Synthesis and evaluation of N-(benzofuran-5-yl)aromaticsulfonamide derivatives as novel HIF-1 inhibitors that possess anti-angiogenic potential

Article abstract of DOI:10.1016/j.bmc.2016.06.021

Hypoxia-inducible factor-1 (HIF-1) as a key mediator in tumor metastasis, angiogenesis, and poor patient prognosis has been recognized as an important cancer drug target. A novel series of N-(benzofuran-5-yl)aromaticsulfonamide derivatives were synthesized and evaluated as HIF-1 inhibitor. Among these compounds, 7q exhibited specific inhibitory effects on HIF-1 by downregulating the expression of HIF-1α under hypoxic conditions. It inhibited the HIF-1 transcriptional activity (IC₅₀?=?12.5?±?0.7?μM) and secretion of VEGF (IC₅₀?=?18.8?μM) in MCF-7 cells. Meanwhile, it also significantly suppressed hypoxia-induced migration of HUVEC cells in nontoxic concentrations. Additionally, tube formation assay demonstrated its anti-angiogenesis activity. Finally, the in vivo study indicated that compound 7q could retard angiogenesis in CAM model. These findings supported the HIF-1 inhibitory effect and anti-angiogenic potential of this class of compounds as HIF-1 inhibitor.

Full text of DOI:10.1016/j.bmc.2016.06.021

Accepted Manuscript
Systhesis and evaluation of N-(benzofuran-5-yl) aromaticsulfonamide deriva-
tives as novel HIF-1 inhibitors that possess anti-angiogenic potential
Jinlian Wei, Yingrui Yang, Yali Li, Xiaofei Mo, Xiaoke Guo, Xiaojin Zhang,
Xiaoli Xu, Zhengyu Jiang, Qidong You
PII:
S0968-0896(16)30444-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.021
BMC 13077
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 May 2016
8 June 2016
11 June 2016
Please cite this article as: Wei, J., Yang, Y., Li, Y., Mo, X., Guo, X., Zhang, X., Xu, X., Jiang, Z., You, Q., Systhesis
and evaluation of N-(benzofuran-5-yl) aromaticsulfonamide derivatives as novel HIF-1 inhibitors that possess anti-
angiogenic potential, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.06.021
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Systhesis and evaluation of N-(benzofuran-5-yl) aromaticsulfonamide derivatives as novel HIF-1
inhibitors that possess anti-angiogenic potential
Jinlian Wei,a,b Yingrui Yang,a,b Yali Li,a,b Xiaofei Mo,a,b Xiaoke Guo, a,b Xiaojin Zhang,a,b,c Xiaoli Xu,a,b
Zhengyu Jiang, a,b and Qidong You, a,b, 
aState Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing 210009, China
bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
cDepartment of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing 210009, China

Bioorganic & Medicinal Chemistry
Systhesis and evaluation of N-(benzofuran-5-yl) aromaticsulfonamide derivatives as novel HIF-1
inhibitors that possess anti-angiogenic potential
Jinlian Wei,a,b Yingrui Yang,a,b Yali Li,a,b Xiaofei Mo,a,b Xiaoke Guo, a,b Xiaojin Zhang,a,b,c Xiaoli Xu,a,b Zhengyu
Jiang, a,b and Qidong You, a,b, 
aState Key Laboratory of Natural Medicines, and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing
210009, China
bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
cDepartment of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing 210009, China
ARTICLE INFO
ABSTRACT
Hypoxia-inducible factor-1 (HIF-1) as a key mediator in tumor metastasis, angiogenesis, and
poor patient prognosis has been recognized as an important cancer drug target. A novel series of
N-(benzofuran-5-yl) aromaticsulfonamide derivatives were synthesized and evaluated as HIF-1
inhibitor. Among these compounds, 7q exhibited specific inhibitory effects on HIF-1 by
downregulating the expression of HIF-1α under hypoxic conditions. It inhibited the HIF-1
transcriptional activity (IC50 = 12.5 ± 0.7 μM) and secretion of VEGF (IC50 = 18.8 μM) in MCF-
7 cells. Meanwhile, it also significantly suppressed hypoxia-induced migration of HUVEC cells
in nontoxic concentrations. Additionally, tube formation assay demonstrated its anti-
angiogenesis activity. Finally, the in vivo study indicated that compound 7q could retard
angiogenesis in CAM model. These findings supported the HIF-1 inhibitory effect and anti-
angiogenic potential of this class of compounds as HIF-1 inhibitor.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
N-(benzofuran-5-yl) aromaticsulfonamide derivatives
HIF-1
Transcriptional activity
Migration
Anti-angiogenesis
2016 Elsevier Ltd. All rights reserved.
inhibiting HIF (FIH), whose catalysis is also O2-dependent.11
1. Introduction
There is an increasing number of focuses on transcription
factors which are found to be associated with various cellular
progress.1 Hypoxia is a hallmark of many malignant tumors and
However, under hypoxic conditions, HIF-1α is stabilized without
being hydroxylated by PHD and FIH, translocates into the
nuclear and heterodimerizes with HIF-1β, and then recruits
transcriptional coactivator proteins.12-13 The complex HIF-
1α/HIF-1β/coactivator protein initiates an adaptive hypoxic
transcription of a vast variety of target genes through binding
with their hypoxia-responsive elements (HREs), including those
protein products that are correlated with angiogenesis, metabolic
reprogramming, invasion and metastasis of cancer cells.2 It has
been well established that HIF-1α plays a significant role in
multiple processes of cancer development, therefore, it is
recognized as a promising target for cancer therapy.
is regulated by hypoxia-inducible factor-1 (HIF-1),
a
transcription factor orchestrating the expression of many genes
that code for proteins involved in angiogenesis, glucose
metabolism, cell proliferation and metastasis.2 Overexpression of
HIF-1 in tumor cells is closely connected with the more resistant
to radio- and chemo-therapies, the increased risk of metastasis,
the more aggressive phenotype and the strengthened immune
suppression.3 Consequently, inhibition of HIF-1 pathway may
contribute to specific cancer therapies, especially those cancers
tightly correlated to hypoxia.4,5
Among diverse cancer types, breast cancer stands out
because of its increasing incidence rate and high mortality
worldwide.14 Breast cancer has become the second leading cause
of death in female patients only after lung cancer.15 Evidences
supported that HIF-1 was correlated with poor prognosis in
human breast cancer due to the high metastasis and HIF-1
expression.2,16 It has been reported that expression of HIF-1
target genes was increased in the triple-negative breast cancer.17-
HIF-1 is a bHLH-PAS (basic-helix-loop-helix-PER-ARNT-
SIM) heterodimer consisting of two subunits: HIF-1α and HIF-1β
(also known as the aryl hydrocarbon receptor nuclear
translocator, ARNT).6 The level of HIF-1α is O2-dependent while
HIF-1β is constitutively expressed.7 Under normal oxygen
conditions, HIF-1α is hydroxylated by prolyl hydroxylase (PHD)
at residues of Pro402 and Pro564, and is subjected to proteasome
degradation through the following recognition of von Hippel-
Lindau (pVHL).8-10 Besides, Asn803 of HIF-1α C-terminal
transactivation domain (C-TAD) is hydroxylated by the factor-
———
18
Breast cancer cell lines, such as MCF-7 and MDA-MB-231,
have been used as classical tools for HIF-1 pathway research.19
So far, several HIF-1 inhibitors have been discovered, such as
 Corresponding author. Tel. / fax: +86-25-8327-1216; e-mail: youqd@163.com

topotecan,20 bortezomib,21 YC-1,22 and PX-478 (Fig. 1).23
Topotecan and bortezomib have been approved for marketing,24-
2.2 Pharmacology
2.2.1. N-(benzofuran-5-yl) aromaticsulfonamide derivatives
inhibit HIF-1 transcriptional activity
25
YC-1 and PX-478 are undergoing clinical trials on patients
expressing high levels of HIF-1α in their tumors.26-27 However,
the chemotype of HIF-1α inhibitor is still limited, discovery of
new HIF-1α inhibitor with better efficiency is still an important
task for medicinal chemists.
