neuronal damage, thus had a wide range of benefits to brain
injury caused by cerebral ischemia/reperfusion.10–12
However, scutellarin (1) has low water-solubility,13 and the
bioavailability of scutellarin (1) was very low with the absolute
oral bioavailability in Beagle dog was rarely 0.4%.14 Interestingly,
some researchers found that scutellarin (1) was mainly absorbed
in the form of its hydrolyzed product scutellarein (2) (Fig. 1) by
⇑
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).
http://dx.doi.org/10.1016/j.bmc.2015.09.047
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Shi, Z.-H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.047

2
Z.-H. Shi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
OH
OH
OH
O
O
HO
O
O
O
HO
HO
O
O
HO
OH
HO
OH
OH
Scutellarein (2)
Scutellarin (1)
Figure 1. Chemical structures of scutellarin (1) and scutellarein (2).
intestinal,15 furthermore, in the clinical trials,14 a large amount of
scutellarein (2) was found in urine and plasma after oral adminis-
tration of breviscapine, indicating that scutellarin (1) was firstly
hydrolyzed into aglycone when reaching colon and was then
absorbed as scutellarein (2) as the real bioactive component in
the body. Pharmacodynamics confirmed that scutellarein (2) had
better protective effect than scutellarin (1) in rat cerebral
ischemia,16 scutellarein (2) can prevent thrombosis and platelet
aggregation, and improve the characteristics of hemorheology in
rats.17 In our previous studies, we found that scutellarein (2) had
stronger scavenging capacities toward DPPH, ABTS+Å, ÅOH free
radicals than scutellarin (1), and had better protective effect on
H2O2-induced cytotoxicity in PC12 cells.18,19 These results sug-
gested that scutellarein (2) can be a promising lead compound
for the discovery of potent agent with thrombin-inhibitory and
antioxidant activities for the treatment of ischemic cerebrovascu-
lar disease.
Amination of the substrates has considerable importance for
the synthesis and modification of biologically active compounds.20
This technique provides convenient access to many useful syn-
thetic building blocks, because the resulting amino group can be
easily converted to various functionalities, particularly, to quater-
nary ammonium salts to increase water solubility. In our previous
studies, we synthesized some scutellarein derivatives where the
amino side chain was introduced at the C-7 hydroxyl position.19
Herein, we describe the synthesis and in vitro biological evaluation
of scutellarein derivatives with electrophilic substitution at C-40,
together with a discussion of structure–activity relationships. The
thrombin inhibition activity of all the new derivatives were evalu-
ated through the analyzation of thrombin time (TT), activated par-
tial thromboplastin time (APTT), prothrombin time (PT) and
fibrinogen (FIB). The antioxidant activities of these target products
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, and the
at the room temperature, the benzophenone ketal group in 7a–7e
was removed to afford the target compounds 8a–8e.
Subsequently, we want to synthesize the other three series of
scutellarein derivatives 16a–16c, 21a–21c and 26a–26b based on
Scheme 1, however, it did not work, so these compounds were
obtained as shown in Scheme 2, considering that the different reac-
tion conditions could be used to remove benzyl and ketal protect-
ing groups. Firstly, benzyl bromide was used to protect the
hydroxyl group selectively at C-40 position in compound 3 to afford
9. Fortunately, the benzophenone ketal group was removed under
the hydrolysis condition with AcOH in H2O to give 10 in 96% yield.
Taken into account that the methoxymethyl and benzyl groups
could be deprotected by different reaction conditions, methoxy-
methyl group can be removed in acidic condition whilst benzyl
group is labile under hydrogenation, chloromethyl methyl ether
was then selected to protect the two ortho phenolic hydroxyl
groups at C-6 and C-7 positions in 10. Compound 10 reacted with
chloromethyl methyl ether and K2CO3 in acetone solution affording
11 with free phenolic hydroxyl group at C-5 position.25 The benzyl
group at C-40 position in 11 could be selectively removed under
hydrogenation condition, this method gave 12 in 91% yield with
the catalyst of 10% Pd/C in a mixed solution of EtOH and THF.
TLC analysis showed that this reaction was finished after 8 h and
no side product was detected. The reaction of 12 with RCl
14a–14c, 19a–19c and 24a–24b using K2CO3 and KI in DMF led
to 15a–15c, 20a–20c and 25a–25b, respectively. The side chains
14a–14c were obtained by the reaction of 4a–4c with chloroacetyl
chloride 13 in refluxing benzene. 19a–19c were produced from
18a–18c by SOCl2 catalyzed chlorination, which were obtained
after alkylation of 4a–4c and 2-chloro-1-ethanol (17), 24a–24b
were synthesized from 1-chloro-3-hydroxypropan (22) in the same
way of 19a–19c. Finally, HCl catalyzed removing of the methoxy-
methyl protecting group afforded the scutellarein derivatives
16a–16c, 21a–21c and 26a–26b in high yield.25
water
spectrophotometer.
solubility
were
assessed
by
ultraviolet
(UV)
2.2. Biological activity
2.2.1. Anti-thrombic activity
2. Results and discussion
2.1. Chemistry
Because the antithrombotic activity can be assessed by measur-
ing the prolongation of the plasma clotting time of thrombin time
(TT), activated partial thromboplastin time (APTT), INR increase-
ment of prothrombin time (PT), and reduction of fibrinogen (FIB)
content according to our previous studies,25 so the thrombin activ-
ity of different compounds was investigated for TT, APTT, PT 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 hydro-
lyzed 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.
The 40-O-amide-scutellarein derivatives 8a–8e were synthe-
sized as shown in Scheme 1. Under nitrogen protection, scutel-
larein (2) was obtained by refluxing scutellarin (1) with 6N HCl
in EtOH solution.20,21 Then at 175 °C and using diphenyl ether as
solvent according to our developed method,22–24 2 was treated
with dichlorodiphenylmethane to produce the desired compound
3 in high yield after reacting for 30 min. Subsequently, 3 was con-
verted into 7a–7e after the reaction with acyl chlorides 6a–6e
using K2CO3 and KI as the catalysts in N,N-dimethylformamide
(DMF).19 The side chains 6a–6e were synthesized from 4a–4e
and bis(trichloromethyl) carbonate (5). Under hydrogenation con-
ditions in THF/EtOH using the catalyst of 10% palladium on carbon
In the series of scutellarein derivatives 8a–8e, the most active
compound was 8b, it prolonged TT and PT, decreased FIB content
Please cite this article in press as: Shi, Z.-H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.047

Z.-H. Shi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
OH
OH
iii
OH
OH
ii
O
O
HO
HO
O
O
HO
HO
O
O
O
O
O
O
O
Ph
Ph
i
OH
HO
OH
OH
OH
2
3
1
O
O
v
O
R1
+
R1H
O
R1
Cl3CO
OCCl3
N
R1
Cl
6a-6e
4a-4e
5
HO
HO
O
O
O
O
O
O
O
Ph
Ph
iv
H3C
N
CH3
CH3
CH3
CH3
CH3
CH3
R1 = a:
b:
c:
d
e:
N
:
N
O
,
N
N CH3
OH
OH
O
,
,
,
7a-7e
8a-8e
H3C
Scheme 1. Reagents and conditions: (i) HCl, EtOH, N2, reflux, 36 h, 17%; (ii) dichlorodiphenylmethane, diphenyl ether, 175 °C, 30 min, 85%; (iii) 6a–6e, K2CO3, KI, DMF, 25 °C,
12 h, 78.3–86.2%; (iv) H2, 10% Pd/C, THF/EtOH, 8 h, 91.5–93.4%; (v) DMF, CCl4, reflux, 6 h.
OCH2Ph
ii
OCH2Ph
OCH2Ph
OH
i
O
O
HO
HO
O
O
MOMO
MOMO
O
O
O
O
O
O
O
O
Ph
Ph
Ph
Ph
iii
OH
10
OH
11
OH
OH
3
9
OR
OR
OH
MOMO
MOMO
O
O
HO
HO
O
MOMO
MOMO
O
O
O
Cl
vi
iv
v
Cl
R1
19a 19c
R1
Cl
RCl
=
R1
14a-14c
24a 24b
-
-
OH
OH
O
OH
12
CH3
CH3
CH3
15a-15c
20a-20c
25a-25b
16a-16c
21a-21c
26a-26b
a
R1
=
:
b
c
O
:
:
N
N
N
,
,
CH3
O
O
ix
OH
viii
OH
Cl
vii
+
R1H +
R1H
4a-4c
Cl
R1
18a-18c
R1
19a-19c
Cl
Cl
Cl
R1
14a 14c
17
4a-4c
13
-
viii
ix
R1H +
4a-4b
Cl
OH
R1
23a-23b
OH
R1
24a-24b
Cl
22
Scheme 2. Reagents and conditions: (i) PhCH2Br, K2CO3, DMF, 25 °C, 12 h, 93%; (ii) AcOH/H2O 4:1, reflux, 1.5 h, 96%; (iii) chloromethyl methyl ether (MOMCl), K2CO3, acetone,
reflux, 6 h, 89%; (iv) 10% Pd/C, H2, THF/EtOH, 8 h, 91%; (v) RCl, K2CO3, KI, DMF, 25 °C, 12 h, 61.8–68.1%; (vi) HCl, Et2O/CH2Cl2 1:1, 25 °C, 6 h, 83.8–89.3%; (vii) benzene, reflux,
1 h; (viii) toluene, reflux, 3 h; (ix) SOCl2, toluene, reflux, 2 h.
Table 1
8b was replaced by fatty amine groups such as dimethylamine
and diisopropylamine to produce 8a and 8d, all the four indicators
were shown to be less active than 8e, this result indicated that the
morpholine ring in the side chain was very important for the
antithrombotic activity.
