Synthesis and biological evaluation of scopoletin derivatives

Article abstract of DOI:10.1016/j.bmc.2012.10.059

A series of new scopoletin derivatives were designed and synthesized. Their anti-proliferative effect was initially evaluated against various human cancer cell lines. Among the tested compounds, A1, A2, and D6 showed significant anti-proliferative activities. Angiogenesis was detected by endothelial cell migration assay and tube formation study. The results showed that A1, A2, and D6 inhibited the vascular endothelial growth factor (VEGF)-stimulated proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro. Moreover, they inhibited the vessel growth in the chorioallantoic membrane in vivo. This inhibition was correlated with a significant decrease in the VEGF-triggered phosphorylated forms of ERK1/2 and Akt. In summary, these findings strongly suggested that these scopoletin derivatives might be structurally novel angiogenesis inhibitors.

Full text of DOI:10.1016/j.bmc.2012.10.059

Bioorganic & Medicinal Chemistry 21 (2013) 84–92
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of scopoletin derivatives
Xueting Cai a, Jie Yang a, Jinpei Zhou b, Wuguang Lu a, Chunping Hu a, Zhenhua Gu a, Jiege Huo a,
Xiaoning Wang a, Peng Cao a,
⇑
a Laboratory of Cellular and Molecular Biology, Jiangsu Province Academy of Traditional Chinese Medicine, 100# Shizi Street, Hongshan Road, Nanjing 210028, Jiangsu, China
b Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new scopoletin derivatives were designed and synthesized. Their anti-proliferative effect was
initially evaluated against various human cancer cell lines. Among the tested compounds, A1, A2, and D6
showed significant anti-proliferative activities. Angiogenesis was detected by endothelial cell migration
assay and tube formation study. The results showed that A1, A2, and D6 inhibited the vascular endothe-
lial growth factor (VEGF)-stimulated proliferation, migration, and tube formation of human umbilical
vein endothelial cells in vitro. Moreover, they inhibited the vessel growth in the chorioallantoic mem-
brane in vivo. This inhibition was correlated with a significant decrease in the VEGF-triggered phosphor-
ylated forms of ERK1/2 and Akt. In summary, these findings strongly suggested that these scopoletin
derivatives might be structurally novel angiogenesis inhibitors.
Received 19 September 2012
Revised 29 October 2012
Accepted 31 October 2012
Available online 15 November 2012
Keywords:
Scopoletin derivative
Angiogenesis
Invasion
Ó 2012 Elsevier Ltd. All rights reserved.
Metastasis
1. Introduction
medicine (TCM) for treating various rheumatoid diseases. Sco pos-
sesses a wide range of biological activities such as anti-inflamma-
Angiogenesis has gained increasing attention because of its cru-
cial function in the initiation and progression of many diseases such
as cancer and RA. Hence, anti-angiogenic intervention is becoming
a promising approach for the treatment of these diseases. For exam-
ple, Bevacizumab (Avastin, anti-VEGF monoclonal antibody) and
Vatalanib (VEGFR inhibitor) have been clinically approved for treat-
ment of many malignant tumors by anti-angiogenic pathways.
Many TCMs that are often used for both cancer and RA treatment
contain bioactive constituents with anti-angiogenic properties.
For example, epigallocatechin-3-gallate (EGCG), which is a main
polyphenolic constituent of green tea, functions as an angiogenesis
inhibitor by modulating the protease activity during endothelial
morphogenesis;1 sinomenine, which is the main alkaloid constitute
that is present in Sinomenium acutum and has been used to treat
RA for over one thousand years in China, is able to alleviate angio-
genesis in vitro and in vivo;2 scopoletin, which is a coumarin com-
pound with multiple bioactivities, is the main active constituent of
Erycibe obtusifolia Benth stems that has long been used for RA treat-
ment in TCM. Preliminary pharmacological experiments found that
scopoletin can inhibit angiogenesis. Therefore, a series of new
scopoletin derivatives was designed and synthesized.
tory, hypouricemic, and antioxidant activities.3 Reports have
indicated that several coumarin-type compounds block angiogen-
esis by inhibiting the endothelial cell growth.4,5 Previous data also
demonstrated that scopoletin and its derivatives have anti-tumor
and anti-angiogenic activities.6 These findings pave the way for fu-
ture studies on the anti-angiogenic potential and related mecha-
nisms of scopoletin derivatives.
2. Results and discussion
2.1. Chemistry
Scopoletin (6-methoxy-7-hydroxycoumarin) was synthesized
according to previous method.6 Briefly, 2,4-dihydroxy-5-methoxy-
benzaldehyde was obtained in a one-step reaction from 2,4,5-tri-
methoxybenzaldehyde by reaction with aluminium(III) chloride
in dichloromethane, followed by acid hydrolysis. Treatment of 5
with malonic acid in pyridine for 24 h at room temperature (rt)
using phenylamine as catalysts afforded 7-hydroxy-6-methoxy-2-
oxo-2H-chromene-3-carboxylic acid in 86% yield. Then heating
7-hydroxy-6-methoxy-2-oxo-2H-chromene-3-carboxylic acid in a
pyridine/ethylene glycol mixture (1:1.1) to reflux for 3 h gave
scopoletin.
Scopoletin (6-methoxy-7-hydroxycoumarin, Sco) is one of the
main coumarin constituents that occur in the stems of Erycibe
obtusifolia Benth, which is commonly used in traditional Chinese
The design of class A and class B compounds: the reaction in the
presence of scopoletin and alkyl or aromatic halides gave the prod-
ucts A1–A7 or B1–B6. Dehydrohalogenation was happened under
⇑
Corresponding author. Tel./fax: +86 25 85608666.
E-mail addresses: cao_lab@126.com, pcao79@yahoo.com (P. Cao).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.059

X. Cai et al. / Bioorg. Med. Chem. 21 (2013) 84–92
85
the conditions of potassium carbonate as acid binding agent,
generating a series of derivative.
Millicell chamber filter (Millipore, USA). However, the addition of
scopoletin derivatives (25 mol/L) to the top chamber significantly
l
The design of class C compounds: the reaction in the presence
of B1 (7-(2-bromoethoxy)-6-methoxycoumarin) and different
secondary amine gave the products C1–C6.
reduced the number of migrated cells (Fig. 2A). Comparatively,
scopoletin did not show the same inhibitory effect at the same
concentration.
The design of class D compounds: the reaction in the presence
of B5 (7-(oxiran-2-methoxy)-6-methoxycoumarin) and different
secondary amine gave the products D1–D6.
2.5. A1, A2, and D6 suppressed tube formation of HUVECs
The maturation of the migrated endothelial cells into a tube-like
structure is critical for the formation of functional vessels. We eval-
uated the effect of the scopoletin derivatives on the formation of
the tube-like structures by plating HUVECs with VEGF165 (10 ng/mL)
on the matrigel. At 5 h of plating, the migrated endothelial cells
rapidly aligned with one another, and formed into tube-like
structures. This process required cell–matrix interaction, intercel-
lular communication, and cell motility. However, the migration
effect of VEGF165 was significantly inhibited by A1, A2, and D6.
Incomplete sprouting and branching or broken network between
the tubes of HUVECs were observed (Fig. 2B). These results (the
migration assay and tube formation) indicated that scopoletin
derivatives (A1, A2, and D6) have the ability to block VEGF-induced
in vitro angiogenesis far better than scopoletin (no inhibitory effect
at the same concentration).