The N-(benzofuran-5-yl) aromaticsulfonamide derivatives
7a-u were tested for their inhibitory effect on HIF-1
transcriptional activity in MCF-7 cells under hypoxic conditions
(1% O2) using dual luciferase reporter gene assay (Table 1). PX-
478 was used as a positive control. Different chemical groups,
including methyl, chloro, bromo, trifluoromethyl, tert-butyl and
nitro (7a~7p) were introduced onto the benzene ring of
benzenesulfonamide. Generally, these groups did not affect the
HRE activity obviously, indicating that mono-substitution was
not favorable to the HIF-1 inhibition. Meanwhile, compounds
with ortho-substituted phenyl (7c, 7f, 7i, 7l, 7o) exhibited better
HIF-1 inhibitory potency, compared to the corresponding meta-
(7b, 7e, 7h, 7k, 7n) and para- (7a, 7d, 7g, 7j, 7m) ones (Table 1).
There was an enhancement in HIF-1 inhibition when 3,4-
dimethoxylphenyl (7q) was introduced to the structure of N-
(benzofuran-5-yl) sulfonamide, with an IC50 value of 12.5 ± 0.7
μM. 2,5-disubstituted methoxylphenyl (7r) was also introduced,
with an IC50 value of 15.7 ± 1.6 μM, suggesting that
dimethoxylphenyl might be better options. To examine the
importance of the benzenesulfonamide moiety, the benzene-ring
was replaced by other rings including 2-naphthyl (7s), 2-thienyl
(7t) and 3-pyridyl (7u). 7s maintained its activity in HRE assays,
consistent with that of compound 7p. It indicated larger and more
hydrophobic groups were preferred at benzenesulfonamide
moiety. On the contrary, substitution with aromatic heterocycles
reduced the HIF-1 transcriptional activity, suggesting that polar
groups were popular in further optimization. The antiproliferative
activities of 7a-u toward human breast cancer cell lines were
weaker than their HIF-1 transcription inhibitory activities (Table
1), revealing that N-(benzofuran-5-yl) aromaticsulfonamide
derivatives 7a-u suppressed HIF-1 transcriptional activity
without cytotoxicity. The results were the same with those
previously reported HIF-1 inhibitors, such as YC-1 and PX-478,
which inhibited HIF-1 transcriptional activity at lower
concentrations and blocked tumor cell proliferation at much
higher concentrations.26-27 It is probably due to the hypoxia
microenvironment itself, inhibiting HIF-1 pathway could
primarily affect progresses of angiogenesis, migration and
invasion, while the tumor growth could take place later.2 Together,
compound 7q with the best HIF-1 inhibitory effect (IC50 = 12.5 ±
0.7 μM, slightly more potent than positive control PX-478, IC50 =
20.2 ± 4.4 μM) was selected for further biological evaluation.
Figure 1. Structures of representative HIF-1 inhibitors.
We have carried out a screening of our in-house compound
collection for searching compounds that can inhibit HIF-1α
transcriptional activity in MCF-7 cells under hypoxic condition
by a cell-based HRE reporter assay. Compound 7a, with N-
(benzofuran-5-yl) benzenesulfonamide core, showed inhibitory
effect on HIF-1α transcriptional activity. Considering this was a
new chemotype for HIF-1α inhibition, it was then recognized as
the starting compound for further structural optimization.
2. Results and discussion
2.1 Chemistry
Some novel N-(benzofuran-5-yl) aromaticsulfonamide
derivatives were designed by changing the substituents on other
benzene-ring or replacing the benzene-ring with naphthyl, thienyl
or pyridyl in order to investigate its preliminary structure-activity
of HIF-1 inhibition. The target compounds were synthesized as
shown in Scheme 1. 4-methoxyaniline (1) was treated with Ac2O,
and the protected aniline 2 was converted to N-(3-acetyl-4-
hydroxyphenyl) acetamide 3 by Friedel-Crafts acylation. 4 was
formatted through nucleophilic substitution of
3
and
BrCH2COOEt under alkaline conditions. Furan ring 5 was
obtained through a cyclization via microwave. Deacetylation of 5
in the acidic condition generated intermediate 6. The final
products of N-(benzofuran-5-yl) aromaticsulfonamides 7a-u
were generated through condensation reactions of 6 with
different sulfonyl chloride.
Scheme 1. Synthesis of N-(benzofuran-5-yl) aromaticsulfonamide 7a-u. Reagents and conditions: (a) DCM, Ac2O, N2, reflux, 2 h,

94%; (b) DCM, AcCl2, AlCl3, reflux, 12 h, 78%; (c) DMF, BrCH2COOEt, K2CO3, KI, 80 oC, 4 h, 87%; (d) DMF, K2CO3, microwave,
145 oC, 7 min, 59%; (e) EtOH, 3M HCl, N2, reflux, 6 h, 71%; (f) DCM, RSO2Cl, pyridine, rt., 12 h, 56% ~ 82%.
2.2.2. 7q suppresses HIF-1α and VEGF
mechanism of 7q, we first examined the effects of 7q on the
expression of HIF-1α in hypoxia. As shown by the data, the
levels of HIF-1α mRNA were unchanged without 7q treatment in
both normoxia and hypoxia, but were decreased after the
treatment of 7q in hypoxia in a dose-dependent manner (Fig. 2a).
Compared with little expression in normoxia, HIF-1α protein
level was greatly boosted under hypoxia,33-34 which can be
reduced by 7q in a concentration-dependent manner in MCF-7
cells (Fig. 2b-c).