In the series of scutellarein derivatives 16a–16c, in which there
was one more methylene group compared to 8a–8c, 16a showed
the same antithrombotic activity compared to 8a, 16a only
increased APTT and decreased FIB. 16b and 16c showed less
antithrombotic activity compared to 8b and 8c, respectively, three
indicators in 16b which were TT, APTT and PT were shown to be
less active than 8b, and no indicator in 16c showed stronger activ-
ity compared to 8c.
When the carbonyl group in 16a–16c was changed into methy-
lene group, three compounds 21a–21c were obtained, these com-
pounds showed equal antithrombotic activity to 16a–16c, 21a
and 21c only showed increased TT and APTT compared to 16a
and 16c, and 21b only showed increased APTT and decreased FIB
compared to 16b.
The thrombin inhibition activity of scutellarein derivatives
Compd (100
l
M)
Plasma coagulation parameters
TT (s)
APTT (s)
PT (s)
FIB (g/l)
1
2
8a
8b
8c
8d
8e
16a
16b
16c
21a
21b
21c
26a
26b
23.08 0.87
24.40 1.59
24.30 1.15
24.62 1.97
24.36 0.16
24.57 0.81
24.52 0.94
22.27 2.20
19.90 1.20
20.92 0.72
22.97 2.80
19.70 0.35
22.03 0.86
19.90 2.20
19.60 1.71
34.18 2.25
36.65 3.60
27.78 1.21
28.28 1.59
28.20 1.40
27.97 2.31
26.87 3.36
29.25 0.70
27.10 1.86
26.40 2.72
29.97 1.12
29.05 1.35
30.45 0.63
26.58 1.19
28.25 3.10
6.28 0.08
5.93 1.21
8.10 0.27
8.55 0.15
7.67 0.19
7.97 0.19
7.03 0.41
7.17 0.17
7.33 0.07
7.45 0.11
6.82 0.45
6.70 0.25
6.90 0.17
6.15 0.26
6.63 0.08
6.13 0.15
6.84 0.10
5.56 0.67
5.52 0.18
5.21 0.22
5.65 0.49
5.42 0.63
5.14 0.22
5.49 0.25
5.37 0.29
5.11 0.15
5.23 0.28
5.72 0.22
5.28 0.23
5.42 0.29
Data represent mean SD. n = 4.
When the carbon chain in 21a and 21b extended to produce 26a
and 26b, the antithrombotic activity decreased significantly, which
could be seen that no one indicator showed stronger activity com-
pared to 21a and 21b.
The above antithrombotic activity indicated that using formyl
group as a linker between the scutellarein ring and the side chain
compared to scutellarein (2). Interestingly, when the morpholine
ring in 8b was replaced by 4-methylpiperazine to afford 8e, the
antithrombotic activity decreased significantly, this result could
be seen that the TT, APTT and PT of 8e decreased compared with
those indicators in 8b. Furthermore, when the morpholine ring in
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Z.-H. Shi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
was very important to keep the antithrombotic activity, when the
formyl group was changed into acetyl, ethyl and propyl groups, the
antithrombotic activity decreased, respectively.
duce formazan, the amount of which directly related to the num-
ber of living cells. So the neuroprotective effects of the
synthesized scutellarein derivatives were evaluated by the ability
to protect PC12 cells against H2O2-induced cytotoxicity using
MTT assay method.18
The results of the inhibition rate against cell damage were sum-
marized in Table 2. Comparison of the data reported in Table 2
indicated compounds 8b, 8d and 8e showed stronger inhibitory
effect than scutellarein (2) against H2O2-induced cytotoxicity in
2.2.2. Antioxidants
The in vitro antioxidant activities26 of these scutellarein deriva-
tives were assessed by measuring their scavenging capacities
toward DPPH assay and the ability to protect PC12 cells against
H2O2-induced cytotoxicity.
PC12 cells, in a concentration of 50
94.25%, 93.36% and 92.48% inhibition rate against cell damage,
and in a concentration of 25 M, 8b, 8d and 8e showed 73.89%,
71.68% and 68.58% inhibition rate against cell damage, respec-
tively, while the inhibiting rates of scutellarein (2) were 91.15%
and 64.60% at 50 and 25 lM, respectively. The less active com-
pounds were 8c and 8a, these two compounds exhibited 75.22%
and 71.23% inhibition rate against cell damage in a concentration
lM, 8b, 8d and 8e showed
2.2.2.1. DPPH radical-scavenging activity.
DPPH assay27
measured the hydrogen-donating ability of antioxidants to convert
the stable DPPH free radical into 1,1-diphenyl-2-picrylhydrazine.
The reaction was accompanied by a change in color from deep-yel-
low 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 scutellarein derivatives
8a–8e were very active in the DPPH assay, with three compounds
8b, 8d and 8e showed stronger antioxidant activities compared to
l
of 50
lM, 51.33% and 48.67% inhibition rate against cell damage
in a concentration of 25
lM, respectively. The protective activity
scutellarin (1) (IC50 = 27.93
l
l
M) and scutellarein (2) (IC50 = 22.58 -
M), with their IC50s against DPPH were 19.27 M, 17.03 M and
M, respectively. In the series of 16a–16c, in which there
of 16a–16c against H2O2-induced cytotoxicity on PC12 cells
decreased, with their inhibition rates were below 70% in a concen-
l
l
18.87
l
tration of 50
lost its protective activity against cell damage with its inhibition
rate below 60% in a concentration of 50 M and below 40% in a
concentration of 25 M.
lM. Unfortunately, compounds 21a–21c and 26a–26b
was one more methylene group compared to 8a–8c, the antioxi-
dant activity decreased significantly, with their IC50s were
l
80.70
lM, 83.26
lM and 81.28
l
M, respectively. In the two series
l
of 21a–16c and 26a–26b, in which there was no carbonyl group
in the amide side chain, the antioxidant activity was very low with
their IC50s were above 200 lM.
These antioxidant activities showed the similar structure–activ-
ity relationship compared to the antithrombotic biology, for exam-
ple, the presence of the methanamide group in the side chain was
very important to protect the PC12 cells against H2O2-induced
oxidative injury, while the introduction of ethanamide, ethylamine
and propylamine groups would decrease the protective activity
against H2O2-induced cytotoxicity on PC12 cells.
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.28 Active mitochondria of living cells can cleave MTT to pro-
2.2.3. Solubility
Table 2
The in vitro antioxidatant activity in DPPH assay, protective effect against H2O2-
induced cytotoxicity in PC12 cells, and water solubility of the scutellarein derivatives
The aqueous solubility of the synthesized scutellarein deriva-
tives was determined using UV spectrophotometer.29–31 As pre-
sented in Table 2, the introduction of morpholinyl ring showed
the best water solubility among the four series of scutellarein
derivatives, the values of 8b, 16b, 21b and 26b in water solubility
Compd DPPH assay
IC50 M)
PC12 cells assay
Concentrations Inhibiting
Water solubility
(l
Solubility
g/ml)
Fold
of compd (
lM)
rate (%)
(
l
increasea
were 15.34 lg/ml, 9.45 lg/ml, 9.40 lg/ml and 8.56 lg/ml, and
1
27.93
22.58
37.17
19.27
32.60
17.03
18.87
80.70
83.26
81.28
214.8
263.6
205.1
231.1
274.4
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
50
25
79.20
53.10
91.15
64.60
71.23
48.67
94.25
73.89
75.22
51.33
93.36
71.68
92.48
68.58
68.14
46.46
64.60
43.36
66.37
44.69
56.19
35.84
48.23
32.30
58.41
38.50
52.21
33.63
44.25
28.32
7.62
6.85
9.56
showed 2.34-fold, 1.38-fold, 1.37-fold and 1.25-fold compared to
scutellarein (2) respectively. Furthermore, the introduction of
methyl piperazinyl ring in 8e also showed remarkable increase in
2
1.00
1.40
2.34
1.35
1.27
2.20
1.36
1.38
1.31
1.35
1.37
1.27
1.24
1.25
8a
solubility with its value was 15.06 lg/ml and exhibited 2.20-fold.
The replacement of alicyclic amine by the aliphatic amine showed
good water solubility, such as the dimethyl amine substituted
derivative 8a, 16a, 21a and 26a, their values in water solubility
8b
15.34
9.23
8.67
15.06
9.32
9.45
8.95
9.28
9.40
8.68
8.50
8.56
8c
were 9.56 lg/ml, 9.32 lg/ml, 9.28 lg/ml and 8.50 lg/ml, and only
8d
showed 1.40-fold, 1.36-fold, 1.35-fold and 1.24-fold compared to
scutellarein (2), respectively. These results indicated that the intro-
duction of the alicyclic amine such as the morpholinyl ring and the
methyl piperazinyl ring would increase the aqueous solubility of
the synthesized scutellarein derivatives.
8e
16a
16b
16c
21a
21b
21c
26a
26b
3. Conclusion
Scutellarin (1) has been used clinically to treat acute cerebral
infarction and paralysis induced by cerebrovascular diseases such
as hypertension, cerebral thrombosis, cerebral haemorrhage for
many years in China,9 it has showed the effectiveness on dilating
blood vessels, improving microcirculation, increasing cerebral
blood flow, and inhibiting platelet aggregation.32 However, scutel-
larin (1) has pharmacokinetic problems such as low solubility in
water and fast metabolism which results in its low bioavailability.
Amination of the substrates has considerable importance for the
a
Fold increase in water solubility relative to scutellarein (2).