2.2. Effect of scopoletin and scopoletin derivatives on
endothelial cell proliferation
The anti-proliferative activity of scopoletin and 25 scopoletin
derivatives were tested with human umbilical vein endothelial
cells (HUVEC) (Fig. 1). As shown in Table 1, the scopoletin deriva-
tives A1, A2, B5, and D6 exerted the strongest anti-proliferative ef-
fect with an IC50 of 16.5, 9.8, 4.8, and 24.2 lM. The anti-angiogenic
activity of A1, A2, and D6 were further studied, whereas B5 was
not used because of its instability.
2.3. Effect of scopoletin, A1, A2, and D6 against the proliferation
of normal cells and tumor cells
We performed the cell proliferation assay to evaluate the cyto-
toxicity of scopoletin derivatives against both normal (peripheral
blood mononuclear cells, PBMC) and tumor cell lines (human pan-
creatic carcinoma cell line PANC-1, human pancreatic adenocarci-
noma cell line BxPC-3, human breast adenocarcinoma cell line
MDA-MB-231, human breast ductal carcinoma cell line MDA-MB-
435, human hepatic carcinoma HepG2, human colorectal carci-
noma cell line HCT 116, human gastric carcinoma cell line
MKN45, human lung carcinoma cell line A549, human epithelial
carcinoma cell line A431, human glioma cell line SHG-44, human
chronic myelogenous leukemia cell line K562, human promyelo-
cytic leukemia cell line HL-60, and human T lymphoma cell line
HuT 78). The concentration of the scopoletin derivatives with
50% inhibition (IC50) on the cells was determined, and the results
are summarized in Table 2. The results showed that A2 possessed
higher cytotoxicity to MDA-MB-231 cells while A1 was the most
effective on killing PANC-1 cells. D6 showed higher cytotoxicity
to many cancer cell lines than A1 and A2.
2.6. A1, A2, and D6 inhibited angiogenesis in vivo
The anti-angiogenic activity of the scopoletin derivatives on the
chorioallantoic membrane (CAM) was examined using a chicken
embryo model to investigate its responses to angiogenesis. Using
this model, we additionally examined the potential in vivo anti-
angiogenic activity of the scopoletin derivatives. New blood vessels
formed on CAMs in the control group, whereas the addition of A1,
A2, and D6 at 5 nmol per egg incubated for 48 h showed a notable
restraint. At 10 nmol per egg, the inhibition became more promi-
nent (Fig. 3). These results demonstrate that A1, A2, and D6 were
able to suppress angiogenesis in the chicken embryos.
2.7. A1, A2 and D6 inhibited expression of MMPs
Gelatinases such as MMP-2 and MMP-9 have important func-
tions in tumor invasion and cell migration.8 In the present study,
we detected the protein expression of these two MMPs in PANC-
1 cells treated by scopoletin and scopoletin derivatives. In the
control group, the cultured PANC-1 constitutively expressed the
MMP-9 protein, whereas in the treated group, the protein expres-
sion of MMP-9 and MMP-2 were gradually down-regulated (Fig. 4).
2.4. A1, A2, and D6 reduced VEGF-stimulated HUVEC migration
The migration of the endothelial cells is a prerequisite for angi-
ogenesis. VEGF is a potent stimulator for the migration of endothe-
lial cell.7 We determined the effect of A1, A2, and D6 on HUVEC
migration stimulated with VEGF165 using the Boyden Chamber as-
say because scopoletin derivatives can inhibit HUVEC proliferation
in a concentration-dependent manner.6 After VEGF165 stimulation
for 20 h, a large number of cells migrated to the lower side of the
2.8. Influence of A1, A2, and D6 on VEGF signaling pathways
The p44/42 mitogen-activated protein kinase (ERK1/2 MAPK)
cascade is the main pathway responsible for the VEGF-induced
Figure 1. HUVEC. (A) photograph of HUVECs (200Â); (B) characterization of HUVECs by assessing the Factor VIII Related Antigen (von Willebrand factor, vWF) expression
(400Â); (C) negative control group (immunocytochemical reaction with secondary antibody) (400Â).

86
X. Cai et al. / Bioorg. Med. Chem. 21 (2013) 84–92
Table 1
Anti-proliferation activity of scopoletin and scopoletin derivatives (A1–D6) on HUVECs
A
B
OCH3
OCH3
R1
O
O
O
O
O
O R
O
R2
C
D
OCH3
O
O CH3
O
R
O
O
R
O
O
OH
Compound
Structure
R1
R2
Inhibition rate (%)
At 100
2.1 0.2
IC50 (lM)
At 10
l
M
lM
Scopoletin
0.0 1.5
13.2 9.4
62.6 5.6
4.1 2.3
7.6 6.2
3.6 1.2
0.0 4.6
3.4 2.2
/
A1
A2
A3
A4
A5
A6
A7
A
A
A
A
A
A
A
Br
Cl
NO2
OCH3
F
H
H
H
H
H
Cl
H
97.5 0.7
83.8 1.6
27.2 5.3
85.6 0.2
16.6 2.2
42.8 2.5
83.6 0.2
16.8 1.4
10.0 0.8
/
37.5 0.1
/
/
Cl
H
62.4 3.3
B1
B2
B
B
4.9 1.6
0.3 4.4
5.6 1.1
3.3 0.9
/
/
H2C
CN
—CH2CH2CH2CH2Br
O
B3
B
0.1 1.5
9.4 1.0
/
H2C
B4
B5
B6
B
B
B
–CH2CH2Br
–CH2CH2Cl
–CH2CH2CH2Cl
11.2 0.9
62.2 0.1
1.2 2.9
16.9 1.9
95.5 0.5
79.5 0.4
/
5.3 0.5
42.4 1.1
CH3
CH3
HN
N
CH
C1
C2
C
C
0.1 1.6
0.1 4.9
0.1 4.0
3.1 0.3
/
/
CH2CH3
CH2 CH3
C3
C4
C5
C6
C
C
C
C
0.0 1.5
0.0 2.5
1.9 1.1
0.1 2.9
1.9 0.5
12.0 0.5
7.9 0.7
6.1 0.7
/
/
/
/
N
N
N
O
N
CH3
N
CH3
CH3
HN
CH
D1
D2
D
D
0.1 2.8
0.5 2.3
6.3 0.7
0.3 0.1
/
/
CH2CH3
N
CH2 CH3
D3
D4
D5
D
D
D
0.4 5.2
0.1 1.4
0.5 3.7
0.1 0.1
3.4 0.6
0.3 2.6
/
/
/
N
N
N
O
N
CH3

X. Cai et al. / Bioorg. Med. Chem. 21 (2013) 84–92
87
Table 1 (continued)
Compound
Structure
D
R1
R2
Inhibition rate (%)
At 100 lM
IC50 (lM)
At 10
lM
D6
31.4 0.7
80.7 2.3
24.2 1.6
N
Table 2
IC50 cytotoxicity values (lM) of scopoletin derivatives against PBMC and tumor cell lines
Compound PBMC PANC-1
BxPC-3
MDA-MB-
231
MDA-MB-
435
HepG2
HCT 116 MKN45
A549
A431
SHG-44
K562
>100
HL-60
>100
HuT 78
>100
Scopoletin >100 >100
>100
>100
47.0 1.4
>100
>100
>100
92.4 2.2
63.0 0.7
>100
90.4 1.1 >100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
A1
A2
D6
>100 63.7 1.6 >100
>100 19.8 2.1 >100
>100 >100
70.0 1.2 90.4 1.5 93.7 2.4
52.5 1.2 65.7 0.7 62.1 1.5
56.3 0.5 59.5 1.3 99.2 1.9 >100
83.9 1.4 26.1 1.1 78.9 1.7 42.7 0.4 93.9 0.9 42.8 1.6 43.7 1.6 30.2 1.3 46.6 1.0
35.0 1.3 51.6 1.2
Figure 2. Scopoletin derivatives (A1, A2, and D6) inhibited VEGF-induced migration and tube formation on HUVECs. (A) HUVECs were plated onto 24-well Millicell chamber
and stimulated with VEGF165 in the presence or absence of scopoletin derivatives (A1, A2, and D6). After 20 h, the cells migrated into the bottom of the membrane and were
photographed (100Â); (B) HUVECs were seeded onto 96-well plate previously coated with matrigel and stimulated with VEGF165 in the presence or absence of scopoletin
derivatives (A1, A2 and D6) for 5 h. The tube-like structure was photographed using a microscope (200Â). Compared with the control group, ⁄P <0.01.