The levels of HIF-1 mainly depended on the regulation of
HIF-1α, which is oxygen sensitive. Many HIF-1α inhibitors have
been developed and their mechanisms of action have been fully
demonstrated, such as to decrease HIF-1α mRNA levels,28 to
inhibit HIF-1α protein synthesis,29 to promote HIF-1α
degradation,30 to block HIF-1α binding to DNA,31 and to
suppress HIF-1α transcriptional activity.32 In order to explore the
Table 1. In vitro inhibition of HIF-1α transcriptional activity in cell-based HRE reporter assay under hypoxia conditions and
cytotoxicity under hypoxia conditions.
Antiproliferative activity IC50 (μM) b
HRE-Luc
ID
R
IC50 (μM)a
45.4 ± 8.3
32.7 ± 2.8
19.4 ± 1.8
50.0 ± 5.8
36.6 ± 0.5
20.8 ± 2.1
29.9 ± 3.3
25.6± 2.8
19.9 ± 1.6
41.7 ± 3.9
36.3 ± 5.8
16.5 ± 2.0
36.2 ± 5.1
29.4 ± 2.5
24.4 ± 3.7
27.1 ± 1.9
12.5 ± 0.7
15.5 ± 2.6
22.6 ± 3.2
45.4 ± 4.5
34.1 ± 2.9
20.2 ± 4.4
MCF-7
53.9 ± 7.7
101.2 ± 9.9
105.2 ± 8.2
81.2 ± 5.4
83.9 ± 8.1
>200
56.5 ± 2.3
57.0 ± 0.4
185.4 ± 23.6
48.7 ± 1.4
51.4 ± 0.2
93.9 ± 0.9
69.6 ± 7.9
74.8 ± 3.5
156.9 ± 10.1
22.1 ± 2.3
52.0 ± 3.8
52.3 ± 7.2
20.8 ± 3.1
92.3 ± 10.2
>200
MDA-MB-231
25.2 ± 1.8
29.5 ± 1.5
89.2 ± 7.0
53.9 ± 2.1
57.3 ± 1.0
>200
31.1 ± 4.0
33.7 ± 3.4
178.7 ± 9.5
24.3 ± 4.2
30.2 ± 2.8
82.4 ± 5.6
33.1 ± 1.6
59.9 ± 8.7
80.4 ± 7.3
22.4 ± 1.7
35.4 ± 3.1
26.7 ± 2.7
21.6 ± 1.6
44.1 ± 1.3
140.8 ± 12.7
62.8 ± 9.0
MDA-MB-468
24.0 ± 1.2
25.3 ± 5.2
53.4 ± 2.8
44.0 ± 1.0
53.7 ± 4.7
137.4 ± 2.2
26.4 ± 3.6
30.7 ± 1.9
138.4 ± 21.3
27.2 ± 3.6
28.9 ± 4.2
74.9 ± 3.2
24.0 ± 3.8
30.1 ± 4.1
96.9 ± 6.2
8.1 ± 1.1
4-methylphenyl
3- methylphenyl
2- methylphenyl
4-chlorophenyl
3- chlorophenyl
2- chlorophenyl
4-bromophenyl
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
3- bromophenyl
2- bromophenyl
4-(trifluoromethyl)phenyl
3-(trifluoromethyl)phenyl
2-(trifluoromethyl)phenyl
4-nitrophenyl
3- nitrophenyl
2- nitrophenyl
4-tert-butylphenyl
3,4-dimethoxylphenyl
2,5-dimethoxylphenyl
2-naphthyl
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
PX-478
20.5 ± 3.0
20.7 ± 2.1
15.7 ± 0.9
34.3 ± 4.0
62.9 ± 3.9
45.1± 3.6
2-thienyl
3-pyridyl
19.7 ± 1.1

Figure 2. The effects of compound 7q on the expression levels of HIF-1α and VEGF in MCF-7 cells. (a) HIF-1α mRNA was measured
by RT-PCR analyses in MCF-7 cells after 7q treatment for 24h under normixia or hypoxia conditions. β-actin was used as internal
control. Bars are the mean ± SD (n = 3). Statistical significance: *: P < 0.05, **: P < 0.01, and ***: P < 0.001 versus hypoxia control. (b)
HIF-1α protein expression was detected by immunoblot analysis with the specific antibody. β-actin was used as an internal control. (c)
For quantity of (b), images were analyzed using ImageJ software. Bars are the mean ± SD (n = 3). The comparisons were made relative
to hypoxia alone group, and the different levels of significance were indicated as *P < 0.05 and **P < 0.01 and ***: P < 0.001. (d)
mRNA levels of VEGF was detected by RT-PCR. β-actin was used as an internal control. Bars are the mean ± SD (n = 3). Statistical
significance: *: P < 0.05, **: P < 0.01, and ***: P < 0.001 versus hypoxia control. (e) The secretion of VEGF protein in MCF-7 cells
was obviously suppressed by 7q. The cells were incubated for 24 h with or without treatment. Bars are the mean ± SD (n = 3).
Statistical significance: **: P < 0.01, and ***: P < 0.001 versus hypoxia control.
biological of 7q, we subsequently performed wound healing
migration assays to determine 7q on HUVEC migration. To
avoid direct cytotoxic effects, the cell viability of HUVEC was
assessed by MTT at first. As shown by the data (Fig. 3a), 7q
could not induce obvious cytotoxicity for HUVEC cells. In the
next wound healing assay, the number of migrating HUVEC cells
decreased in a concentration-dependent manner (Fig. 3b-c).
These results suggested that nontoxic doses of 7q possess anti-
migratory potential.
Figure 3. The effects of 7q on hypoxia-induced migration on
HUVEC cells. (a) The cell survival of HUVECs with different
concentrations of 7q treatment (24 h) was detected using MTT
assay. All error bars are the mean ± SD (n = 3). (b) HUVEC cells
migration was measured by wound healing assay. The cells were
treated 7q at different concentrations. The straight lines indicate
the scratched wound gaps. (c) For quantity of (b), images were
analyzed using ImageJ software. Bars are the mean ± SD (n = 3).
The comparisons were made relative to the control group of 24 h,
and the different levels of significance were indicated as **: P <
0.01 and ***: P < 0.001.
During hypoxia, HIF-1α accumulates and translocates into
nuclei where it activates the transcription of downstream genes,
such as VEGF. Thus, in order to confirm the effect of 7q on HIF-
1 activation, further studies were focused on the changes of the
mRNA and protein levels of VEGF after 7q treatment. As shown
by the data, both mRNA levels of VEGF were remarkably
suppressed in a dose-dependent fashion after 7q treatment (Fig.
2d), indicating that the transcriptional activation of HIF-1α was
inhibited. The effect of compound 7q on hypoxia-induced VEGF
secretion was then measured using ELISA (enzyme-linked
immunosorbent assay) method. Data confirmed that 7q
significantly inhibited the hypoxia-induced secretion of VEGF in
MCF-7 cells in a concentration-dependent manner, and the IC50
value was calculated as 18.8 μM (Fig. 2e). Taken together, these
findings suggested that 7q suppressed HIF-1 transcriptional
activity affecting both HIF-1α mRNA levels and HIF-1α protein
accumulation, and blocked the expression of its downstream
target gene VEGF.