Please cite this article in press as: Shi, Z.-H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.047

Z.-H. Shi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
synthesis and modification of biologically active compounds,20
because the resulting amino group can be easily converted to qua-
ternary ammonium salts to increase water solubility. In this study,
we took scutellarein (2) as a promising lead compound, which is
one major metabolite of scutellarin (1) in vivo, designed and syn-
thesized new scutellarein derivatives with electrophilic substitu-
tion at C-40, to improve biological activities of scutellarein (2). In
particular, compound 8b, which there was a morpholine ring in
the side chain at the C-40 position, demonstrated stronger antico-
agulant and antioxidant activity, better water solubility compared
with scutellarein (2), which warrants it as a promising agent for
the treatment of ischemic cerebrovascular disease.
solid appeared was filtered, and then purified by column chro-
matography on silica gel with 25% ethyl acetate in petroleum ether
as eluent to afford 3 (13.35 g, 85%) as a yellow solid, mp 284–
286 °C. 1H NMR (DMSO-d6, 300 MHz) d: 6.69 (s, 1H, C3-H), 6.82
0
0
(s, 1H, C8-H), 6.96 (d, J = 8.7 Hz, 2H, C3 C5 -H), 7.47–7.50 (m, 6H,
0
0
ArH), 7.58–7.61 (m, 4H, ArH), 7.94 (d, J = 8.7 Hz, 2H, C2 C6 -H),
10.41 (s, 1H, C4 -OH), 12.99 (s, 1H, C5-OH). 13C NMR (75 MHz,
0
DMSO-d6) d: 94.50 (C8), 103.32 (C3), 104.93 (C10), 116.45 (2 ꢁ C
(C30,C50)), 119.16 (C), 121.21 (C10), 126.29 (C6), 126.34 (2 ꢁ C(C20,
C60)), 126.50 (2 ꢁ C), 129.05 (4 ꢁ C), 129.14 (4 ꢁ C), 139.26
(2 ꢁ C), 141.94 (C9), 152.77 (C5), 152.95 (C7), 161.80 (C40),
164.00 (C2), 182.32 (C4). ESI-MS: m/z 449 [MꢀH]ꢀ.
4.2.3. Synthesis of 7a–7e
4. Experimental
DMF (7.73 ml, 0.1 mol) was added into a solution of imine (4a–
4c) (1 mol) in CCl4 (500 ml) at 25 °C, 30 min later, a solution of
triphosgene (119.1 g, 0.4 mol) in CCl4 (200 ml) was added drop-
wise. This mixture was refluxed gently for 6 h and then allowed
to cool to room temperature. After concentration under reduced
pressure, the crude residue was purified by vacuum distillation
to afford the imino chlorides (6a–6e) which were then used
directly in the next step.
Compound 3 (100 mg, 0.27 mmol) was added into anhydrous
DMF (20 mL) at 0 °C, treated with K2CO3 (44 mg, 0.32 mmol) and
KI (11 mg, 0.07 mmol) followed by imino chlorides (6a–6e)
(0.32 mmol), then the reaction mixture was warmed to room tem-
perature. After 12 h, the mixture was poured into water (50 mL),
and then extracted with ethyl acetate (50 mL ꢁ 3), the organic
layer was washed with brine (100 mL), dried over Na2SO4, filtered
and concentrated under reduced pressure. The appeared crude
material was then purified by column chromatography on silica
gel using 50% ethyl acetate in petroleum ether as eluent to afford
7a–7e as yellow solids.
4.1. General methods
Scutellarin (1) was purchased from Sichuan Mianning Jiexiang
Materials Co. Ltd (Chengdu, China). Reagents and solvents were
purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai,
China). All non-aqueous reactions were carried out under nitrogen
protection using flame-dried glassware, the anhydrous solvents
were transferred via syringe or stainless steel cannula. Organic sol-
vents were concentrated below 45 °C by Büchi rotary evaporator at
about 20 mm Hg. Using UV light of 254 nm as the detection wave-
length, all reactions were monitored by TLC, performed on 0.15–
0.20 mm silica gel plates. Chromatographic separation was carried
out on silica gel (160–200 mesh) using mixtures of petroleum
ether (60–90) and ethyl acetate as eluent. The melting points
(Mp) were measured on a WRS-1B apparatus and were not cor-
rected. The 1H NMR and 13C NMR spectra were carried out on Bru-
ker AV-300 (300 MHz) in DMSO-d6, chemical shifts were expressed
as d values in ppm relative to tetramethylsilane as standard, and
peak abbreviations were expressed as follows: s, singlet; d,
doublet; m, multiplet. Mass spectra (MS) were performed on
Waters Synapt HDMS spectrometer equipped with an electrospray
ionization source (ESI).
4.2.3.1. 4-(9-Hydroxy-8-oxo-2,2-diphenyl-8H-[1,3]dioxolo[4,5-g]
chromen-6-yl)phenyl dimethylcarbamate (7a). 78.3% yield, yel-
low solid, mp 211–213 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.94 (s,
3H, CH3), 3.07 (s, 3H, CH3), 7.04 (s, 1H, C3-H), 7.12 (s, 1H, C8-H),
7.33–7.38 (m, 4H, ArH), 7.47–7.59 (m, 10H, ArH), 8.11 (d,
J = 8.7 Hz, 2H, C2 C6 -H), 12.98 (s, 1H, C5-OH). 13C NMR (75 MHz,
DMSO-d6) d: 39.05 (2 ꢁ C(CH3)), 94.52 (C8), 103.31 (C3), 104.95
(C10), 119.15 (C), 121.46 (2 ꢁ C(C30,C50)), 126.21 (C10), 126.30
(C6), 126.49 (2 ꢁ C), 128.33 (2 ꢁ C(C20,C60)), 129.04 (4 ꢁ C), 129.13
(4 ꢁ C), 139.27 (2 ꢁ C), 141.95 (C9), 152.76 (C5), 152.94 (C7),
155.06 (CO), 155.77 (C40), 164.01 (C2), 182.33 (C4). ESI-MS: m/z
522 [M+H]+.
4.2. Synthesis of scutellarein derivatives
0
0
4.2.1. 5,6,7-Trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-
4-one (2)
Concentrated HCl (120 mL) was added into a stirring mixture of
1 (10 g, 21.65 mmol) and water (10 mL) in ethanol (120 mL), the
reaction mixture was refluxed for 36 h under nitrogen, and then
was allowed to cool to room temperature. Water (1000 mL) was
added, the solid appeared was filtered, and then purified by col-
umn chromatography on silica gel with 50% ethyl acetate in petro-
leum ether as eluent to afford 2 (1.05 g, 17% yield) as a yellow solid,
mp 160–162 °C. 1H NMR (DMSO-d6, 300 MHz) d: 6.57 (s, 1H, C3-H),
4.2.3.2. 4-(9-Hydroxy-8-oxo-2,2-diphenyl-8H-[1,3]dioxolo[4,5-g]-
chromen-6-yl)phenyl morpholine-4-carboxylate (7b).
85.4%
yield, yellow solid, mp 193–195 °C. 1H NMR (300 MHz, DMSO-d6)
d: 3.12–3.15 (m, 4H, morpholine CH2NCH2), 3.54–3.57 (m, 4H, mor-
pholine CH2OCH2), 7.04 (s, 1H, C3-H), 7.13 (s, 1H, C8-H), 7.38 (d,
0
0
6.74 (s, 1H, C8-H), 6.91 (d, J = 8.8 Hz, 2H, C3 C5 -H), 7.90 (d,
0
0
J = 8.8 Hz, 2H, C2 C6 -H), 8.71 (s, 1H, C6-OH), 10.29 (s, 1H, C7-OH),
0
0
J = 8.7 Hz, 2H, C3 C5 -H), 7.45–7.49 (m, 6H, ArH), 7.54–7.58 (m, 4H,
10.44 (s, 1H, C4 -OH), 12.78 (s, 1H, C5-OH). 13C NMR (75 MHz,
0
ArH), 8.11 (d, 2H, J = 8.7 Hz, C2 C6 -H), 12.97 (s, 1H, C5-OH). 13C
NMR (75 MHz, DMSO-d6) d: 47.96 (2 ꢁ C(CH2)), 67.11 (2 ꢁ C(CH2)),
94.53 (C8), 103.30 (C3), 104.94 (C10), 119.16 (C), 121.45 (2 ꢁ C
(C30,C50)), 126.22 (C10), 126.31 (C6), 126.48 (2 ꢁ C), 128.32 (2 ꢁ C
(C20,C60)), 129.05 (4 ꢁ C), 129.14 (4 ꢁ C), 139.26 (2 ꢁ C), 141.96
(C9), 152.77 (C5), 152.93 (C7), 155.14 (CO), 155.75 (C40), 164.00
(C2), 182.31 (C4). ESI-MS: m/z 564 [M+H]+.
0
0
DMSO-d6) d: 98.53 (C8), 102.27 (C3), 103.22 (C10), 115.76 (2 ꢁ C
(C30,C50)), 121.37 (C10), 124.91 (C6), 128.47 (2 ꢁ C(C20,C60)),
145.42 (C9), 152.98 (C5), 153.28 (C7), 160.99 (C40), 163.47 (C2),
181.95 (C4). ESI-MS: m/z 287 [M+H]+.
4.2.2. 9-Hydroxy-6-(4-hydroxyphenyl)-2,2-diphenyl-8H-1,3-
dioxolo[4,5-g][1]benzopyran-8-one (3)
Scutellarein (2) (10 g, 34.94 mmol) was added into a solution of
dichlorodiphenylmethane (12.4 g, 52.29 mmol, 1.5 equiv) in diphe-
nyl ether (200 mL), the reaction mixture was heated at 175 °C
under nitrogen, and then was allowed to cool to room temperature
after 30 min. Petroleum ether (1000 mL) was added dropwise, the
4.2.3.3. 4-(9-Hydroxy-8-oxo-2,2-diphenyl-8H-[1,3]dioxolo[4,5-
g]chromen-6-yl)phenyl diethylcarbamate (7c). 82.1% yield,
yellow solid, mp 196–198 °C. 1H NMR (300 MHz, DMSO-d6) d:
1.18–1.21 (t, 6H, 2 ꢁ CH3), 3.32–3.34 (m, 4H, 2 ꢁ CH2), 7.03 (s,
0
0
1H, C3-H), 7.12 (s, 1H, C8-H), 7.33 (d, J = 8.7 Hz, 2H, C3 C5 -H),
Please cite this article in press as: Shi, Z.-H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.047

6
Z.-H. Shi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7.46–7.49 (m, 6H, ArH), 7.54–7.58 (m, 4H, ArH), 8.11 (d, J = 8.7 Hz,
(2 ꢁ C(CH2)), 98.53 (C8), 102.26 (C3), 103.24 (C10), 121.65 (2 ꢁ C
(C30,C50)), 124.93 (C6), 127.02 (C10), 128.44 (2 ꢁ C(C20,C60)),
145.44 (C9), 153.28 (C7), 154.82 (C40), 154.88 (CO), 155.97 (C5),
163.41 (C2), 181.96 (C4). ESI-MS: m/z 400 [M+H]+.