proliferation of endothelial cells, whereas the stress-activated pro-
tein kinase-2 (p38 MAPK) pathway is principally responsible for
conveying VEGF signals to microfilaments, which induces the actin
cytoskeleton that regulates cell migration to rearrange. Both path-
ways are the key down-streams of VEGF signal transductions.9,10
Our results showed that A1, A2, and D6 antagonized the VEGF-
induced angiogenesis in HUVECs. This result prompted us to inves-
tigate if the anti-angiogenic effect of the scopoletin derivatives
results from its interaction with the VEGF signaling pathways.
Therefore, we examined the effects of A1, A2, and D6 on ERK1/2,
Akt, src, and p38 MAPK. As shown in Figure 5, ERK1/2, Akt were
phosphorylated by the addition of the exogenous VEGF to HUVECs.
However, pre-treatment with A1, A2, or D6 blocked the VEGF-
induced phosphorylation of Akt without affecting the src and
p38. The p-ERK1/2 was down-regulated by A1 and D6, but had
no effect on A2. In all cases, the total steady-state protein levels
remained unchanged, which suggests that several VEGF receptors
may be blocked by A1, A2, and D6 and leads to the interruption
of VEGF-triggered signaling.
3. Conclusions
Angiogenesis is a highly regulated process that involves a com-
plex cascade of events.11,12 VEGF is an endothelial cell-specific
mitogenic and chemotactic agent,13 and its inhibitory effects on
endothelial cells should block the whole process of angiogenesis.
Based on this target, a series of scopoletin and scopoletin deriva-
tives were synthesized and tested their anti-angiogenesis activities.

88
X. Cai et al. / Bioorg. Med. Chem. 21 (2013) 84–92
Figure 5. Effect of scopoletin derivatives (A1, A2, and D6) on VEGF-stimulated
activation of related signaling pathways. Western blot assays were performed to
examine the phosphorylation levels of src, ERK1/2, Akt, and p38 MAPK.
FTIR-8400S spectrophotometer. NMR spectra: Bruker ACF-300 Q
apparatus at 300 MHz for 1H NMR (internal standard, TMS). Mass
spectrometry: Hewlett-Packard 1100 LC/MSD spectrometer; in
m/z. Elemental analyses: CHN–O-Rapid instrument.
Figure 3. Scopoletin derivatives (A1, A2, and D6) inhibited angiogenesis in vivo.
Photographs are representative pictures from three independent experiments.
Cytotoxicity assay indicated that most of scopoletin derivatives
showed potent cytotoxicity against both tumor cells and HUVEC
compared to scopoletin, especially A1, A2, and D6. Compared with
scopoletin, A1, A2, and D6 exhibited significant inhibition of both
proliferation and VEGF-induced migration and invasion at concen-
trations without evident cytotoxicity. In conclusion, the prelimin-
ary in vitro activities and in vivo activities of these compounds
possess potential for design of better future molecules targeting
tumor angiogenesis. Further studies on their mechanism of anti-
angiogenesis activity are in progress.
4.1.2. Preparation of 2,4-dihydroxy-5-methoxybenzaldehyde
To
a stirred suspension of aluminum(III) chloride (80 g,
0.60 mol) in dry dichloromethane (400 mL), a solution of 2,4,5-tri-
methoxybenzaldehyde (20 g, 0.1 mol) in dry dichloromethane
(100 mL) was added dropwise, then the mixture was refluxed for
4 h. The reaction mixture was cooled and poured onto 500 g of
ice to which 100 ml of concentrated hydrochloric acid was added.
The organic layer was separated and the aqueous phase was ex-
tracted twice with dichloromethane (200 mL). The combined or-
ganic layer was washed with saturation salt solution, dried over
magnesium sulfate, evaporated and recrystallized from toluene
to give 2,4-dihydroxy-5-methoxybenzaldehyde as yellow solid
(15.79 g, 92.12%), mp 152–153 °C.
4. Experimental protocols
1H NMR (CDCl3, 300 MHz) d, ppm: 9.66 (s, 1H, CHO), 11.31 (s,
1H, 2-OH), 6.87 (s, 1H, 4-OH), 6.51 (s, 1H, H6), 6.40 (s, 1H, H3),
3.90 (s, 3H, OCH3).
4.1. Synthesis and characterization
4.1.1. General
All reagents were purchased from the Shanghai Chemical Re-
agent Company. Column chromatography: silica gel 60 (200–300
mesh). Thin-layer chromatography: silica gel 60 at F254 plates
(250 mm; Qingdao Ocean Chemical Company, China). Melting
point: capillary tube; uncorrected. IR spectra: Shimadzu
4.1.3. Preparation of 7-hydroxy-6-methoxy-2-oxo-2H-
chromene-3-carboxylic acid
The flask was charged with 9.49 g (56 mmol) 2, 4-dihydroxy-5-
methoxybenzaldehyde, 13.5 g, (130 mmol) malonic acid, 1 ml
Figure 4. Gelatin zymography of MMP-9 and MMP-2. The bands indicated MMP-9 activity. MMP-9 and MMP-2 collected from the culture medium of the same number of
PANC-1 cells cultured with scopoletin or scopoletin derivatives for 24 h. Compared with the control group, ⁄P <0.01.

X. Cai et al. / Bioorg. Med. Chem. 21 (2013) 84–92
89
phenylamine and 30 mL pyridine. The resulting solution was stir-
red at room temperature for over 24 h and then acidified to pH 4
using dilute HCl. The precipitate was collected by suction filtration
and followed by recrystallization from ethanol to give the pale yel-
low solid (11.49 g, 86.2%). Mp 231–232 °C.
4.1.5.5. Preparation of 7-[2-(4-fluoro-phenyl)-2-oxo-ethoxy]-6-
methoxy-2H-chromen-2-one (A5). Yield, 76.2%; white crystals,
mp: 206–208 °C; 1H NMR (CDCl3, 300 MHz, ppm): 3.94 (s, 3H,
OCH3), 5.41 (s, 2H, COCH2O), 6.31 (d, 1H, J = 9.0 Hz, C3-H), 6.70 (s,
1H, C5-H), 6.90 (s, 1H, C8-H), 7.63 (d, 1H, J = 9.0 Hz, C4-H), 7.26,
1H NMR (DMSO-d6): d: 3.80 (s, 3H, OCH3), 4.04 (s, 1H, OH), 6.76
(s, 1H, C5-H), 7.54 (s, 1H, C8-H), 8.61 (s, 1H, C4-H), 11.06 (s, 1H,
COOH); EI-MS (m/z): 236 [M]+.