Figure 4. Effect of 7q on the hypoxia-enhanced HUVECs tube
formation. (a) HUVECs tube formation was measured after 7q
treatment for 24 h. The experiments were performed as described
in capillary-like tube formation. (b) Quantification of the effect
of 7q on tube formation is shown in column diagram. Bars are
the mean ± SD (n = 3). Statistical significance: *: P < 0.05, **: P
< 0.01, and ***: P < 0.001 versus hypoxia control.
2.2.4. 7q inhibits hypoxia-induced tube formation
Although the process of angiogenesis is very complex, tube
formation of endothelial cells is one of the key steps.36 Thus, we
settled down to investigate how 7q affected tube formation using
two dimensioned Matrigel assay. As shown in Fig. 4a, tube-like
structures were robustly formed under hypoxia conditions
compared with normoxia conditions. After 7q treatment, the
formation of tubular structures were greatly reduced in a
2.2.3. 7q reduces hypoxia-induced cell migration
It is well known that HIF-1 activation can promote cancer
cell migration and angiogenesis through activating transcription
of specific genes, such as VEGF.35 To explore the HIF-1 related

concentration-dependent manner (Fig. 4b). These results
revealed that 7q can inhibit angiogenesis in vitro.
5-yl) aromaticsulfonamide was identified and verified. The best
compound 7q, would provide a promising lead for further
optimization in discovering new HIF-1 inhibitors.
4. Experimental section
4.1 Chemistry
Unless stated otherwise, all reagents and solvents were used
as received from commercial suppliers. Reactions in microwave
reactors were performed by single-node heating in an Initiator
1
(Biotage). H NMR and 13C NMR spectra were recorded with a
Bruker Avance 300 MHz spectrometer at 300 MHz and 75 MHz,
respectively, at 303 K using TMS as an internal standard. High
resolution mass spectra (HRMS) were recorded on a Water Q-
Tof micro mass spectrometer. HPLC analysis for reaction
monitoring and purity determination was performed on an
Agilent1100 LC system by the standard method on an Amethyst
C18-P column (4.6 × 150 mm, 5 μm, Merck); λ = 254 nm;
mobile phase: A (acetonitrile)/B (H2O), 80:20 v/v.
4.1.1. Synthesis of N-(4-methoxyphenyl) acetamide (2)
Acetic anhydride (16.58 g, 162.40 mmol) was slowly
dripped into a stirred solution of 4-methoxyaniline (1) (10 g,
81.20 mmol) in CH2Cl2 (120 mL) using dropping funnel. After
the dripping of acetic anhydride, the reaction mixture was purged
with N2 gas and stirred at 30 oC for 2 h, and then the mixture was
evaporated to give silver-white crude product 12.60 g (76.27
Figure 5. Effect of 7q on the hypoxia-enhanced angiogenesis in
vivo using CAM assay. (a) Different concentrations of 7q were
placed on the exposed CAM for 3 days to inhibit angiogenesis. (b)
Quantification of the effect of 7q on tube formation is shown in
column diagram. Bars are the mean ± SD (n = 3). Statistical
significance: ***: P < 0.001 versus hypoxia control.
1
mmol, 94%). H NMR (300 MHz, DMSO-d6): δ 9.72 (s, 1H),
7.48 – 7.45 (m, 2H), 7.21 – 6.70 (m, 2H), 3.98 (s, 3H), 1.20 (s,
3H). Used directly for following reaction without further
purifying.
2.2.5. 7q inhibits hypoxia-induced angiogenesis in vivo
4.1.2. Synthesis of N-(3-acetyl-4-hydroxyphenyl) acetamide (3)
To assess the anti-angiogenesis activity of 7q, different
angiogenesis models were employed. Here, we additionally
performed the Chicken chorioallantoic membrane (CAM) assay
to verify the anti-angionesis capacity of 7q. From this model, we
focused on the process of new blood vessel formation and vessel
responses to 7q. From the data in Fig. 5a, more and thicker new
blood vessels were formed in response to hypoxic stimulus.
When different concentrations of 7q (10, 25 and 50 μM) was
used, the quantity of vessels was reduced, and the thick ones
vanished. It indicated that 7q inhibited the in vivo angiogenesis in
a concentration-dependent manner (Fig. 5b).
The crude 2 (5.10 g, 30.87 mmol) was dissolved in
dichloromethane (120 mL) with stirring and cooling, to which
aluminium chloride and acetyl chloride were added quickly.
Then the mixture was heated to reflux for 12h. After cooling to
room temperature, the supernatant liquid of the mixture was
poured into 100 mL ice-cold water, while the underlayer residue
was stirred with 4% aqueous HCl for another half an hour. The
precipitate was filtrated through buchner funnel and dried to give
a yellow-green solid of 4.68g (24.22 mmol, 78%). 1H NMR (300
MHz, DMSO-d6): δ 11.55 (s, 1H), 9.93 (s, 1H), 8.06 (d, J = 2.58
Hz, 1H), 7.65 (dd, J = 2.6, 8.9 Hz, 1H), 6.91 (d, J = 8.9 Hz, 1H),
2.58 (s, 3H), 2.01 (s, 3H); HRMS (ESI): calcd. For C10H12NO3
[M+H]+ 194.0812, found 194.0815.
3. Conclusion
HIF-1 is a promising target of cancer therapy. Based on this
target, numbers of chemical inhibitors have been disclosed. In the
current study, we identified 7a and developed its derivatives
containing N-(benzofuran-5-yl) aromaticsulfonamide core as
HIF-1 inhibitors. Among all the compounds, 7q showed the best
inhibitory effect against HIF-1 activity (IC50 = 12.5 ± 0.7 μM).
Western blot assay and RT-PCR analysis indicated that 7q
downregulated the expression of HIF-1α in hypoxia, and further
blocked its transcription activation on VEGF both in mRNA
levels and protein levels (IC50 = 18.8 μM). Because VEGF is
associated with cell angiogenesis and migration, the suppressed
functionality of VEGF by 7q treatment led to decreased HUVEC
migration and tube formation. In addition, 7q exhibited
significant blockade in angiogenesis in CAM model. In
conclusion, a new chemotype as HIF-1 inhibitor, N-(benzofuran-
4.1.3. Synthesis of ethyl 2-(4-acetamido-2-acetylphenoxy)
acetate (4)
To a solution of N-(3-acetyl-4-hydroxyphenyl) acetamide 3
(4.68 g, 24.25 mmol) in DMF (50 mL), ethyl bromoacetate and
potassium carbonate were added. The mixture was refluxed for
4h. Then the mixture was poured into water (200 mL), and the
precipitate was filtered off and dried to give white solid (5.88 g,
yield 87%). 1H NMR (300 MHz, DMSO-d6): δ 9.95 (s, 1H), 7.80
– 7.66 (m, 2H), 7.05 (d, J = 8.9 Hz, 1H), 4.89 (s, 2H), 4.17 (q, J =
7.1 Hz, 2H), 2.60 (s, 3H), 2.00 (s, 3H), 1.21 (t, J = 7.1 Hz, 4H);
HRMS (ESI): calcd. For C14H18NO5 [M+H]+ 280.1179, found
280.1175.