2H, C2 C6 -H), 12.98 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d:
12.98 (2 ꢁ C(CH3)), 42.76 (2 ꢁ C(CH2)), 94.52 (C8), 103.29 (C3),
104.93 (C10), 119.15 (C), 121.44 (2 ꢁ C(C30,C50)), 126.23 (C10),
126.33 (C6), 126.47 (2 ꢁ C), 128.31 (2 ꢁ C(C20,C60)), 129.04
(4 ꢁ C), 129.15 (4 ꢁ C), 139.27 (2 ꢁ C), 141.95 (C9), 152.76 (C5),
152.94 (C7), 155.02 (CO), 155.76 (C40), 164.01 (C2), 182.32 (C4).
ESI-MS: m/z 550 [M+H]+.
0
0
4.2.4.3.
4-(5,6,7-Trihydroxy-4-oxo-4H-chromen-2-yl)phenyl
diethylcarbamate (8c). 93.4% yield, yellow solid, mp 115–
117 °C. 1H NMR (300 MHz, DMSO-d6) d: 1.21–1.24 (t, 6H, 2 ꢁ CH3),
3.41–3.44 (m, 4H, 2 ꢁ CH2), 6.61 (s, 1H, C3-H), 6.90 (s, 1H, C8-H),
0
0
0
0
4.2.3.4. 4-(9-Hydroxy-8-oxo-2,2-diphenyl-8H-[1,3]dioxolo[4,5-g]
chromen-6-yl)phenyl diisopropylcarbamate (7d). 86.2% yield,
yellow solid, mp 205–207 °C. 1H NMR (300 MHz, DMSO-d6) d:
1.26 (d, 12H, 4 ꢁ CH3), 3.96–4.05 (m, 2H, CH), 7.03 (s, 1H, C3-H),
7.32 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.09 (d, J = 8.7 Hz, 2H, C2 C6 -H),
8.79 (s, 1H, C6-OH), 10.51 (s, 1H, C7-OH), 12.65 (s, 1H, C5-OH). 13C
NMR (75 MHz, DMSO-d6) d: 12.96 (2 ꢁ C(CH3)), 42.78 (2 ꢁ C
(CH2)), 98.55 (C8), 102.25 (C3), 103.23 (C10), 121.67 (2 ꢁ C(C30,
C50)), 124.91 (C6), 127.01 (C10), 128.46 (2 ꢁ C(C20,C60)), 145.44
(C9), 153.28 (C7), 154.84 (C40), 155.00 (CO), 155.98 (C5), 163.42
(C2), 181.95 (C4). ESI-MS: m/z 386 [M+H]+.
0
0
7.13 (s, 1H, C8-H), 7.33 (d, J = 8.7 Hz, 2H, C3 C5 -H), 7.44–7.49 (m,
0
0
6H, ArH), 7.53–7.58 (m, 4H, ArH), 8.09 (d, J = 8.7 Hz, 2H, C2 C6 -H),
12.98 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 21.16 (4 ꢁ C
(CH3)), 46.94 (2 ꢁ C(CH)), 94.50 (C8), 103.31 (C3), 104.94 (C10),
119.16 (C), 121.46 (2 ꢁ C(C30,C50)), 126.25 (C10), 126.35 (C6),
126.48 (2 ꢁ C), 128.32 (2 ꢁ C(C20,C60)), 129.05 (4 ꢁ C), 129.16
(4 ꢁ C), 139.28 (2 ꢁ C), 141.97 (C9), 152.77 (C5), 152.93 (C7),
155.03 (CO), 155.77 (C40), 164.02 (C2), 182.34 (C4). ESI-MS: m/z
578 [M+H]+.
4.2.4.4.
4-(5,6,7-Trihydroxy-4-oxo-4H-chromen-2-yl)phenyl
diisopropylcarbamate (8d). 93.1% yield, yellow solid, mp 243–
245 °C. 1H NMR (300 MHz, DMSO-d6) d: 1.26 (d, 12H, 4 ꢁ CH3),
3.96–4.05 (m, 2H, 2 ꢁ CH), 6.61 (s, 1H, C3-H), 6.90 (s, 1H, C8-H),
0
0
0
0
7.30 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.17 (d, J = 8.7 Hz, 2H, C2 C6 -H),
8.80 (s, 1H, C6-OH), 10.53 (s, 1H, C7-OH), 12.65 (s, 1H, C5-OH).
13C NMR (75 MHz, DMSO-d6) d: 21.18 (4 ꢁ C(CH3)), 46.96 (2 ꢁ C
(CH)), 98.53 (C8), 102.25 (C3), 103.23 (C10), 121.66 (2 ꢁ C(C30,
C50)), 124.91 (C6), 127.02 (C10), 128.45 (2 ꢁ C(C20,C60)), 145.44
(C9), 153.29 (C7), 154.82 (C40), 155.01 (CO), 155.99 (C5), 163.41
(C2), 181.95 (C4). ESI-MS: m/z 414 [M+H]+.
4.2.3.5. 4-(9-Hydroxy-8-oxo-2,2-diphenyl-8H-[1,3]dioxolo[4,5-g]-
chromen-6-yl)phenyl 4-methylpiperazine-1-carboxylate (7e).
82.9% yield, yellow solid, mp 217–219 °C. 1H NMR (300 MHz,
DMSO-d6) d: 3.12–3.15 (t, 4H, 2 ꢁ CH2), 3.54–3.57 (t, 4H,
2 ꢁ CH2), 3.67 (s, 3H, CH3), 7.04 (s, 1H, C3-H), 7.13 (s, 1H, C8-H),
0
0
7.38 (d, J = 8.7 Hz, 2H, C3 C5 -H), 7.44–7.49 (m, 6H, ArH), 7.53–
0
0
7.58 (m, 4H, ArH), 8.11 (d, J = 8.7 Hz, 2H, C2 C6 -H), 12.97 (s, 1H,
C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 44.56 (C(CH3)), 48.95
(2 ꢁ C(CH2)), 55.11 (2 ꢁ C(CH2)), 94.49 (C8), 103.30 (C3), 104.93
(C10), 119.15 (C), 121.44 (2 ꢁ C(C30,C50)), 126.24 (C10), 126.32
(C6), 126.45 (2 ꢁ C), 128.30 (2 ꢁ C(C20,C60)), 129.04 (4 ꢁ C),
129.15 (4 ꢁ C), 139.27 (2 ꢁ C), 141.98 (C9), 152.75 (C5), 152.92
(C7), 154.86 (CO), 155.78 (C40), 164.03 (C2), 182.31 (C4). ESI-MS:
m/z 577 [M+H]+.
4.2.4.5. 4-(5,6,7-Trihydroxy-4-oxo-4H-chromen-2-yl)phenyl 4-
methylpiperazine-1-carboxylate (8e). 92.8% yield, yellow solid,
mp 152–154 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.24 (s, 3H, CH3),
2.36–2.39 (t, 4H, 2 ꢁ CH2), 3.42–2.45 (t, 4H, 2 ꢁ CH2), 6.28 (s, 1H,
0
0
C3-H), 6.91 (s, 1H, C8-H), 7.35 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.18 (d,
0
0
J = 8.7 Hz, 2H, C2 C6 -H), 8.77 (s, 1H, C6-OH), 10.50 (s, 1H, C7-OH),
12.25 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 44.57 (C
(CH3)), 48.98 (2 ꢁ C(CH2)), 55.12 (2 ꢁ C(CH2)), 98.54 (C8), 102.25
(C3), 103.23 (C10), 121.66 (2 ꢁ C(C30,C50)), 124.94 (C6), 127.01
(C10), 128.43 (2 ꢁ C(C20,C60)), 145.45 (C9), 153.29 (C7), 154.82
(C40), 154.89 (CO), 155.97 (C5), 163.42 (C2), 181.96 (C4). ESI-MS:
m/z 413 [M+H]+.
4.2.4. Synthesis of 8a–8e
Compound 7a–7e (100 mg) was stirred with 10% Pd/C catalyst
(2 mg) in EtOH (50 mL) and THF (50 mL) for 8 h under hydrogen
at atmospheric pressure, then the Pd/C catalyst was removed by
filtration over celite. After the filtrate was evaporated, the crude
material was purified by column chromatography on silica gel
using 50% ethyl acetate in petroleum ether as eluent to afford
8a–8e as yellow solids.
4.2.5. 6-(4-(Benzyloxy)phenyl)-9-hydroxy-2,2-diphenyl-8H-
[1,3]dioxolo[4,5-g]chromen-8-one (9)
K2CO3 (107 mg, 0.77 mmol, 1.75 equiv) was added to a solution
of 3 (200 mg, 0.44 mmol) in DMF (10 mL) followed by the addition
of benzyl bromide (0.078 mL, 0.66 mmol, 1.5 equiv). This mixture
was stirred at 0 °C for 2 h and then was warmed to room temper-
ature. After 12 h, the mixture was dissolved in water (100 mL),
extracted with ethyl acetate (50 mL ꢁ 3), then the organic layer
was washed with brine (100 mL), dried over Na2SO4, and concen-
trated under reduced pressure to give the crude solid, which was
then purified by column chromatography on silica gel using 33%
ethyl acetate in petroleum ether as eluent to afford 9 (221 mg,
0.41 mmol, 93% yield) as a yellow solid, mp 226–228 °C. 1H NMR
(300 MHz, DMSO-d6) d: 5.22 (s, 2H, CH2), 6.95 (s, 1H, C3-H), 7.08
4.2.4.1.