8.03 (dd, 4H, ArH); IR (KBr, m): 3415, 3061, 1732, 1601, 1568,
1388, 1279, 1237, 1147 cmÀ1; MS (ESI, m/z): [MÀH]À 327.
4.1.5.6. Preparation of 7-[2-(3,4-dichloro-phenyl)-2-oxo-eth-
oxy]-6-methoxy-2H-chromen-2-one (A6). Yield, 51.5%; white
crystals, mp: 199–201 °C; 1H NMR (CDCl3, 300 MHz, d ppm): 3.91
(s, 3H, OCH3), 5.35 (s, 2H, COCH2O), 6.32 (d, 1H, J = 9.6 Hz, C3-H),
6.71 (s, 1H, C5-H), 6.90 (s, 1H, C8-H), 7.63 (d, 1H, J = 9.6 Hz, C4-H),
4.1.4. Preparation of 7-hydroxy-6-methoxy-2H-chromen-2-one
3.5 g (14.8 mmol) 7-hydroxy-6-methoxy-2-oxo-2H-chromene-
3-carboxylic acid was refluxed in 18 ml ethylene glycol and
20 ml pyridine for 3.5 h. After cooling the reaction, the mixture
was acidified to pH 4 using a solution of HCl and extracted with
CH2Cl2. The CH2Cl2 layers were pooled, dried (Na2SO4), and evapo-
rated to give the product, followed by recrystallization from etha-
nol. Yellow solid (2.55 g, 89.5%); mp 201–202 °C.
7.40, 7.65 (dd, 4H, ArH); IR (KBr,
m): 3435, 1727, 1700, 1612,
1388, 1277, 1145 cmÀ1; MS (ESI, m/z): [M]+ 378.
4.1.5.7. Preparation of 6-methoxy-7-(2-oxo-2-phenyl-ethoxy)-
2H-chromen-2-one (A7). Yield, 93.5%; white crystals, mp: 175–
177 °C; 1H NMR (CDCl3, 300 MHz, d ppm): 3.95 (s, 3H, OCH3),
5.47 (s, 2H, COCH2O), 6.30 (d, 1H, J = 9.3 Hz, C3-H), 6.70 (s, 1H,
C5-H), 6.91 (s, 1H, C8-H), 7.63 (d, 1H, J = 9.3 Hz, C4-H), 7.56–7.97
1H NMR (DMSO-d6): d: 3.79 (s, 3H, OCH3), 6.16 (d, 1H, J = 9.4 Hz,
C3-H), 6.74 (s, 1H, C5-H), 7.11 (s, 1H, C8-H), 7.82 (1H, d, J = 9.4 Hz,
C4-H), 10.29 (s, 1H, OH); EI-MS (m/z): 193 [M+H]+, 215 [M+Na]+.
4.1.5. General procedures for the synthesis of the compounds
A1–A7 and B1–B5
(m, 5H, ArH); IR (KBr, m): 3438, 3061, 1710, 1562, 1510, 1386,
1250, 1153 cmÀ1; MS (ESI, m/z): [M]+ 310.
0.3 g (1.56 mmol) scopoletin, 1.87 mmol alkyl or aromatic ha-
lides and 0.32 g (2.34 mmol) K2CO3 were combined and refluxed
in 20 mL aceton for 2 h, then removal of the solvent under reduced
pressure. The crude product was washed with water, filtrered and
dried, followed by recrystalliztion from petroleum ether and ethyl
acetate.
4.1.5.8. Preparation of 7-(2-bromoethoxy)-6-methoxy-2H-
chromen-2-one (B1). Yellow power, yield, 83.6%, mp 180–184 °C;
1H NMR (CDCl3, 300 MHz) d, ppm: 3.91 (3H, s, OCH3), 6.32 (1H,
d, J = 9.3 Hz, H3), 7.63 (1H, d, J = 9.6 Hz, H4), 6.84 (1H, s, H5),
6.89 (1H, s, H8), 4.23 (2H, t, OCH2), 3.71 (2H, t, BrCH2); IR (KBr,
cmÀ1): 3296, 3081, 2940, 1714, 1614, 1559, 1509, 1459, 1421,
1383, 1281, 1142, 1018; MS (EI, m/z): [M]+ 298.
4.1.5.1. Preparation of 7-[2-(4-bromo-phenyl)-2-oxo-ethoxy]-6-
methoxy-2H-chromen-2-one (A1).
Yield, 82.3%; white crys-
tals, mp: 208–210 °C; 1H NMR (CDCl3, 300 MHz, d ppm): 3.94 (s,
3H, OCH3), 5.40 (s, 2H, COCH2O), 6.31 (d, 1H, J = 9.3 Hz, C3-H),
6.70 (s, 1H, C5-H), 6.90 (s, 1H, C8-H), 7.69 (d, 1H, J = 9.3 Hz, C4-H),
4.1.5.9.
Preparation
of
7-(2-chloro-ethoxy)-6-methoxy-
2H-chromen-2-one (B2).
Yield, 747%; yellow crystals, mp:
160–162 °C; 1H NMR (CDCl3, 300 MHz, d ppm): 3.87 (t, 2H, ClCH2),
3.91 (s, 3H, OCH3), 4.33 (t, 2H, OCH2), 6.32 (d, 1H, J = 9.3 Hz, C3-H),
6.84 (s, 1H, C5-H), 6.88 (s, 1H, C8-H), 7.62 (d, 1H, J = 9.3 Hz, C4-H);
7.66, 7.88 (dd, 4H, ArH); IR (KBr,
m): 3143, 3061, 1733, 1688,
1614, 1568, 1516, 1391, 1278, 1249, 1148 cmÀ1; MS (ESI, m/z):
[M+Na]+ 413.
IR (KBr, m
): 3413, 2928, 1724, 1460, 1385, 1144, 1098 cmÀ1; MS
(ESI, m/z): [M]+ 254.
4.1.5.2. Preparation of 7-[2-(4-chloro-phenyl)-2-oxo-ethoxy]-6-
methoxy-2H-chromen-2-one (A2). Yield, 87.2%; white crystals,
mp: 218–220 °C; 1H NMR (CDCl3, 300 MHz, d ppm): 3.95 (s,
3H, OCH3), 5.41 (s, 2H, COCH2O), 6.31 (d, 1H, J = 9.0 Hz, C3-H),
6.71 (s, 1H, C5-H), 6.91 (s, 1H, C8-H), 7.64 (d, 1H, J = 9.0 Hz, C4-H),
4.1.5.10. Preparation of 7-(3-chloropropoxy)-6-methoxy-
2H-chromen-2-one (B3).