4.1.4. Synthesis of ethyl 5-acetamido-3-methylbenzofuran-2-
carboxylate (5)
1.31 (t, J = 7.1, 3H); HRMS (ESI): calcd. For C19H19NO5S
[M+H]+ 374.1057 found 374.1053; HPLC (80% acetonitrile in
water): tR = 6.0 min, 99.7%.
To a solution of ethyl 2-(4-acetamido-2-acetylphenoxy)
acetate 4 (2.00 g, 7.16 mmol) in DMF (10 mL), potassium
carbonate (1.48 g, 10.74 mmol) was added. The resulting mixture
was heated under microwave irradiation (350W) for 7 min. The
reaction mixture was then poured into water. The precipitate was
filtered off and dried to obtain white solid (1.10 g, yield 59%); 1H
NMR (300 MHz, DMSO-d6): δ 10.10 (s, 1H), 8.11 (d, J = 1.6 Hz,
1H), 7.65 – 7.46 (m, 2H), 4.34 (q, J = 7.1 Hz, 2H), 2.49 (s, 3H),
2.06 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H); HRMS (ESI): calcd. For
C14H16NO4 [M+H]+ 262.1074, found 262.1072.
4.1.6.4. Ethyl 3-methyl-5-((4- chlorophenyl) sulfonamido)
benzofuran-2-carboxylate (7d). Yield, 69%; a pink powder; 1H-
NMR (300 MHz, DMSO-d6): δ 10.40(s, 1H), 7.74-7.56 (m, 5H),
7.45 (d, J = 1.8 Hz, 1H), 7.18 (dd, J = 8.9, 2.0 Hz, 1H), 4.34 (q, J
= 7.1 Hz, 2H), 2.46(s, 3H), 1.33 (t, J = 7.1 Hz, 3H); HRMS (ESI):
calcd. For C18H16ClNO5S [M+H]+ 394.0510, found 394.0525;
HPLC (80% acetonitrile in water): tR = 8.0 min, 99.9%
4.1.6.5. Ethyl 3-methyl-5-((3-chlorophenyl) sulfonamido)
benzofuran-2-carboxylate (7e). Yield, 81%; a white powder; 1H
NMR (300 MHz, DMSO-d6) δ 10.41 (s, 1H), 7.77 – 7.50 (m, 5H),
7.47 – 7.37 (m, 1H), 7.22 – 7.09 (m, 1H), 4.33 (q, J = 7.1 Hz,
2H), 2.46 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H); HRMS (ESI): calcd.
For C18H16ClNO5S [M+H]+ 394.0510, found 394.0504; HPLC
(80% acetonitrile in water): tR = 6.4 min, 98.6%.
4.1.5. Synthesis of ethyl 5-acetamido-3-methylbenzofuran-2-
carboxylate (6)
To a solution of 3M hydrochloric acid (20 mL) in ethanol
(30 mL), 5 (1.00 g, 3.83 mmol) was added. The mixture was
heated to reflux for 6 h with N2 gas purging. The clear solution
obtained was cooled to room temperature and condensed under
4.1.6.6. Ethyl 3-methyl-5-((2-chlorophenyl) sulfonamido)
benzofuran-2-carboxylate (7f). Yield, 80%; a white powder; 1H
NMR (300 MHz, DMSO-d6) δ 10.66 (s, 1H), 8.06 – 7.96 (m, 1H),
7.69 – 7.37 (m, 5H), 7.23 (dd, J = 8.9, 2.2 Hz, 1H), 4.32 (q, J =
7.10 Hz, 2H), 2.43 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H); HRMS (ESI):
calcd. For C18H16ClNO5S [M+H]+ 394.0510, found 394.0519;
HPLC (80% acetonitrile in water): tR = 5.7 min, 98.5%.
1
reduced pressure to give a brown solid (0.69 g, yield 71 %). H
NMR (300 MHz, DMSO-d6): δ 10.56 (brs, 2H), 7.84 – 7.71 (m,
2H), 7.56 – 7.44 (m, 1H), 4.35 (q, J = 7.1 Hz, 2H), 2.53 (s, 3H),
1.34 (t, J = 7.1 Hz, 3H); HRMS (ESI): calcd. for C12H14NO3
[M+H]+ 220.0968, found 220.0964.
4.1.6. Synthesis of products 7a-u
4.1.6.7. Ethyl 3-methyl-5-(4-bromophenyl) sulfonamido)
benzofuran-2-carboxylate (7g). Yield, 81.7%; a white powder;
1H NMR (300 MHz, DMSO-d6): δ 10.39 (s, 1H), 7.80 – 7.69 (m,
2H), 7.68 – 7.52 (m, 3H), 7.47 – 7.39 (m, 1H), 7.17 (dd, J = 8.9,
2.2 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 2.46 (s, 3H), 1.32 (t, J =
7.1 Hz, 3H). 13C NMR(75 MHz, Chloroform-d): δ 159.30, 150.95,
141.21, 138.44, 133.03, 132.31, 128.87, 128.68, 126.75, 124.91,
122.74, 113.65, 112.70, 60.84, 14.08, 8.98. HRMS (ESI): calcd.
For C18H16BrNO5S [M+H]+ 438.0005, found 437.9998; HPLC
(80% acetonitrile in water): tR = 4.5 min, 96.8%.
4.1.6.1. Ethyl 3-methyl-5-((4-methylphenyl) sulfonamido)
benzofuran-2-carboxylate (7a).
To a solution of hydrochloride of ethyl 5-amino-3-
methylbenzofuran-2-carboxylate 5 (0.22 g, 0.90 mmol) in
dichloromethane (30 mL) was added pyridine (0.29 g, 3.61
mmol) and 4-methyl benzenesulfonyl chloride (0.21 g, 1.08
mmol). The mixture was stirred overnight at room temperature,
and the reaction mixture was washed with dilute HCl and water.
The organic phase was dried over Na2SO4, concentrated in vacuo
and purified by silica gel column chromatography to give 7a, a
white powder of (0.27 g, yield 82%). 7b-e were synthesized in a
similar way.
4.1.6.8. Ethyl 5-((3-bromophenyl) sulfonamido) -3-
methylbenzofuran-2-carboxylate (7h). Yield, 57%; a white
1
powder; H NMR (300 MHz, DMSO-d6) δ 10.39 (s, 1H), 7.88 –
1
Compound 7a: Yield, 82%; a white powder; H NMR (300
7.77 (m, 2H), 7.73 – 7.35 (m, 4H), 7.15 (d, J = 7.3 Hz, 1H), 4.32
(q, J = 7.0 Hz, 2H), 2.45 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H); HRMS
(ESI): calcd. For C18H16BrNO5S [M+H]+ 438.0005, found
437.9999; HPLC (80% acetonitrile in water): tR = 6.5 min,
97.8%.