4-(5,6,7-Trihydroxy-4-oxo-4H-chromen-2-yl)phenyl
dimethylcarbamate (8a). 92.3% yield, yellow solid, mp 263–
265 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.93 (s, 3H, CH3), 3.06 (s,
3H, CH3), 6.61 (s, 1H, C3-H), 6.90 (s, 1H, C8-H), 7.32 (d, J = 8.7 Hz,
2H, C3 C5 -H), 8.07 (d, J = 8.7 Hz, 2H, C2 C6 -H), 8.78 (s, 1H, C6-OH),
10.53 (s, 1H, C7-OH), 12.65 (s, 1H, C5-OH). 13C NMR (75 MHz,
DMSO-d6) d: 39.01 (2 ꢁ C(CH3)), 98.54 (C8), 102.26 (C3), 103.23
(C10), 121.66 (2 ꢁ C(C30,C50)), 124.92 (C6), 127.01 (C10), 128.45
(2 ꢁ C(C20,C60)), 145.43 (C9), 153.29 (C7), 154.83 (C40), 155.01
(CO), 155.98 (C5), 163.42 (C2), 181.96 (C4). ESI-MS: m/z 358 [M
+H]+.
0
0
0
0
0
0
(s, 1H, C8-H), 7.22 (d, J = 8.7 Hz, 2H, C3 C5 -H), 7.32–7.48 (m, 11H,
0
0
ArH), 7.55–7.61 (m, 4H, ArH), 8.04 (d, J = 8.7 Hz, 2H, C2 C6 -H),
13.10 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 71.47 (C
(CH2)), 94.52 (C8), 103.33 (C3), 104.94 (C10), 114.83 (2 ꢁ C(C30,
C50)), 119.17 (C), 119.92 (C10), 125.95 (2 ꢁ C(C20,C60)), 126.30
(C6), 126.51 (2 ꢁ C), 127.15 (2 ꢁ C), 127.82 (C), 129.01 (2 ꢁ C),
129.06 (4 ꢁ C), 129.15 (4 ꢁ C), 139.27 (2 ꢁ C), 141.28 (C), 141.96
(C9), 152.79 (C5), 152.94 (C7), 163.99 (C40), 164.02 (C2), 182.34
(C4). ESI-MS: m/z 541 [M+H]+.
4.2.4.2. 4-(5,6,7-Trihydroxy-4-oxo-4H-chromen-2-yl)phenyl mor-
pholine-4-carboxylate (8b). 91.5% yield, yellow solid, mp 234–
235 °C. 1H NMR (300 MHz, DMSO-d6) d: 3.43–3.46 (m, 4H,
2 ꢁ CH2), 3.63–3.66 (m, 4H, 2 ꢁ CH2), 6.62 (s, 1H, C3-H), 6.92 (s,
0
0
1H, C8-H), 7.37 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.09 (d, J = 8.7 Hz, 2H,
0
0
C2 C6 -H), 8.78 (s, 1H, C6-OH), 10.54 (s, 1H, C7-OH), 12.65 (s, 1H,
C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 47.98 (2 ꢁ C(CH2)), 67.13
Please cite this article in press as: Shi, Z.-H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.047

Z.-H. Shi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
4.2.6. 2-(4-(Benzyloxy)phenyl)-5,6,7-trihydroxy-4H-chromen-4-
one (10)
10 °C. 30 min later, this mixture was refluxed gently for 1 h and
then allowed to cool to room temperature. The organic layer was
washed with brine (10 mL), dried over Na2SO4, filtered and concen-
trated under reduced pressure. The crude residue was purified by
vacuum distillation to afford the imino chlorides (14a–14c) which
were then used directly in the next step.
2-Chloro-1-ethanol (17) (0.05 mol, 3.4 ml) was added dropwise
into the solution of imine (4a–4c) (0.1 mol) in toluene (20 ml) at
25 °C, this mixture was refluxed gently for 3 h and then allowed
to cool to room temperature. The mixture was filtered, washed
with toluene. Then SOCl2 (10 ml) was added into the filtrate, this
mixture was refluxed gently for 2 h, and concentrated under
reduced pressure. The residue was recrystallized from anhydrous
alcohol to afford 19a–19c as white solids which were then used
directly in the next step. 24a–24b were synthesized from 1-
chloro-3-hydroxypropan (22) and imine (4a–4c) in the same way
of 19a–19c.
Compound 12 (100 mg, 0.27 mmol) was added into anhydrous
DMF (20 mL) at 0 °C, treated with K2CO3 (44 mg, 0.32 mmol) and
KI (11 mg, 0.07 mmol) followed by 14a–14c, 19a–19c and 24a–
24b (0.32 mmol), then the reaction mixture was warmed to room
temperature. After 12 h, the mixture was poured into water
(50 mL), and then extracted with ethyl acetate (50 mL ꢁ 3), the
organic layer was washed with brine (100 mL), dried over Na2SO4,
filtered and concentrated under reduced pressure. The appeared
crude material was then purified by column chromatography on
silica gel using 50% ethyl acetate in petroleum ether as eluent to
afford 15a–15c, 20a–20c and 25a–25b as yellow solids.
Compound 9 (150 mg, 0.28 mmol) was added to a solution of
water (4 mL) in AcOH (16 mL), this solution was refluxed for
1.5 h under nitrogen and then allowed to cool. The mixture was
poured into water (100 mL), extracted with ethyl acetate
(50 mL ꢁ 3), then the organic layer was washed with brine
(100 mL), dried over Na2SO4, filtered and concentrated under
reduced pressure. The obtained crude material was purified by col-
umn chromatography on silica gel using 25% ethyl acetate in pet-
roleum ether as eluent to afford 10 (100 mg, 0.27 mmol, 96%
yield) as a yellow solid, mp 234–236 °C. 1H NMR (300 MHz,
DMSO-d6) d: 5.28 (s, 2H, CH2), 6.81 (s, 1H, C3-H), 6.92 (d, 2H,
0
0
J = 8.7 Hz, C3 C5 -H), 7.00 (s, 1H, C8-H), 7.35–7.45 (m, 3H, ArH),
0
0
7.51–7.54 (m, 2H, ArH), 7.94 (d, 2H, J = 8.7 Hz, C2 C6 -H), 8.74 (s,
1H, C6-OH), 10.35 (s, 1H, C7-OH), 12.69 (s, 1H, C5-OH). 13C NMR
(75 MHz, DMSO-d6) d: 71.46 (C(CH2)), 98.54 (C8), 102.28 (C3),
103.24 (C10), 114.86 (2 ꢁ C(C30,C50)), 119.95 (C10), 124.93 (C6),
125.97 (2 ꢁ C(C20,C60)), 127.14 (2 ꢁ C), 127.83 (C), 129.02 (2 ꢁ C),
141.28 (C), 145.43 (C9), 152.98 (C5), 153.27 (C7), 163.47 (C2),
163.97 (C40), 181.96 (C4). ESI-MS: m/z 375 [MꢀH]ꢀ.
4.2.7. 2-(4-(Benzyloxy)phenyl)-5-hydroxy-6,7-bis(methoxymeth-
oxy)-4H-chromen-4-one (11)
Chloromethyl methyl ether (0.082 mL, 1.08 mmol) was added
into a solution of 10 (100 mg, 0.27 mmol) in dry acetone (20 mL)
followed by the addition of K2CO3 (156 mg, 1.13 mmol). This mix-
ture was refluxed gently for 6 h and then allowed to cool to room
temperature. The reaction mixture was filtered, washed with ace-
tone, and then concentrated under reduced pressure. The crude
residue was purified by column chromatography on silica gel using
20% ethyl acetate in petroleum ether as eluent to afford 11
(112 mg, 0.24 mmol, 89%) as a yellow solid, mp 174–176 °C. 1H
NMR (300 MHz, DMSO-d6) d: 3.45 (s, 3H, CH3), 3.54 (s, 3H, CH3),
5.17 (s, 2H, CH2), 5.24 (s, 2H, CH2), 5.37 (s, 2H, CH2), 6.62 (s, 1H,
4.2.9.1. 2-(4-(5-Hydroxy-6,7-bis(methoxymethoxy)-4-oxo-4H-
chromen-2-yl)phenoxy)-N,N-dimethylacetamide (15a).
68.1% yield, yellow solid, mp 186–187 °C. 1H NMR (300 MHz,
DMSO-d6) d: 3.01 (s, 6H, 2 ꢁ CH3), 3.31 (s, 3H, CH3), 3.54 (s, 3H,
CH3), 4.94 (s, 2H, CH2), 5.18 (s, 2H, CH2), 5.38 (s, 2H, CH2), 6.62
0
0
(s, 1H, C3-H), 6.96 (s, 1H, C8-H), 7.10 (d, J = 8.7 Hz, 2H, C3 C5 -H),
C3-H), 7.00 (s, 1H, C8-H), 7.23 (d, J = 8.7 Hz, 2H, C3 C5 -H), 7.34–
8.06 (d, J = 8.7 Hz, 2H, C2 C6 -H), 12.70 (s, 1H, C5-OH). 13C NMR
(75 MHz, DMSO-d6) d: 38.13 (2 ꢁ C(CH3)), 56.22 (C(CH3)), 56.37
(C(CH3)), 64.22 (C(CH2)), 94.16 (C(CH2)), 94.23 (C(CH2)), 96.65
(C8), 102.25 (C3), 102.87 (C10), 115.03 (2 ꢁ C(C30,C50)), 121.00
(C10), 127.12 (2 ꢁ C(C20,C60)), 129.89 (C6), 144.78 (C9), 150.77
(C5), 155.32 (C7), 163.33 (C40), 163.45 (C2), 166.02 (CO), 181.97
(C4). ESI-MS: m/z 460 [M+H]+.