Yield, 65.2%; yellow crystals, mp:
118–120 °C; 1H NMR (CDCl3, 300 MHz) d, ppm: 3.89 (3H, s,
OCH3), 6.30 (1H, d, J = 9.6 Hz, H-3), 7.63 (1H, d, J = 9.6 Hz, H-4),
6.86 (1H, s, H-5), 6.90 (1H, s, H-8), 4.23 (2H, t, OCH2), 2.36 (2H,
m, C–CH2–C), 3.78 (2H, t, ClCH2); IR (KBr, cmÀ1): 3414, 3067,
2926, 2857, 1716, 1613, 1560, 1509, 1383, 1257, 1141, 1091; MS
(EI, m/z): [M]+ 268.
7.52, 7.96 (dd, 4H, ArH); IR (KBr,
m): 3414, 3059, 1732, 1695,
1614, 1568, 1518, 1388, 1278, 1250, 1150 cmÀ1; MS (ESI, /z):
[MÀH]À 343.
4.1.5.3. Preparation of 6-methoxy-7-[2-(4-nitro-phenyl)-2-oxo-
ethoxy]-2H-chromen-2-one (A3). Yield, 76.1%; white crystals,
mp: 230–232 °C; 1H NMR (CDCl3, 300 MHz, d ppm): 3.93 (s,
3H, OCH3), 5.60 (s, 2H, COCH2O), 6.38 (d, 1H, J = 9.6 Hz, C3-H),
6.75 (s, 1H, C5-H), 6.91 (s, 1H, C8-H), 7.66 (d, 1H, J = 9.6 Hz, C4-H),
4.1.5.11. Preparation of 7-(4-bromo-butoxy)-6-methoxy-
2H-chromen-2-one (B4).
Yield, 68.8%; white crystals, mp: 68–
70 °C; 1H NMR (CDCl3, 300 MHz, d ppm): 2.07 (S, 4H, CH2CH2), 2.36
(m, 2H, CCH2C), 3.51 (t, 2H, BrCH2), 3.89 (s, 3H, OCH3), 4.23 (t, 2H,
OCH2), 6.30 (d, 1H, J = 9.3 Hz, C3-H), 6.80 (s, 1H, C5-H), 6.86 (s, 1H,
8.17, 8.38 (dd, 4H, ArH); IR (KBr,
m): 3417, 3059, 1732,
1696, 1613, 1567, 1519, 1387, 1278, 1150 cmÀ1; MS (ESI, m/z):
[MÀH]À 354.
C8-H), 7.63 (d, 1H, J = 9.3 Hz, C4-H); IR (KBr, m): 3453, 2922, 2862,
1735, 1561, 1424, 1275, 1143, 1090 cmÀ1; MS (ESI, m/z): [M]+ 326.
4.1.5.4. Preparation of 6-methoxy-7-[2-(4-methoxy-phenyl)-
4.1.5.12. Preparation of 6-methoxy-7-(oxiran-2-ylmethoxy)-
2H-chromen-2-one (B5). Yield, 77.4%; white crystals, mp: 147–
150 °C; 1H NMR (CDCl3, 300 MHz) d, ppm: 3.91 (3H, s, OCH3),
6.31 (1H, d, J = 9.3 Hz, H3), 7.63 (1H, d, J = 9.3 Hz, H4), 6.87 (1H, s,
H5), 6.89 (1H, s, H8), 2.93, 2.97 (2H, d, J = 3.9 Hz, CH2), 3.42 (1H,
m, CH), 4.06–4.39 (2H, d, J = 5.7 Hz, C–CH2–O); IR (KBr, cmÀ1):
3417, 3010, 1713, 1615, 1560, 1511, 1387, 1276, 1247, 1168; MS
(EI, m/z): [M]+ 248.
2-oxo-ethoxy]-2H-chromen-2-one (A4).
Yield, 58.8%; white
crystals, mp: 199–201 °C; 1H NMR (CDCl3, 300 MHz, d ppm): 3.94
(s, 3H, OCH3), 5.41 (s, 2H, COCH2O), 6.29 (d, 1H, J = 9.3 Hz, C3-H),
6.69 (s, 1H, C5-H), 6.90 (s, 1H, C8-H), 7.62 (d, 1H, J = 9.3 Hz, C4-H),
7.00, 7.99 (dd, 4H, ArH); IR (KBr,
m): 3414, 3030, 1711, 1684,
1601, 1566, 1515, 1390, 1273, 1240, 1147 cmÀ1; MS (ESI, m/z):
[MÀH]À 339.

90
X. Cai et al. / Bioorg. Med. Chem. 21 (2013) 84–92
4.1.6. General procedures for the synthesis of the target
compounds C1–C6
4.1.7. General procedures for the synthesis of the target
compounds D1–D6
A mixture of 0.3 g (0.001 mmol) B1, 0.0012 mol amine were dis-
solved in 20 ml ethanol. And the mixture was heated under reflux
for 24 h, after which it was cooled to room temperature. After
evaporation of solvent, residual was subjected to silica gel column,
and chromatographed with eluent (ethyl acetate/hexane 1:2) to
yield the desired product.
A mixture of 0.248 g (0.001 mmol) of B6 and 0.0012 mol amine
were dissolved in 20 mL of ethanol. The mixture was heated in re-
flux for 24 h and cooled at room temperature. After the solvent
evaporated, the residual mixture was subjected to silica gel column
chromatography with an eluent (ethyl acetate/hexane 1:2) to ob-
tain the desired product.
4.1.7.1. Preparation of 7-(2-hydroxy-3-(isopropylamino)-
4.1.6.1. Preparation of 7-(2-(diisopropylamino)ethoxy)-6-meth-
propoxy)-6-methoxy-2H-chromen-2-one (D1).
Yield, 72.7%;
oxy-2H-chromen-2-one (C1).
Yield, 72.2%; white crystals,
yellowish powder, mp: 142–144 °C; 1H NMR (CDCl3, 300 MHz) d,
ppm: 3.89 (3H, s, OCH3), 6.30 (1H, d, J = 9.3 Hz, H-3), 7.63 (1H, d,
9.6 Hz, H4), 6.86 (1H, s, H5), 6.95 (1H, s, H-8), 1.20 (6H, d, 2CH3),
2.87 (1H, m, (CH3)2CH), 2.95–2.97 (2H, d, N–CH2–C), 4.27 (1H, m,
CH–OH), 4.12 (2H, d, J = 4.2 Hz, CH2–O); IR (KBr, cmÀ1): 3417,
3078, 2969, 2822, 1717, 1616, 1561, 1513, 1383, 1276, 1149; MS
(ESI, m/z): [M+H]+ 308.
mp: 205–208 °C; 1H NMR (CDCl3, 300 MHz) d, ppm: 3.92 (3H, s,
OCH3), 6.30 (1H, d, J = 9.6 Hz, H3), 7.61 (1H, d, J = 9.3 Hz, H4), 6.84
(1H, s, H5), 6.87 (1H, s, H8), 1.21 (6H, d, J = 6.3 Hz, NCH (CH3)2),
3.03 (1H, m, NCH (CH3)2), 3.16 (2H, t, N–CH2–C), 4.25 (2H, t, C–
CH2–O); IR (KBr, cmÀ1): 3417, 3019, 2957, 2886, 1716, 1614,
1560, 1511, 1456, 1383, 1275, 1155, 1098; MS (ESI, m/z): [M+H]+
278.
4.1.7.2. Preparation of 7-(3-(diethylamino)-2-hydroxypropoxy)-
4.1.6.2. Preparation of 7-(2-(diethylamino)ethoxy)-6-methoxy-
6-methoxy-2H-chromen-2-one (D2).