MHz, DMSO-d6): δ 10.23 (s, 1H), 7.65 – 7.57 (m, 2H), 7.54 (d, J
= 8.9 Hz, 1H), 7.41 (d, J = 2.2 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H),
7.17 (dd, J = 8.9, 2.2 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 2.45 (s,
3H), 2.31 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H); HRMS (ESI): calcd.
For C19H19NO5S [M+H]+ 374.1057, found 374.1059; HPLC
(80% acetonitrile in water): tR = 5.5 min, 99.0%.
4.1.6.9. Ethyl 5-((2-bromophenyl) sulfonamido) -3-
methylbenzofuran-2-carboxylate (7i). Yield, 69%; a white
1
powder; H NMR (300 MHz, DMSO-d6) δ 10.66 (s, 1H), 8.05
4.1.6.2. Ethyl 3-methyl-5-((3-methylphenyl) sulfonamido)
benzofuran-2-carboxylate (7b). Yield, 78%; a white powder; 1H
NMR (300 MHz, DMSO-d6): δ10.25 (s, 1H), 7.60 – 7.33 (m,
6H), 7.18 (dd, J = 8.9, 2.2 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 2.46
(s, 3H), 2.32 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H); HRMS (ESI):
calcd. For C19H19NO5S [M+H]+ 374.1057, found 374.1053;
HPLC (80% acetonitrile in water): tR = 12.6 min, 97.0%.
(dd, J = 7.3, 2.3 Hz, 1H), 7.86 – 7.76 (m, 1H), 7.61 – 7.45 (m,
3H), 7.40 (d, J = 2.2 Hz, 1H), 7.23 (dd, J = 9.0, 2.3 Hz, 1H), 4.32
(q, J = 7.1 Hz, 2H), 2.43 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H); HRMS
(ESI): Calcd. For C18H16BrNO5S [M+H]+ 438.0005, found
437.9999; HPLC (80% acetonitrile in water): tR = 5.5 min,
98.6%.
4.1.6.10. Ethyl 3-methyl-5-((4-(trifluoromethyl) phenyl)
4.1.6.3. Ethyl 3-methyl-5-((2-methylphenyl) sulfonamido)
benzofuran-2-carboxylate (7c). Yield, 78%; a white powder;1H
NMR (300 MHz, DMSO-d6): δ 10.41 (s, 1H), 7.84 (d, J = 7.8 Hz,
1H), 7.55-7.45 (m, 2H), 7.45 – 7.25 (m, 3H), 7.18 (dd, J = 8.9,
2.2 Hz, 1H), 4.31 (q, J = 7.1 Hz, 2H), 2.58 (s, 3H), 2.42 (s, 3H),
sulfonamido) benzofuran-2-carboxylate (7j). Yield, 57%; a
1
white powder; H NMR (300 MHz, DMSO-d6) δ 10.53 (s, 1H),
7.92 (brs, 4H), 7.57 (d, J = 8.9 Hz, 1H), 7.43 (d, J = 1.9 Hz, 1H),
7.17 (dd, J = 8.9, 2.1 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 2.45 (s,
3H), 1.32 (t, J = 7.1 Hz, 3H); HRMS (ESI): calcd. For

C19H16F3NO5S [M+H]+ 428.0774, found 428.0761; HPLC (80%
acetonitrile in water): tR = 2.9 min, 97.7%.
4.1.6.17.
Ethyl
3-methyl-5-((3,
4-dimethoxylphenyl)
sulfonamido) benzofuran-2-carboxylate (7q). Yield, 90%; a
white powder; H NMR (300 MHz, DMSO-d6) δ 10.12 (s, 1H),
1
4.1.6.11. Ethyl 3-methyl-5-((3-(trifluoromethyl) phenyl)
7.55 (d, J = 8.9 Hz, 1H), 7.44 (d, J = 2.1 Hz, 1H), 7.33 – 7.22 (m,
2H), 7.18 (dd, J = 9.0, 2.2 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 4.33
(q, J = 7.1 Hz, 2H), 3.74 (d, J = 13.1 Hz, 6H), 2.46 (s, 3H), 1.32
(t, J = 7.1, 3H); 13C NMR(75 MHz, Chloroform-d): δ 160.30,
150.74, 152.14, 148.97, 141.91, 132.29, 130.09, 129.61, 125.39,
123.60, 121.40, 115.35, 112.63, 110.31, 109.60, 61.32, 56.10,
56.07, 14.34, 9.37; HRMS (ESI): calcd. For C20H21NO7S [M+H]+
420.1111, found 420.1116; HPLC (80% acetonitrile in water): tR
= 4.6 min, 94.7%.
sulfonamido) benzofuran-2-carboxylate (7k). Yield, 71%; a
white powder; H NMR (300 MHz, DMSO-d6) δ 10.46 (s, 1H),
1
8.05 – 7.92 (m, 3H), 7.77 (t, J = 7.7 Hz, 1H), 7.57 (d, J = 8.9 Hz,
1H), 7.41 (d, J = 2.2 Hz, 1H), 7.14 (dd, J = 8.9, 2.3 Hz, 1H), 4.32
(q, J = 7.1 Hz, 2H), 2.44 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H); HRMS
(ESI): calcd. For C19H16F3NO5S [M+H]+ 428.0774, found
428.0770. HPLC (80% acetonitrile in water): tR = 6.2 min,
98.1%.
4.1.6.12. Ethyl 3-methyl-5-((2-(trifluoromethyl) phenyl)
4.1.6.18.
Ethyl
3-methyl-5-((2,
5-dimethoxylphenyl)
sulfonamido) benzofuran-2-carboxylate (7l). Yield, 79%; a
sulfonamido) benzofuran-2-carboxylate (7r). Yield, 62%; a
white powder; H NMR (300 MHz, DMSO-d6) 10.42 (s, 1H),
1
white powder; H NMR (300 MHz, DMSO-d6) δ 10.66 (s, 1H),
1
8.14 – 8.05 (m, 1H), 7.98 (m, 1H), 7.83 (m, 2H), 7.58 (d, J = 8.91
Hz, 1H), 7.44 (d, J = 2.2 Hz, 1H), 7.22 (dd, J = 8.9, 2.2 Hz, 1H),
4.33 (q, J = 7.1 Hz, 2H), 2.44 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H);
HRMS (ESI): calcd. For C19H17F3NO5S [M+H]+ 428.0774,
found 428.0769; HPLC (80% acetonitrile in water): tR = 8.7 min,
98.2%.
7.88 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 2.1 Hz, 1H), 7.62 – 7.57
(m, 2H), 7.45 (d, J = 1.7 Hz, 2H), 4.66 (q, J = 7.1 Hz, 2H),
4.17 (s, 3H), 4.02 (s, 3H), 2.78 (s, 3H), 1.65 (t, J = 7.1 Hz,
3H); HRMS (ESI): calcd. For C20H21NO7S [M+H]+ 420.1111,
found 405.1110; HPLC (80% acetonitrile in water): tR = 4.5 min,
97.7%.