0
0
0
0
0
0
7.49 (m, 5H, ArH), 8.04 (d, J = 8.7 Hz, 2H, C2 C6 -H), 12.69 (s, 1H,
C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 56.22 (C(CH3)), 56.36 (C
(CH3)), 71.48 (C(CH2)), 94.16 (C(CH2)), 94.23 (C(CH2)), 96.67 (C8),
102.25 (C3), 102.89 (C10), 114.87 (2 ꢁ C(C30,C50)), 119.96 (C10),
125.98 (2 ꢁ C(C20,C60)), 127.13 (2 ꢁ C), 127.84 (C), 129.01 (2 ꢁ C),
129.87 (C6), 141.29 (C), 144.78 (C9), 150.76 (C5), 155.32 (C7),
163.45 (C2), 163.98 (C40), 181.97 (C4). ESI-MS: m/z 465 [M+H]+.
4.2.9.2.
5-Hydroxy-6,7-bis(methoxymethoxy)-2-(4-(2-mor-
4.2.8. 5-Hydroxy-2-(4-hydroxyphenyl)-6,7-bis(methoxymeth-
oxy)-4H-chromen-4-one (12)
pholino-2-oxoethoxy)phenyl)-4H-chromen-4-one (15b).
62.3% yield, yellow solid, mp 176–178 °C. 1H NMR (300 MHz,
DMSO-d6) d: 3.47 (s, 3H, CH3), 3.48–3.52 (m, 4H, 2 ꢁ CH2), 3.54
(s, 3H, CH3), 3.58–3.63 (m, 4H, 2 ꢁ CH2), 4.99 (s, 2H, CH2), 5.18
(s, 2H, CH2), 5.37 (s, 2H, CH2), 6.63 (s, 1H, C3-H), 6.96 (s, 1H, C8-
Compound 11 (100 mg, 0.22 mmol) was stirred with 10% Pd/C
catalyst (2 mg) in EtOH (50 mL) and THF (50 mL) for 8 h under
hydrogen at atmospheric pressure, then the Pd/C catalyst was
removed by filtration over celite. After the filtrate was evaporated,
the crude material was purified by column chromatography on sil-
ica gel using 50% ethyl acetate in petroleum ether as eluent to
afford 12 (76 mg, 0.20 mmol, 91%) as a yellow solid, mp 151–
152 °C. 1H NMR (300 MHz, DMSO-d6) d: 3.44 (s, 3H, CH3), 3.54 (s,
3H, CH3), 5.17 (s, 2H, CH2), 5.37 (s, 2H, CH2), 6.61 (s, 1H, C3-H),
0
0
0
0
H), 7.13 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.03 (d, J = 8.7 Hz, 2H, C2 C6 -
H), 12.69 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 44.44
(2 ꢁ C(CH2)), 56.23 (C(CH3)), 56.36 (C(CH3)), 64.18 (C(CH2)),
66.33 (2 ꢁ C(CH2)), 94.18 (C(CH2)), 94.21 (C(CH2)), 96.65 (C8),
102.25 (C3), 102.89 (C10), 115.13 (2 ꢁ C(C30,C50)), 121.01 (C10),
127.25 (2 ꢁ C(C20,C60)), 129.89 (C6), 144.78 (C9), 150.77 (C5),
155.32 (C7), 163.19 (C40), 163.47 (C2), 167.03 (CO), 181.95 (C4).
ESI-MS: m/z 502 [M+H]+.
0
0
6.87 (s, 1H, C8-H), 6.94 (d, J = 8.6 Hz, 2H, C3 C5 -H), 7.96 (d,
0
0
0
J = 8.6 Hz, 2H, C2 C6 -H), 10.41 (s, 1H, C4 -OH), 12.74 (s, 1H, C5-
OH). 13C NMR (75 MHz, DMSO-d6) d: 56.21 (C(CH3)), 56.38 (C
(CH3)), 94.17 (C(CH2)), 94.22 (C(CH2)), 96.66 (C8), 102.26 (C3),
102.88 (C10), 115.77 (2 ꢁ C(C30,C50)), 121.36 (C10), 128.48 (2 ꢁ C
(C20,C60)), 129.88 (C6), 144.77 (C9), 150.78 (C5), 155.31 (C7),
160.98 (C40), 163.46 (C2), 181.96 (C4). ESI-MS: m/z 375 [M+H]+.
4.2.9.3. N,N-Diethyl-2-(4-(5-hydroxy-6,7-bis(methoxymethoxy)-
4-oxo-4H-chromen-2-yl)phenoxy)acetamide (15c).
67.7%
yield, yellow solid, mp 181–182 °C. 1H NMR (300 MHz, DMSO-d6)
d: 1.02–1.07 (t, 3H, CH3), 1.15–1.20 (t, 3H, CH3), 3.24–3.36 (m,
4H, 2 ꢁ CH2), 3.44 (s, 3H, CH3), 3.54 (s, 3H, CH3), 4.94 (s, 2H,
CH2), 5.18 (s, 2H, CH2), 5.37 (s, 2H, CH2), 6.62 (s, 1H, C3-H), 6.95
4.2.9. Synthesis of 15a–15c, 20a–20c and 25a–25b
0
0
Chloroacetyl chloride 13 (0.06 mol, 4.0 ml) was added dropwise
into the solution of imine (4a–4c) (0.06 mol) in benzene (20 ml) at
(s, 1H, C8-H), 7.10 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.03 (d, J = 8.7 Hz,
2H, C2 C6 -H), 12.69 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6)
0
0
Please cite this article in press as: Shi, Z.-H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.047

8
Z.-H. Shi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
d: 12.33 (2 ꢁ C(CH3)), 42.02 (2 ꢁ C(CH2)), 56.22 (C(CH3)), 56.39 (C
(CH3)), 64.25 (C(CH2)), 94.18 (C(CH2)), 94.23 (C(CH2)), 96.67 (C8),
102.27 (C3), 102.89 (C10), 114.01 (2 ꢁ C(C30,C50)), 121.02 (C10),
127.99 (2 ꢁ C(C20,C60)), 129.87 (C6), 144.79 (C9), 150.78 (C5),
155.32 (C7), 163.02 (C40), 163.47 (C2), 166.92 (CO), 181.97 (C4).
ESI-MS: m/z 488 [M+H]+.
4.2.9.8. 5-Hydroxy-6,7-bis(methoxymethoxy)-2-(4-(3-morpho-
linopropoxy)phenyl)-4H-chromen-4-one (25b). 62.4% yield,
yellow solid, mp 203–205 °C. 1H NMR (300 MHz, DMSO-d6) d:
1.91–1.93 (m, 2H, CH2), 2.34–2.37 (m, 2H, CH2), 2.38–2.41 (m,
4H, 2 ꢁ CH2), 3.44–3.47 (m, 4H, 2 ꢁ CH2), 3.48 (s, 3H, CH3), 3.54
(s, 3H, CH3), 4.11–4.13 (m, 2H, CH2), 5.17 (s, 2H, CH2), 5.36 (s,
2H, CH2), 6.62 (s, 1H, C3-H), 6.95 (s, 1H, C8-H), 7.13 (d, J = 8.7 Hz,
0
0
0
0
4.2.9.4. 2-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-hydroxy-6,7-
bis(methoxymethoxy)-4H-chromen-4-one (20a). 64.5% yield,
yellow solid, mp 191–193 °C. 1H NMR (300 MHz, DMSO-d6) d:
2.29 (s, 6H, 2 ꢁ CH3), 2.71–2.75 (t, 2H, CH2), 3.45 (s, 3H, CH3),
3.54 (s, 3H, CH3), 4.16–4.21 (t, 2H, CH2), 5.17 (s, 2H, CH2), 5.36 (s,
2H, CH2), 6.62 (s, 1H, C3-H), 6.96 (s, 1H, C8-H), 7.15 (d, J = 8.7 Hz,
2H, C3 C5 -H), 8.05 (d, J = 8.7 Hz, 2H, C2 C6 -H), 12.70 (s, 1H, C5-
OH). 13C NMR (75 MHz, DMSO-d6) d: 28.33 (C(CH2)), 51.10 (C
(CH2)), 53.75 (2 ꢁ C(CH2)), 56.23 (C(CH3)), 56.39 (C(CH3)), 66.85
(C(CH2)), 66.89 (2 ꢁ C(CH2)), 94.18 (C(CH2)), 94.23 (C(CH2)),
96.67 (C8), 102.27 (C3), 102.86 (C10), 114.38 (2 ꢁ C(C30,C50)),
120.18 (C10), 127.57 (2 ꢁ C(C20,C60)), 129.87 (C6), 144.78 (C9),
150.76 (C5), 155.32 (C7), 159.87 (C40), 163.45 (C2), 181.95 (C4).
ESI-MS: m/z 502 [M+H]+.
0
0
0
0
2H, C3 C5 -H), 8.05 (d, J = 8.7 Hz, 2H, C2 C6 -H), 12.72 (s, 1H,
C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 46.45 (2 ꢁ C(CH3)), 56.23
(C(CH3)), 56.37 (C(CH3)), 58.32 (C(CH2)), 66.88 (C(CH2)), 94.19 (C
(CH2)), 94.23 (C(CH2)), 96.67 (C8), 102.26 (C3), 102.86 (C10),
114.22 (2 ꢁ C(C30,C50)), 120.37 (C10), 127.36 (2 ꢁ C(C20,C60)),
129.89 (C6), 144.76 (C9), 150.77 (C5), 155.33 (C7), 159.87 (C40),
163.45 (C2), 181.97 (C4). ESI-MS: m/z 446 [M+H]+.