Yield, 97.7%; yellowish
powder, mp: 82–84 °C; 1H NMR (CDCl3, 300 MHz) d, ppm: 3.89
(3H, s, OCH3), 6.31 (1H, d, J = 9.6 Hz, H3), 7.63 (1H, d, J = 9.6 Hz,
H4), 6.85 (1H, s, H5), 6.88 (1H, s, H8), 1.13 (6H, t, 2CH3), 2.70 (4H,
m, 2N–CH2–CH3), 2.77 (2H, d, N–CH2–C), 4.19 (1H, m, CH–OH),
4.07 (2H, d, J = 15.3 Hz, C–CH2–O); IR (KBr, cmÀ1): 3427, 3084,
2962, 2824, 1721, 1615, 1560, 1512, 1384, 1276, 1156, 1110; MS
(ESI, m/z): [M+H]+ 322.
2H-chromen-2-one (C2).
Yield, 68.7%; yellowish crystals, mp:
233–235 °C; 1H NMR (CDCl3, 300 MHz) d, ppm: 3.81 (3H, s,
OCH3), 6.24 (1H, d, J = 9.3 Hz, H-3), 7.56 (1H, d, J = 9.6 Hz, H-4),
6.80 (1H, s, H-5), 6.80 (1H, s, H-8), 1.43 (6H, t, 2CH3), 3.30 (4H,
m, 2 N–CH2–CH3), 3.42 (2H, t, N–CH2–C), 4.56 (2H, t, C–CH2–O);
R (KBr, cmÀ1): 3428, 3050, 2946, 1721, 1614, 1565, 1513, 1387,
1278, 1146; MS (ESI, m/z): [M+H]+ 291.
4.1.7.3. Preparation of 7-(2-hydroxy-3-morpholinopropoxy)-
4.1.6.3. Preparation of 6-methoxy-7-(2-morpholinoethoxy)-2H-
chromen-2-one (C3). Yield, 81.97%; yellowish crystals, mp: 136–
139 °C; 1H NMR (CDCl3, 300 MHz) d, ppm: 3.92 (3H, s, OCH3),
6.25 (1H, d, J = 9.6 Hz, H-3), 7.56 (1H, d, J = 9.3 Hz, H-4), 6.99 (1H,
s, H-5), 7.00 (1H, s, H-8), 2.96–3.76 (8H, m, morpholine), 3.20
(2H, t, N–CH2), 4.24 (2H, t, CH2–O); IR (KBr, cmÀ1): 3494, 3050,
2946, 1721, 1614, 1565, 1513, 1387, 1278, 1146; MS (ESI, m/z):
[M+H]+ 306.
6-methoxy-2H-chromen-2-one (D3).
Yield, 74.6%; yellowish
powder, mp: 120–122 °C; 1H NMR (CDCl3, 300 MHz) d, ppm: 3.97
(3H, s, OCH3), 6.31 (1H, d, J = 9.3 Hz, H3), 7.63 (1H, d, J = 9.6 Hz,
H4), 6.86 (1H, s, H5), 6.89 (1H, s, H8), 4.27 (2H, d, J = 3.9 Hz, C–
CH2–O), 4.52 (1H, m, CH–OH), 2.42 (2H, d, J = 6.3 Hz, N–CH2–C),
2.50–3.57 (8H, m, morpholine); IR (KBr, cmÀ1): 3421, 2929, 2854,
1721, 1651, 1560, 1509, 1385, 1276, 1115; MS (ESI, m/z): [M+H]+
336.
4.1.6.4. Preparation of 6-methoxy-7-(2-(4-methylpiperazin-1-
4.1.7.4. Preparation of 7-(2-hydroxy-3-(4-methylpiperazin-
yl)ethoxy)-2H-chromen-2-one (C4).
Yield, 75.7%; yellow
1-yl)propoxy)-6-methoxy-2H-chromen-2-one (D4).
Yield,
powder, mp: 198–201 °C; 1H NMR (CDCl3, 300 MHz) d, ppm: 3.82
(3H, s, OCH3), 6.24 (1H, d, J = 9.6 Hz, H3), 7.56 (1H, d, J = 9.3 Hz,
H4), 6.76 (1H, s, H5), 6.78 (1H, s, H8), 2.40 (3H, s, CH3), 2.76–3.58
(8H, m, piperazine), 2.87 (2H, t, N–CH2–C), 4.12 (2H, t, CH2–O);
IR (KBr, cmÀ1): 3450, 1943, 2837, 1712, 1618, 1566, 1513, 1385,
1297, 1151; MS (ESI, m/z): [M+H]+ 319.
50.4%; yellowish powder, mp: 98–100 °C; 1H NMR (CDCl3,
300 MHz) d, ppm: 3.89 (3H, s, OCH3), 6.30 (1H, d, J = 9.3 Hz, H3),
7.63 (1H, d, J = 9.6 Hz, H4), 6.85 (1H, s, H5), 6.88 (1H, s, H8), 2.30
(3H, s, CH3), 2.63–2.71 (8H, m, piperazine), 2.71 (2H, d, N–CH2–
C), 4.18 (1H, m, CH–OH), 4.07 (2H, d, J = 5.7 Hz, CH2–O); IR (KBr,
cmÀ1): 3402, 3083, 2932, 2805, 1720, 1614, 1560, 1511, 1384,
1277, 1154; MS (ESI, m/z): [M+H]+ 349.
4.1.6.5. Preparation of 6-methoxy-7-(2-(pyrrolidin-1-yl)eth-
oxy)-2H-chromen-2-one (C5). Yield, 69.2%; yellow powder, mp:
212–215 °C; 1H NMR (CDCl3, 300 MHz) d, ppm: 3.82 (3H, s,
OCH3), 6.21 (1H, d, J = 9.6 Hz, H3), 7.54 (1H, d, J = 9.6 Hz, H4), 6.79
(1H, s, H5), 6.80 (1H, s, H8), 1.93–2.99 (8H, m, pyrrolidine), 3.21
(2H, t, N–CH2–C), 4.34 (2H, t, CH2–O); IR (KBr, cmÀ1): 3463,
3026, 2964, 2930, 1711, 1613, 1564, 1513, 1387, 1278, 1145; MS
(ESI, m/z): [M+H]+ 290.
4.1.7.5.
Preparation
of
7-(2-hydroxy-3-(pyrrolidin-1-yl)-
Yield, 94.0%;
propoxy)-6-methoxy-2H-chromen-2-one (D5).
yellowish powder, mp: 105–108 °C; 1H NMR (CDCl3, 300 MHz) d,
ppm: 3.89 (3H, s, OCH3), 6.30 (1H, d, J = 9.3 Hz, H3), 7.63 (1H, d,
J = 9.6 Hz, H4), 6.85 (1H, s, H5), 6.88 (1H, s, H8), 2.30 (3H, s, CH3),
1.83–2.63 (8H, m, pyrrolidine), 4.20 (1H, m, CH–OH), 4.08 (2H, d,
J = 4.2 Hz, CH2–O), 2.77–2.84 (2H, d, N–CH2–C); IR (KBr, cmÀ1):
3402, 3081, 2955, 2806, 1719, 1613, 1562, 1512, 1389, 1276,
1142; MS (ESI, m/z): [M+H]+ 320.