4.1.6.13. Ethyl 3-methyl-5-((4-nitrophenyl) sulfonamido)
benzofuran-2-carboxylate (7m). Yield, 60%; a white powder;
1H NMR (300 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.40 – 8.30 (m,
2H), 8.00 – 7.91 (m, 2H), 7.58 (d, J = 8.9 Hz, 1H), 7.46 (d, J =
2.0 Hz, 1H), 7.17 (dd, J = 8.9, 2.2 Hz, 1H), 4.33 (q, J = 7.1 Hz,
2H), 2.45 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H); HRMS (ESI): calcd.
For C18H16N2O7S [M+H]+ 405.0751, found 405.0736; HPLC
(80% acetonitrile in water): tR = 5.0 min, 96.6%.
4.1.6.19.
Ethyl
3-methyl-5-(naphthalene-2-sulfonamido)
benzofuran-2-carboxylate (7s). Yield, 65%; a white powder; 1H
NMR (300 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.41 (d, J = 1.7 Hz,
1H), 8.11 (d, J = 7.2 Hz, 1H), 8.08 – 7.96 (m, 2H), 7.83 – 7.58
(m, 3H), 7.57 – 7.44 (m, 2H), 7.21 (dd, J = 8.8, 2.3 Hz, 1H), 4.33
(q, J = 7.1 Hz, 2H), 2.44 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H); HRMS
(ESI): calcd. For C22H19NO5S [M+H]+ 410.1057, found 410.1061;
HPLC (80% acetonitrile in water): tR = 6.3 min, 98.0%.
4.1.6.14. Ethyl 3-methyl-5-((3-nitrophenyl) sulfonamido)
benzofuran-2-carboxylate (7n). Yield, 57%; a white powder; 1H
NMR (300 MHz, DMSO-d6) δ 10.58 (s, 1H), 8.50 – 8.38 (m,
2H), 8.06 (d, J = 8.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.57 (d, J
= 8.8 Hz, 1H), 7.46 (d, J = 2.1 Hz, 1H), 7.16 (dd, J = 8.6, 2.2 Hz,
1H), 4.32 (q, J = 7.1 Hz, 2H), 2.46 (s, 3H), 1.31 (t, J = 7.1 Hz,
3H); HRMS (ESI): calcd. For C18H16N2O7S [M+H]+ 405.075,
found 405.0766; HPLC (80% acetonitrile in water): tR = 5.3 min,
97.8%.
4.1.6.20.
Ethyl
3-methyl-5-(thiophene-2-sulfonamido)
benzofuran-2-carboxylate (7t). Yield, 78%; a yellow powder;
1H NMR (300 MHz, DMSO-d6) δ 10.42 (s, 1H), 7.87 (dd, J =
4.96, 1.41 Hz, 1H), 7.59 (dd, J = 8.9, 1.9 Hz, 1H), 7.54 – 7.41
(m, 2H), 7.23 (dd, J = 8.9, 2.2 Hz, 1H), 7.14 – 7.07 (m, 1H), 4.34
(q, J = 7.1 Hz, 2H), 2.47 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H); HRMS
(ESI): Calcd. For C16H15NO5S2 [M+H]+ 366.0464, found
366.0472; HPLC (80% acetonitrile in water): tR = 4.8 min,
98.1%.
4.1.6.15. Ethyl 3-methyl-5-((2-nitrophenyl) sulfonamido)
benzofuran-2-carboxylate (7o). Yield, 57%; a red powder; H
4.1.6.21.
Ethyl
3-methyl-5-(pyridine-3-sulfonamido)
1
benzofuran-2-carboxylate (7u). Yield, 73%; a pink powder; 1H
NMR (300 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.88 – 8.72 (m,
2H), 8.11-7.96 (m, 1H), 7.63 – 7.52 (m, 2H), 7.45 (d, J = 2.1 Hz,
1H), 7.17 (dd, J = 8.9, 2.2 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 2.46
(s, 3H), 1.32 (t, J = 7.1 Hz, 3H); HRMS (ESI): calcd. For
C17H16N2O5S [M+H]+ 361.0853, found 361.0855; HPLC (80%
acetonitrile in water): tR = 4.2 min, 95.5%.
NMR (300 MHz, DMSO-d6) δ10.76 (s, 1H), 8.01 – 7.90 (m, 2H),
7.89 – 7.72 (m, 2H), 7.61 (dd, J = 8.9, 1.9 Hz, 1H), 7.47 (s, 1H),
7.23 (dd, J = 8.9, 2.2 Hz, 1H), 4.33 (q, J = 7.1, 1.9 Hz, 2H), 2.47
(s, 3H), 1.32 (t, J = 7.1, 3H); HRMS (ESI): calcd. For
C18H16N2O7S [M+H]+ 405.0751, found 405.0758; HPLC (80%
acetonitrile in water): tR = 5.1 min, 95.7%.
4.1.6.16. Ethyl 3-methyl-5-((4-tert-butylphenyl) sulfonamido)
benzofuran-2-carboxylate (7p). Yield, 68%; a white powder; 1H
NMR (300 MHz, DMSO-d6) δ 10.29 (s, 1H), 7.71 – 7.62 (m,
2H), 7.56- 7.49 (m, 3H), 7.39 (s, 1H), 7.21 (dd, J = 8.9, 2.3 Hz,
1H), 4.32 (q, J = 7.1 Hz, 2H), 2.43 (s, 3H), 1.31 (t, J = 7.1, 3H),
1.23 (s, 9H); 13C NMR(75 MHz, Chloroform-d): δ 160.29,
150.94, 152.30, 142.00, 135.72, 131.96, 127.71, 127.14, 126.01,
125.47, 123.90, 115.68, 112.76, 61.31, 35.15, 31.00, 14.38, 9.37;
HRMS (ESI): calcd. For C22H25NO5S [M+H]+ 416.1526, found
416.1535; HPLC (80% acetonitrile in water): tR = 8.4 min,
97.3%.
4.2 Biology
4.2.1. Luciferase reporter assays
HIF-1 transactivation activity was determined by transiently
transfecting MCF-7 cells with a construct expressing luciferase
under the control of three hypoxia response elements (24-mers)
from the Pgk-1 gene. HRE-luciferase was a gift from Navdeep
Chandel (Addgene plasmid # 26731).37 Plasmid DNA was
prepared using a commercial kit (TIANGEN, Beijing, China).
The empty pGL3 control plasmid and the pRL-SV40 renilla
luciferase containing plasmid used as controls for transfection
efficiency were purchased from Promega (Madison, Wisconsin).