4.2.10. Synthesis of 16a–16c, 21a–21c and 26a–26b
HCl (1 mL) was added dropwise into a solution of 15a–15c,
20a–20c and 25a–25b (1.00 mmol) in CH2Cl2 (5 mL) and Et2O
(5 mL) at 0 °C, then this mixture was warmed to room tempera-
ture. After 6 h, the mixture was poured into water (20 mL), and
then extracted with ethyl acetate (20 mL ꢁ 3), the organic layer
was washed with brine (20 mL), dried over Na2SO4, filtered and
concentrated under reduced pressure. The appeared crude material
was purified by column chromatography on silica gel using 50%
ethyl acetate in petroleum ether as eluent to afford 16a–16c,
21a–21c and 26a–26b as yellow solids.
4.2.9.5. 5-Hydroxy-6,7-bis(methoxymethoxy)-2-(4-(2-morpholi-
noethoxy)phenyl)-4H-chromen-4-one
(20b). 61.8%
yield,
yellow solid, mp 198–199 °C. 1H NMR (300 MHz, DMSO-d6) d:
2.71–2.74 (t, 4H, 2 ꢁ CH2), 3.44 (s, 3H, CH3), 3.53 (s, 3H, CH3),
3.55–3.60 (m, 6H), 4.19–4.21 (t, 2H, CH2), 5.17 (s, 2H, CH2), 5.36
(s, 2H, CH2), 6.62 (s, 1H, C3-H), 6.95 (s, 1H, C8-H), 7.15 (d,
0
0
0
0
J = 8.7 Hz, 2H, C3 C5 -H), 8.05 (d, J = 8.7 Hz, 2H, C2 C6 -H), 12.69 (s,
1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 53.79 (2 ꢁ C(CH2)),
54.82 (C(CH2)), 56.22 (C(CH3)), 56.37 (C(CH3)), 66.88 (2 ꢁ C
(CH2)), 66.92 (C(CH2)), 94.16 (C(CH2)), 94.22 (C(CH2)), 96.67 (C8),
102.25 (C3), 102.89 (C10), 114.36 (2 ꢁ C(C30,C50)), 120.25 (C10),
127.66 (2 ꢁ C(C20,C60)), 129.88 (C6), 144.78 (C9), 150.76 (C5),
155.33 (C7), 159.88 (C40), 163.44 (C2), 181.94 (C4). ESI-MS: m/z
488 [M+H]+.
4.2.10.1. N,N-Dimethyl-2-(4-(5,6,7-trihydroxy-4-oxo-4H-chro-
men-2-yl)phenoxy)acetamide (16a). 88.2% yield, yellow solid,
mp 228–229 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.85 (s, 6H,
2 ꢁ CH3), 4.96 (m, 2H, CH2), 6.60 (s, 1H, C3-H), 6.88 (s, 1H, C8-H),
0
0
0
0
7.10 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.09 (d, J = 8.7 Hz, 2H, C2 C6 -H),
8.96 (s, 1H, C6-OH), 10.63 (s, 1H, C7-OH), 12.36 (s, 1H, C5-OH).
13C NMR (75 MHz, DMSO-d6) d: 38.12 (2 ꢁ C(CH3)), 64.23 (C
(CH2)), 98.67 (C8), 102.24 (C3), 103.55 (C10), 115.02 (2 ꢁ C(C30,
C50)), 121.01 (C10), 124.66 (C6), 127.11 (2 ꢁ C(C20,C60)), 145.57
(C9), 152.86 (C5), 153.87 (C7), 163.34 (C40), 163.44 (C2), 166.03
(CO), 181.96 (C4). ESI-MS: m/z 372 [M+H]+.
4.2.9.6. 2-(4-(2-(Diethylamino)ethoxy)phenyl)-5-hydroxy-6,7-
bis(methoxymethoxy)-4H-chromen-4-one (20c). 63.2% yield,
yellow solid, mp 203–205 °C. 1H NMR (300 MHz, DMSO-d6) d:
1.23–1.28 (t, 6H, 2 ꢁ CH3), 2.51–2.54 (m, 4H, 2 ꢁ CH2), 3.25–3.28
(t, 2H), 3.45 (s, 3H, CH3), 3.54 (s, 3H, CH3), 4.14–4.17 (t, 2H, CH2),
5.09 (s, 2H, CH2), 5.17 (s, 2H, CH2), 6.63 (s, 1H, C3-H), 6.96 (s, 1H,
4.2.10.2. 5,6,7-Trihydroxy-2-(4-(2-morpholino-2-oxoethoxy)phe-
nyl)-4H-chromen-4-one (16b). 86.8% yield, yellow solid, mp
231–232 °C. 1H NMR (300 MHz, DMSO-d6) d: 3.42–3.46 (m, 4H,
2 ꢁ CH2), 3.58–3.62 (m, 4H, 2 ꢁ CH2), 4.99 (s, 2H, CH2), 6.28 (s,
0
0
C8-H), 7.14 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.05 (d, 2H, J = 8.7 Hz,
C2 C6 -H), 12.70 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d:
14.02 (2 ꢁ C(CH3)), 49.58 (2 ꢁ C(CH2)), 54.13 (CH2), 56.23 (C
(CH3)), 56.39 (C(CH3)), 66.33 (C(CH2)), 94.16 (C(CH2)), 94.22 (C
(CH2)), 96.67 (C8), 102.25 (C3), 102.89 (C10), 114.21 (2 ꢁ C(C30,
C50)), 120.25 (C10), 127.36 (2 ꢁ C(C20,C60)), 129.87 (C6), 144.76
(C9), 150.77 (C5), 155.32 (C7), 159.83 (C40), 163.45 (C2), 181.95
(C4). ESI-MS: m/z 474 [M+H]+.
0
0
0
0
1H, C3-H), 6.84 (s, 1H, C8-H), 7.11 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.11
0
0
(d, J = 8.7 Hz, 2H, C2 C6 -H), 8.83 (s, 1H, C6-OH), 10.49 (s, 1H, C7-
OH), 12.35 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 44.43
(2 ꢁ C(CH2)), 64.17 (C(CH2)), 66.34 (2 ꢁ C(CH2)), 98.64 (C8), 102.26
(C3), 103.56 (C10), 115.14 (2 ꢁ C(C30,C50)), 121.00 (C10), 124.67
(C6), 127.26 (2 ꢁ C(C20,C60)), 145.55 (C9), 152.87 (C5), 153.86 (C7),
163.18 (C40), 163.48 (C2), 167.04 (CO), 181.96 (C4). ESI-MS: m/z
414 [M+H]+.
4.2.9.7. 2-(4-(3-(Dimethylamino)propoxy)phenyl)-5-hydroxy-
6,7-bis(methoxymethoxy)-4H-chromen-4-one (25a).
66.9%
yield, yellow solid, mp 199–200 °C. 1H NMR (300 MHz, DMSO-
d6) d: 2.03–2.06 (m, 2H), 2.49 (s, 6H, 2 ꢁ CH3), 2.83–2.86 (m,
2H, CH2), 3.45 (s, 3H, CH3), 3.54 (s, 3H, CH3), 4.13–4.17 (m, 2H,
CH2), 5.17 (s, 2H, CH2), 5.37 (s, 2H, CH2), 6.63 (s, 1H, C3-H), 6.97
4.2.10.3. N,N-Diethyl-2-(4-(5,6,7-trihydroxy-4-oxo-4H-chromen-
2-yl)phenoxy)acetamide (16c). 89.1% yield, yellow solid, mp
235–237 °C. 1H NMR (300 MHz, DMSO-d6) d: 1.16–1.23 (t, 6H,
2 ꢁ CH3), 3.26–3.30 (m, 4H, 2 ꢁ CH2), 4.94 (m, 2H, CH2), 6.28 (s,
0
0
0
0
(s, 1H, C8-H), 7.14 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.06 (d, J = 8.7 Hz,
1H, C3-H), 6.84 (s, 1H, C8-H), 7.08 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.11
2H, C2 C6 -H), 12.69 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6)
d: 28.17 (C(CH2)), 46.38 (2 ꢁ C(CH3)), 54.99 (C(CH2)), 56.22 (C
(CH3)), 56.37 (C(CH3)), 67.01 (C(CH2)), 94.18 (C(CH2)), 94.21 (C
(CH2)), 96.67 (C8), 102.25 (C3), 102.87 (C10), 114.23 (2 ꢁ C(C30,
C50)), 120.18 (C10), 127.52 (2 ꢁ C(C20,C60)), 129.89 (C6), 144.76
(C9), 150.79 (C5), 155.32 (C7), 159.87 (C40), 163.47 (C2), 181.95
(C4). ESI-MS: m/z 460 [M+H]+.
(d, J = 8.7 Hz, 2H, C2 C6 -H), 8.81 (s, 1H, C6-OH), 10.51 (s, 1H, C7-
OH), 12.35 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 12.34
(2 ꢁ C(CH3)), 42.03 (2 ꢁ C(CH2)), 64.26 (C(CH2)), 98.65 (C8),
102.26 (C3), 103.57 (C10), 114.02 (2 ꢁ C(C30,C50)), 121.01 (C10),
124.66 (C6), 127.98 (2 ꢁ C(C20,C60)), 145.57 (C9), 152.86 (C5),
153.88 (C7), 163.03 (C40), 163.46 (C2), 166.91 (CO), 181.96 (C4).
ESI-MS: m/z 400 [M+H]+.
0
0
0
0
Please cite this article in press as: Shi, Z.-H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.047

Z.-H. Shi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
4.2.10.4.
2-(4-(2-(Dimethylamino)ethoxy)phenyl)-5,6,7-trihy-
4.3. Antithrombotic assay
4.3.1. Blood collection
droxy-4H-chromen-4-one (21a). 85.3% yield, yellow solid, mp
143–145 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.87 (s, 6H,
2 ꢁ CH3), 3.51–3.55 (t, 2H, CH2), 4.44–4.47 (t, 2H, CH2), 6.63 (s,
Male New Zealand white rabbits, weighing 2–2.5 kg were
obtained from the experimental animal center of Nanjing Univer-
sity 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 centrifu-
gation at 3000 rpm for 10 min.