4.1.6.6. Preparation of 6-methoxy-7-(2-(piperidin-1-yl)ethoxy)-
2H-chromen-2-one (C6). Yield, 72.8%; yellow powder, mp: 115–
117 °C; 1H NMR (CDCl3, 300 MHz) d, ppm: 3.82 (3H, s, OCH3),
6.24 (1H, d, J = 9.6 Hz, H3), 7.54 (1H, d, J = 9.6 Hz, H4), 6.70 (1H, s,
H-5), 6.79 (1H, s, H8), 4.34 (2H, t, CH2–O), 2.94 (2H, t, N–CH2–C),
1.48–2.65 (10H, m, piperidin); IR (KBr, cm–1): 3291, 3086, 2934,
1715, 1614, 1561, 1510, 1385, 1282, 1141; MS (ESI, m/z): [M+H]+
304.
4.1.7.6.
Preparation
of
7-(2-hydroxy-3-(piperidin-1-yl)-
Yield, 90.4%;
propoxy)-6-methoxy-2H-chromen-2-one (D6).
yellowish powder, mp: 158–160 °C; 1H NMR (CDCl3, 300 MHz) d,
ppm: 3.78 (3H, s, OCH3), 6.60 (1H, d, J = 9.6 Hz, H3), 7.86 (1H, d,
J = 9.3 Hz, H-4), 6.87 (1H, s, H-5), 6.95 (1H, s, H-8), 4.07 (2H, t, C–
CH2–O), 3.89 (1H, m, CH–OH), 2.58 (2H, d, J = 9.3 Hz, N–CH2–C),
1.62–2.76 (10H, m, piperidine); IR (KBr, cmÀ1): 3428, 3000, 2933,

X. Cai et al. / Bioorg. Med. Chem. 21 (2013) 84–92
91
2855, 1710, 1620, 1577, 1515, 1453, 1209, 1123; MS (ESI, m/z):
4.2.5. Cytotoxicity assay
[M+H]+ 334.
The cytotoxicity of the scopoletin derivatives, which have po-
tent HUVEC inhibition ability, was analyzed by MTT assay with
PBMC among the various cancer cell lines. The PBMCs were sepa-
rated from 10 mL of heparinized human blood samples by centrifu-
gation on a human lymphocyte separation medium (Tbdscience
Biotech, Tianjin, China).
4.2. Pharmacology
4.2.1. Materials
The scopoletin and its derivatives were kindly provided by Dr.
Jinpei Zhou. The fertilized chicken eggs were purchased from
QianYuanHao Biological Corporation Limited (Nanjing, China).
The Medium 199 (M199), Dulbecco’s modified Eagle medium
(DMEM), fetal bovine serum (FBS), penicillin streptomycin
(10,000 units/mL penicillin; 10,000 ug/mL streptomycin) were ob-
tained from Gibco (Grand Island, NY, USA). The endothelial cell
growth supplement (ECGS) was purchased from the Upstate Bio-
technology (Lake Placid, NY, USA). The recombinant human
VEGF165 was obtained from Pepro Tech Inc. (Rocky Hill, NJ, USA).
Trypsin, EDTA, and Na2 were obtained from Amresco (Solon, OH,
USA). Heparin sodium was obtained from Wanbang (Jiangsu, China).
Rabbit anti-Factor VIII Related Antigen antibody was purchased from
Zhongshan Goldenbridge Biotechnology (Beijing, China). The anti-
bodies p-ERK1/2, ERK1/2, p-p38, p38, p-Ake, Akt, p-Src, Src, and
GAPDH were obtained from Cell Signaling Technology, USA.
4.2.6. Endothelial cell migration assay
The migration ability of HUVECs was examined by Boyden
Chamber assay. The HUVEC with 80% confluence was washed
once with PBS and was serum-starved in the basal media (with-
out serum and growth supplements) for 12 h. The harvested cells
were counted, and reached a volume of 1 Â 106 cells/mL. DMSO
(control) and scopoletin or scopoletin derivatives (final concentra-
tion: 25
room temperature with 500
l
mol/L) were added to the cell suspension for 30 min at
L of fresh M199 medium (1% FBS)
l
containing 10 ng/mL VEGF165 in the lower wells (24-well plate)
(Corning, NY, USA). The cell migration Millicell chamber
(8.0
lm/6.5 mm Billerico, USA) was inserted at an angle of 45°,
gently pressed down to avoid generating bubbles, and 100
l
L of
the pre-treated cell suspension was added to each chambers.
After 20 h of incubation, the cells were fixed and stained. Five
randomly chosen fields were counted and photographed using a
fluorescence microscope (Zeiss Axio Observer A1, Oberkochen,
Germany).
4.2.2. Culture of tumor cell lines
The human pancreatic cell lines PANC-1 and BxPC-3, human
breast cancer cell lines MDA-MB-231 and MDA-MB-435, human li-
ver cancer cell line HepG2, human colon cancer cell line HCT 116,
human stomach cancer cell line MKN45, human non-small cell
lung cancer cell line A549, human epithelial carcinoma cell line
A431, human glioma cell line SHG-44, human erythromyeloblas-
toid leukemia cell line K562, human promyelocytic leukemia cell
line HL-60, and human T lymphoma cell line HuT 78 were all pur-
chased from the Cell Bank of the Shanghai Institute of Biochemistry
& Cell Biology, Shanghai Institute for Biological Sciences and
Chinese Academy of Sciences. All cells lines were maintained in a
DMEM or RPMI-1640 medium supplemented with 10% FBS,
4.2.7. Tube formation assay
In sterile conditions, the 96-well plates were coated with Matri-
gel at 50 lL/well (BD Biosciences, Bedford, MA, USA) without
introducing air bubbles, and set at 37 °C for 30 min to allow gel-
ling. The HUVEC was pre-incubated in M199 containing 1% FBS
for 6 h, and then harvested after trypsin treatment and was sus-
pended in M199 containing 1% FBS and 10 ng/mL VEGF165 at
5 Â 105 cells/mL. DMSO (control group) and scopoletin or scopole-
100 U/mL penicillin, and 100 lg/mL streptomycin (all available
from Invitrogen, Grand Island, NY, USA), and incubated in a humid-
ified atmosphere of 95% air + 5% CO2 at 37 °C.
tin derivatives (final concentration: 25 lmol/L) were added to the
cells for 30 min before plating onto the Matrigel. After 5 h, the HU-
VECs differentiated and formed into capillary-like structures on
the Matrigel. The enclosed networks of complete tubes from five
randomly chosen fields were counted and photographed using a
microscope (100Â).
4.2.3. Isolation and characterization of endothelial cell
The HUVECs were isolated from the human umbilical cord vein
by trypsin treatment. The human umbilical vein obtained from
healthy newborns of healthy mothers was treated with 0.1% tryp-
sin for 12 min. The harvested endothelial cells were grown in
M199 containing 15% FBS, 0.1 mg/mL heparin, 0.03 mg/mL ECGS,
4.2.8. Chicken chorioallantoic membrane (CAM) assay
The fertilized chicken eggs were incubated for seven days at
a relative humidity of ꢀ55% at 37.5 °C. The eggs were arranged
with the rounded portion positioned upwards at 45° to prevent
microbial growth, and were regularly turned (at least two times
per day). On the experiment day, the eggs were inspected using
an egg candler and only live, fertilized eggs were randomly
divided with ten eggs per group. A 1.5 Â 1.5 cm ‘window’ was
created on the rounded portion of the egg where the air sac
was located. The air sac membrane was carefully punctured
using an injection needle to avoid breaking the blood vessel.