MCF-7 cells in each well of 48-well culture plate were
transfected with 0.4 μg plasmid using 1 μL Lipofectamine 2000
Transfection Reagent (Invitrogen, Calif., USA) according to the
manufacture’s instructions. 6 h later cells were exposed to
hypoxia or normoxia with or without test compounds. After
incubation for 24 h, cells were washed twice with ice-cold PBS
and lysed with 100 μL of Passive Lysis buffer (Promega) and
incubated at room temperature for 10 min. Lysates were
centrifuged and supernatants were stored at -80°C until assayed.
Luciferase assays were done by pipetting 20 μL of cell lysate into
each well of a 96-well plate and analyzed immediately after
addition of luciferase reagent (Promega) by Luminoskan Ascent
(Thermo Scientific, USA). The data were obtained in triplicates
and expressed with the comparison of control.
SYBR Green Master (ROX) (Roche). Primers used for PCR were
as follows: human HIF-1α, 5'-TACTAGCTTTGCAGAATGCTC-
3' (sense) and 5'-GCCTTGTATAGGAGCATTAAC-3' (antisense);
human VEGF, 5'-CCTTGCTGCTCTACCTCCAC-3' (sense) and
5'-CACACAGGATGGCTTGAAGA-3' (antisense); human β-
actin, 5'-TGACGTGGACATCCGCAAAG-3' (sense) and 5'-
CTGGAAGGTGGACAGCGAGG-3'
(antisense).
Relative
expression level was calculated by the ΔΔCT method using a
StepOneTM Real-Time PCR System (Thermo Fisher Scientific),
and normalized with β-actin mRNA level.
4.2.5. VEGF ELISA
3 × 105 cells were seeded onto 6-well plates in serum-free
medium containing various concentrations of compound (0, 2.5,
5, 10, 25 and 50 μM) and incubated for 24 h under hypoxic
conditions. The conditioned medium was collected, and VEGF
levels were determined by VEGF ELISA kit (Neobioscience,
Shenzhen, China) according to the manufacturer’s protocols.
VEGF was expressed as a picogram of VEGF protein per
milliliter medium and per 105 cells.
Inhibition rate (%) = 1- [(RLUtreated - RLUblank) / (RLUcontrol
RLUblank)] × 100%
-
RLU = relative light unit
4.2.2. MTT assay
The in vitro cytotoxicity was determined by MTT assay. The
cells (1 × 104 cells/mL) were seeded into 96-well culture plates.
After overnight incubation, the cells were treated with various
concentrations of compounds or DMSO for 72 h. Then 20.0 μL
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT, Sigma, St. Louis, MO) solution (5 mg/mL in PBS) was
added into each well for 4 h at 37°C. Then the solution of MTT
was removed, followed by the addition of 150.0 μL DMSO to
dissolve the precipitate. The absorbance was measured at 490 nm
using Elx800 absorbance microplate reader (BioTek, Vermont,
USA) and the OD values were obtained. Cell growth inhibitor y
ratio was calculated using the following equation: Inhibitory rate
(%) = [1− (ODtreated − ODblank) / (ODcontrol − ODblank)] × 100%. The
IC50 was then analyzed using GraphPad Prism software.
4.2.6. Wound healing assay
HUVEC cells were seeded in 6 well plates with a density of
3 × 105 cells/well. After 24 hours, confluent monolayers were
gently scratched with a sterile 200 μL tip to create a uniform
wound area. After scratching, cells were incubated in fresh media
with 2.5, 5, 10, 25, and 50 μM of 7q. Cell movement and
migration into the wound area were examined after 24 hours by
Olympus IX51 (Tokyo, Japan) using a 10 × objective. The result
was quantified using the ImageJ software (National Institutes of
Health).
4.2.7. Capillary-like tube formation
Matrigel (12.5 mg/mL, BD Biosciences, MA, USA) was
thawed at 4 °C for overnight, and 50 mL Matrigel was quickly
pipetted onto 96-well plate and allowed to solidify for 30 min.
HUVECs (1 × 104 cells/well) were seeded on the surface of the
matrigel and incubated for 30 min. After adhesion of the cells,
the medium was removed and replaced by fresh medium
supplemented with various concentrations of 7q, and incubated
for another 24 h under hypoxic condition. The tube formation
was photographed with an Olympus IX51 (Tokyo, Japan) using a
10 × objective.
4.2.3. Western blot analysis
After drug treatment for the specified period, the MCF-7
cells were washed with ice-cold PBS for three times and were
harvested by 1 × trypsin. After being centrifuged at 2000 rpm for
5min, the cells were lysed in 80.0 μL of lysis buffer (150.0 mM
NaCl, 1% NP-40, 50.0 mM Tris-HCl, pH 7.5, 1mM NaF, EDTA,
DTT, Leu, and PMSF) for 1 h. Then cells were centrifuged again
at 12000 rpm for 20 min at 4 °C. The supernatant was collected,
and the quantification of the whole cell lysates was determined
by BCA analysis with Varioskan Flash (Thermo, Waltham, MA)
at 567 nm. The lysates were boiled for 5 min in sample loading
buffer (5 ×, Beyotime technology) at a ratio of 4:1. The cell
lysates were subjected to SDS-PAGE, transferred to PVDF
membrane (GE heathcare), and immunoblotted with anti-HIF-1α
antibody (ab51608, Abcam). β-actin was used as an internal
control. After further incubation with HRP-conjugated secondary
antibody, the blots were filmed through the Odyssey infrared
imaging System (LI-COR, Lincoln, Nebraska, USA).
4.2.8. Chicken chorioallantoic membrane (CAM) assay
Anti-angiogenic effect of 7q on CAM was assayed
according to the method described with modification.31 Sterilized
filter paper disks (5 × 5 mm) that were saturated with 15 μL 7q
(0, 10, 25, and 50 μM) were placed on the CAMs. They were
then incubated at 37 °C under normoxia and hypoxia conditions
for another 3 days. Then, an appropriate volume of 10% fat
emulsion (Sino-Swed Pharmaceutical Corp. Ltd, China) was
injected into the embryo chorioallantois for observing the density
and length of vessels toward the CAM face. Neovascular zones
under the filter paper disks were observed and photographedwith
a digital camera at × 5 magnification. The number of newly
grown vessels was counted on digitalized pictures.
4.2.4. Quantitative PCR analysis
Total RNA of MCF-7 cells was isolated using Trizol
(Invitrogen). Concentration and purity of RNA samples were
determined using NanoDrop 2000 spectrophotometer (Thermo
Fisher Scientific). cDNA was reversely transcribed from 1 μg of
the total RNA using PrimeScrpt RT reagent kit (Takara) on a
randomprimer. For the quantitative real-time PCR, DNA
amplification was carried out using the Fast Start Universal
4.2.9. Statistical analysis
All values were expressed as mean ± standard deviation (SD)

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Acknowledgments
This work is supported by 2013ZX09402102-001-005 of the
National Major Science and Technology Project of China
(Innovation and Development of New Drugs); 2012AA020301 of
863 key program; and the Priority Academic Program
Development of Jiangsu Higher Education Institutions.
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