0
0
1H, C3-H), 6.87 (s, 1H, C8-H), 7.17 (d, J = 8.7 Hz, 2H, C3 C5 -H), 8.07
0
0
(d, J = 8.7 Hz, 2H, C2 C6 -H), 8.84 (s, 1H, C6-OH), 10.58 (s, 1H, C7-
OH), 12.72 (s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 46.44
(2 ꢁ C(CH3)), 58.35 (C(CH2)), 66.87 (C(CH2)), 98.66 (C8), 102.25
(C3), 103.58 (C10), 114.23 (2 ꢁ C(C30,C50)), 120.38 (C10), 124.65
(C6), 127.37 (2 ꢁ C(C20,C60)), 145.58 (C9), 152.87 (C5), 153.86
(C7), 159.85 (C40), 163.43 (C2), 181.96 (C4). ESI-MS: m/z 358 [M
+H]+.
4.2.10.5. 5,6,7-Trihydroxy-2-(4-(2-morpholinoethoxy)phenyl)-
4H-chromen-4-one (21b). 83.8% yield, yellow solid, mp
248–250 °C. 1H NMR (300 MHz, DMSO-d6) d: 2.58–2.61 (m, 4H,
2 ꢁ CH2), 2.71–2.73 (t, 2H, CH2), 3.56–3.59 (m, 4H, 2 ꢁ CH2),
4.17–4.20 (t, 2H, CH2), 6.18 (s, 1H, C3-H), 6.74 (s, 1H, C8-H), 7.12
0
0
0
0
(d, J = 8.7 Hz, 2H, C3 C5 -H), 8.07 (d, J = 8.7 Hz, 2H, C2 C6 -H), 12.38
(s, 1H, C5-OH). 13C NMR (75 MHz, DMSO-d6) d: 53.77 (2 ꢁ C
(CH2)), 54.81 (C(CH2)), 66.89 (2 ꢁ C(CH2)), 66.91 (C(CH2)), 98.67
(C8), 102.24 (C3), 103.57 (C10), 114.35 (2 ꢁ C(C30,C50)), 120.24
(C10), 124.64 (C6), 127.67 (2 ꢁ C(C20,C60)), 145.57 (C9), 152.88
(C5), 153.85 (C7), 159.89 (C40), 163.45 (C2), 181.95 (C4). ESI-MS:
m/z 400 [M+H]+.
4.3.2. Plasma antithrombotic assay
TT, APTT, PT and FIB were examined with commercial kits fol-
lowing 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
mined 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. 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. PT was determined by
incubating 40 l PPP solution for 3 min at 37 °C, followed by addi-
tion of 40 l thromboplastin agent and 20 l sample. 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 sample solution. The antithrombotic activity was
lM. TT was deter-
l
l
l
l
4.2.10.6. 2-(4-(2-(Diethylamino)ethoxy)phenyl)-5,6,7-trihydroxy-
4H-chromen-4-one (21c). 87.4% yield, yellow solid, mp
140–141 °C. 1H NMR (300 MHz, DMSO-d6) d: 1.17–1.24 (t, 6H,
2 ꢁ CH3), 3.21–3.25 (m, 4H, 2 ꢁ CH2), 3.52–3.55 (t, 2H, CH2), 4.42–
4.45 (t, 2H, CH2), 6.29 (s, 1H, C3-H), 6.86 (s, 1H, C8-H), 7.19 (d,
l
l
l
l
l
l
l
l
l
0
0
0
0
J = 8.7 Hz, 2H, C3 C5 -H), 8.16 (d, J = 8.7 Hz, 2H, C2 C6 -H), 8.78 (s,
1H, C6-OH), 10.51 (s, 1H, C7-OH), 12.31 (s, 1H, C5-OH). 13C NMR
(75 MHz, DMSO-d6) d: 14.03 (2 ꢁ C(CH3)), 49.59 (2 ꢁ C(CH2)),
56.38 (C(CH3)), 66.34 (C(CH2)), 98.66 (C8), 102.26 (C3), 103.56
(C10), 114.22 (2 ꢁ C(C30,C50)), 120.24 (C10), 124.65 (C6), 127.35
(2 ꢁ C(C20,C60)), 145.56 (C9), 152.87 (C5), 153.86 (C7), 159.84
(C40), 163.44 (C2), 181.94 (C4). ESI-MS: m/z 386 [M+H]+.
l
l
assessed by assaying the prolongation of the plasma clotting time
of TT, APTT, INR increasement of PT, and reduction of FIB content.
4.4. Antioxidant assay
4.4.1. DPPH radical scavenging assay
4.2.10.7. 2-(4-(3-(Dimethylamino)propoxy)phenyl)-5,6,7-trihy-
droxy-4H-chromen-4-one (26a). 89.3% yield, yellow solid, mp
153–155 °C. 1H NMR (300 MHz, DMSO-d6) d: 1.21–1.24 (m, 2H,
CH2), 2.68–2.72 (m, 2H, CH2), 2.75 (s, 6H, 2 ꢁ CH3), 4.16–4.19 (m,
2H, CH2), 6.32 (s, 1H, C3-H), 6.85 (s, 1H, C8-H), 7.15 (d, J = 8.7 Hz,
The DPPHꢂ radical scavenging activity was measured according
to previous studies.20 The solution of the sample (100
lL) in
dimethylsulfoxide (DMSO) was added to 100 lL 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
0
0
0
0
2H, C3 C5 -H), 8.12 (d, J = 8.7 Hz, 2H, C2 C6 -H), 8.84 (s, 1H, C6-OH),
10.47 (s, 1H, C7-OH), 12.33 (s, 1H, C5-OH). 13C NMR (75 MHz,
DMSO-d6) d: 28.16 (C(CH2)), 46.37 (2 ꢁ C(CH3)), 54.98 (C(CH2)),
67.02 (C(CH2)), 98.65 (C8), 102.24 (C3), 103.55 (C10), 114.22
(2 ꢁ C(C30,C50)), 120.17 (C10), 124.66 (C6), 127.53 (2 ꢁ C(C20,C60)),
145.54 (C9), 152.88 (C5), 153.87 (C7), 159.85 (C40), 163.46 (C2),
181.96 (C4). ESI-MS: m/z 372 [M+H]+.
tested compounds had concentrations ranging from 5
lM to
500 M. The reaction leading to the scavenging of DPPH radical
l
was completed within 30 min at 25 °C. The absorbance of the mix-
ture was then measured at 517 nm with a microplate reader. The
inhibition of DPPH radical was expressed as percentage: Scaveng-
ing rate % = [1 ꢀ (A1 ꢀ A2)/A0] ꢁ 100%, where A1 was the absor-
bance 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 concen-
tration of sample that causes 50% loss of the DPPH radical.
4.2.10.8. 5,6,7-Trihydroxy-2-(4-(3-morpholinopropoxy)phenyl)-
4H-chromen-4-one (26b). 85.4% yield, yellow solid, mp 232–
234 °C. 1H NMR (300 MHz, DMSO-d6) d: 1.22–1.25 (m, 2H, CH2),
2.12–2.16 (m, 6H), 3.61–3.65 (m, 4H, 2 ꢁ CH2), 4.11–4.14 (m, 2H,
CH2), 6.28 (s, 1H, C3-H), 6.86 (s, 1H, C8-H), 7.13 (d, J = 8.7 Hz, 2H,
4.4.2. PC12 cells antioxidant assay
0
0
0
0
C3 C5 -H), 8.06 (d, J = 8.7 Hz, 2H, C2 C6 -H), 8.82 (s, 1H, C6-OH),
10.55 (s, 1H, C7-OH), 12.34 (s, 1H, C5-OH). 13C NMR (75 MHz,
DMSO-d6) d: 28.34 (C(CH2)), 51.11 (C(CH2)), 53.74 (2 ꢁ C(CH2)),
66.84 (C(CH2)), 66.88 (2 ꢁ C(CH2)), 98.66 (C8), 102.28 (C3),
103.54 (C10), 114.37 (2 ꢁ C(C30,C50)), 120.17 (C10), 124.67 (C6),
127.56 (2 ꢁ C(C20,C60)), 145.53 (C9), 152.87 (C5), 153.88 (C7),
159.89 (C40), 163.44 (C2), 181.96 (C4). ESI-MS: m/z 414 [M+H]+.
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 ll 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 l
mol Lꢀ1 H2O2 for 1 h
Please cite this article in press as: Shi, Z.-H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.047

10
Z.-H. Shi et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
before the test drugs were added in. Drug concentrations were
the Priority Academic Program Development of Jiangsu Higher
Education Institutions and Project Funded by the Flagship Major
Development of Jiangsu Higher Education Institutions
(PPZY2015A070).
50
20
l
l
mol Lꢀ1 and 25
l of MTT solution for 4 h at 37 °C. The formazan crystals
l of DMSO, and then
l
mol Lꢀ1. The cells were incubated with
formed in intact cells were dissolved in 150
l
vigorously shaking for 10 min. Finally, the absorbance was
assessed at 517 nm. The inhibiting rate of H2O2-induced cytotoxi-
city 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 pres-
ence of H2O2 (model), A0 was the absorbance without sample
and H2O2 (normal).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.09.047.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
4.5. Solubility determination
References and notes
The solubility of scutellarein derivatives in water was deter-
mined using the known method29–31 with minor modifications.
The UV/UV device is composed of a UV source (for UV photodegra-
dation) and a UV absorption detector (for on-line UV measure-
ment). 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 minꢀ1. Under the influence of UV radiation, 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.
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This work was supported by National Natural Science Founda-
tion of China (No. 81274058, 21302225), China Scholarship Council
(No. 201407060046), Natural Science Foundation of Jiangsu Pro-
vince (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), Tech-
nology 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
Please cite this article in press as: Shi, Z.-H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.09.047