100 U/mL penicillin, and 100 lg/mL streptomycin. Cell character-
ization was evaluated by assessing Factor VIII Related Antigen
(von Willebrand factor, vWF) expression, which was detected by
immunocytochemical reaction with the use of the rabbit anti-Fac-
tor VIII Related Antigen antibody. Cell characteristic detection was
achieved using DAB (Zhongshan Goldenbridge Biotechnology, Bei-
jing, China). After 3–5 passages, HUVECs were collected, which
were used in all experiments.
About 50 lL of sterilized water was injected between the air
4.2.4. Endothelial cell proliferation assay
The HUVECs were seeded in 96-well plates consisting of 1 Â 104
cells per well. The medium was replaced by a low-serum medium
(1% fetal bovine serum in M199) containing scopoletin or deriva-
tives. After 24 h of cell treatment, the viability of the HUVECs
were analyzed by MTT [3-(4,5-dimethylthiazol-2-yl)-2 and
5-diphenyltetrazolium bromide] assay as previously described.14
The inhibition percentage of the treated cells was calculated
sac membrane and the CAM so that the air sac membrane
could be easily peeled off. Sterilized filter paper (5 Â 5 mm)
saturated with DMSO (control group) and scopoletin (50 and
100 nmol/egg) or scopoletin derivatives (5 and 10 nmol/egg)
were air-dried and then placed on the CAMs. The eggs were
then covered with parafilm and were placed back to the incu-
bator. After three days, 20% fat emulsion (Chia-tai Tianqing
Pharmaceutical Co., Jiangsu, China) was injected into the CAM,
using the formula:
(A570nm À A630nm control
atives were further assayed.
%
inhibition = 1 À (A570nm À A630nm treated
)
/
and the blood vessels were photographed using a Sony
camera (Sony, Japan).
a100
)
 100%. The IC50 of several scopoletin deriv-

92
X. Cai et al. / Bioorg. Med. Chem. 21 (2013) 84–92
4.2.9. Zymography
5. Statistical analysis
The HUVECs were treated with DMSO (control group) and
scopoletin (100 mol/L) or A1 (10, 25, and 50 mol/L) for 12 h.
l
l
All results shown are expressed as means SEM from triplicate
experiments that were performed in a parallel manner unless
otherwise indicated. Statistical analyses were performed using an
unpaired, two-tailed Student’s t-test. All comparisons were made
relative to the untreated control group, and the significance of dif-
The suspension was collected and centrifuged to detect the activity
of MMP-2 and MMP-9 enzymes. The samples were prepared in the
standard SDS–PAGE treatment buffer but without boiling and
without a reducing agent. For the substrate, 1 mg/mL gelatin was
pre-polymerized on a 10% polyacrylamide gel. After electrophore-
sis, the SDS was removed from the gel and was incubated in a
raising buffer (2.5% Triton X-100, 50 mmol/L Tris–HCl, 0.05%
⁄
ference was indicated as P <0.01.
Acknowledgments
NaN3, 5 mmol/L CaCl2, 1
incubation in an appropriate digestion buffer (50 mmol/L
Tris–HCl, 0.05% NaN3, 5 mmol/L CaCl2, and mol/L ZnCl2,
lmol/L ZnCl2, and pH 7.6), followed by
This work was supported by the National Natural Science Foun-
dation of China (Nos. 81274150, 81202967 and 30873410) and
Jiangsu Province’s Outstanding Leader Program of Traditional Chi-
nese Medicine.
1
l
pH7.6) for 14 h at 37 °C. The zymogram was subsequently stained
with coomassie brilliant blue. The clear bands against the darkly
stained background were photographed using the Gel/Chemi Doc
System (Bio-rad, CA, USA).
References and notes
4.2.10. Western blot
1. Singh, R.; Ahmed, S.; Islam, N.; Goldberg, V. M.; Haqqi, T. M. Arthritis Rheum.
2002, 46, 2079.
2. Kok, T. W.; Yue, P. Y.; Mak, N. K.; Fan, T. P.; Liu, L.; Wong, R. N. Angiogenesis
2005, 8, 3.
3. Moon, P. D.; Lee, B. H.; Jeong, H. J.; An, H. J.; Park, S. J.; Kim, H. R.; Ko, S. G.; Um, J.
Y.; Hong, S. H.; Kim, H. M. Eur. J. Pharmacol. 2007, 555, 218.
4. Nam, N. H.; Kim, Y.; You, Y. J.; Hong, D. H.; Kim, H. M.; Ahn, B. Z. Bioorg. Med.
Chem. Lett. 2002, 12, 2345.
5. Lee, S.; Sivakumar, K.; Shin, W. S.; Xie, F.; Wang, Q. Bioorg. Med. Chem. Lett.
2006, 16, 4596.
The HUVECs were pre-treated with DMSO (control group) and
scopoletin (100 lmol/L) or A1 (10, 25, and 50 lmol/L) for 30 min
and were stimulated with 40 ng/mL VEGF165 for 5 min (for p-
ERK1/2, p-p38 protein detection) or 30 min (for p-Akt, p-Src protein
detection). After stimulation, the cells were collected and lysed on
ice. The lysate was centrifuged at 13,000Âg for 5 min at 4 °C. The
protein concentration in the supernatants was detected using a
Nanodrop 1000 Spectrophotometer (Thermo, NH, USA). An equal
amount of protein was separated on 13% SDS–polyacrylamide gels
(SDS–PAGE) and transferred onto the PVDF membranes (Millipore,
MA, USA). The proteins were detected using specific primary anti-
bodies (p-ERK1/2, ERK1/2, p-p38, p38, p-Ake, Akt, p-Src, Src, and
GAPDH) for 2 h at room temperature, followed by HRP-conjugated
secondary antibodies for 1 h at room temperature. The detection of
the developed proteins was performed using enhanced chemilumi-
nescence (Luminata Crescendo Western HRP substrate, Millipore,
USA). The membranes were then exposed to the film.
6. Zhou, J.; Wang, L.; Wei, L.; Zheng, Y.; Zhang, H.; Wang, Y.; Cao, P.; Niu, A.; Wang,
J.; Dai, Y. Lett. Drug Des. Disc. 2012, 9, 397.
7. Lobov, I. B.; Renard, R. A.; Papadopoulos, N.; Gale, N. W.; Thurston, G.;
Yancopoulos, G. D.; Wiegand, S. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 3219.
8. Mendes, O.; Kim, H. T.; Stoica, G. Clin. Exp. Metastasis 2005, 22, 237.
9. Bullard, L. E.; Qi, X.; Penn, J. S. Invest. Ophthalmol. Vis. Sci. 2003, 44, 1722.
10. Landry, J.; Huot, J. Biochem. Soc. Symp. 1999, 64, 79.
11. Liekens, S.; De Clercq, E.; Neyts, J. Biochem. Pharmacol. 2001, 61, 253.
12. Papetti, M.; Herman, I. M. Am. J. Physiol. Cell Physiol. 2002, 282, C947.
13. Rousseau, S.; Houle, F.; Landry, J.; Huot, J. Oncogene 1997, 15, 2169.
14. Cai, X.; Ye, T.; Liu, C.; Lu, W.; Lu, M.; Zhang, J.; Wang, M.; Cao, P. Toxicol. In Vitro
2011, 25, 1385.