Development of a novel class of mitochondrial ubiquinol-cytochrome c reductase binding protein (UQCRB) modulators as promising antiangiogenic leads

Article abstract of DOI:10.1021/jm500863j

Recently, we identified a novel therapeutic target and a small molecule for regulating angiogenesis. Our study showed that ubiquinol-cytochrome c reductase binding protein (UQCRB) of the mitochondrial complex III plays a crucial role in hypoxia-induced angiogenesis via mitochondrial reactive oxygen species (ROS) mediated signaling. Herein, we developed new synthetic small molecules that specifically bind to UQCRB and regulate its function. To improve the pharmacological properties of 6-((1-hydroxynaphthalen-4-ylamino)dioxysulfone)-2H-naphtho[1,8-bc]thiophen-2-one (HDNT), a small molecule that targets UQCRB, a series of HDNT derivatives were designed and synthesized. Several derivatives showed a significant increase in hypoxia inducible factor 1 (HIF-1) inhibitory potency compared to HDNT. The compounds bound to UQCRB and suppressed mitochondrial ROS-mediated hypoxic signaling, resulting in potent inhibition of angiogenesis without inducing cytotoxicity. Notably, one of these new derivatives significantly suppressed tumor growth in a mouse xenograft model. Therefore, these mitochondrial UQCRB modulators could be potential leads for the development of novel antiangiogenic agents.

Full text of DOI:10.1021/jm500863j

Article
pubs.acs.org/jmc
Development of a Novel Class of Mitochondrial Ubiquinol−
Cytochrome c Reductase Binding Protein (UQCRB) Modulators as
Promising Antiangiogenic Leads
Hye Jin Jung,†,‡,∥ Misun Cho,†,∥ Yonghyo Kim,† Gyoonhee Han,† and Ho Jeong Kwon*,†,§
†Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and
Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
‡Department of Pharmaceutical Engineering, Sun Moon University, No. 100, Kalsan-ri, Tangjeong-myeon, Asan-si, Chungnam
336-708, Republic of Korea
§Department of Internal Medicine, College of Medicine, Yonsei University, Seoul 120-752, Republic of Korea
S
* Supporting Information
ABSTRACT: Recently, we identified a novel therapeutic target and a small molecule for regulating angiogenesis. Our study
showed that ubiquinol−cytochrome c reductase binding protein (UQCRB) of the mitochondrial complex III plays a crucial role
in hypoxia-induced angiogenesis via mitochondrial reactive oxygen species (ROS) mediated signaling. Herein, we developed new
synthetic small molecules that specifically bind to UQCRB and regulate its function. To improve the pharmacological properties
of 6-((1-hydroxynaphthalen-4-ylamino)dioxysulfone)-2H-naphtho[1,8-bc]thiophen-2-one (HDNT), a small molecule that
targets UQCRB, a series of HDNT derivatives were designed and synthesized. Several derivatives showed a significant increase
in hypoxia inducible factor 1α (HIF-1α) inhibitory potency compared to HDNT. The compounds bound to UQCRB and
suppressed mitochondrial ROS-mediated hypoxic signaling, resulting in potent inhibition of angiogenesis without inducing
cytotoxicity. Notably, one of these new derivatives significantly suppressed tumor growth in a mouse xenograft model. Therefore,
these mitochondrial UQCRB modulators could be potential leads for the development of novel antiangiogenic agents.
studies provided the first indication of UQCRB’s role as a
critical mediator of mitochondrial reactive oxygen species
(ROS) mediated hypoxic signaling and the mode of action of
terpestacin at the molecular and organism levels.14,15
Accumulating evidence indicates that cellular oxygen sensing
is required for hypoxia inducible factor (HIF) 1α stabilization,
which is important for tumor cell survival, proliferation, and
angiogenesis.16,17 Under hypoxia, ROS generation at mitochon-
drial complex III inhibits the activity of prolyl hydroxylase
enzymes (PHDs), thereby preventing HIF-1α proteasomal
degradation and allowing its translocation to the nucleus and
dimerization with HIF-1β, initiating the transcription of HIF-1
responsiveness genes.18,19 We found that UQCRB is a crucial
component of a mitochondrial O2 sensor in complex III and
thus regulates the generation of mitochondrial ROS, the key
event in the signaling of cellular hypoxia.14 Moreover, we
demonstrated that terpestacin binding to UQCRB results in
inhibition of hypoxia-induced ROS generation, and conse-
quently, such inhibition blocks HIF activation and tumor
INTRODUCTION
■
Angiogenesis, the formation of new blood vessels, is important
in the pathogenesis of malignant, infectious, fibroproliferative,
and inflammatory diseases.1,2 In particular, tumor angiogenesis
plays a crucial role in tumor propagation, tumor growth, and
metastasis.3−5 Therefore, the specific perturbation of angio-
genesis has been considered a powerful strategy for the
treatment of cancer and other angiogenesis-related human
diseases. Several cellular target proteins that are important for
angiogenesis and their specific inhibitors have been identified.
However, recent clinical studies have revealed that the
inhibition of these target proteins alone is not enough to
block the complex biological processes involved in angiogenesis
and tumor development.6−12 Accordingly, the identification of
novel therapeutic targets for angiogenesis regulation is helpful
to reduce the rate of clinical failure and develop better
antiangiogenic therapeutic agents.
Using a combination of phenotypic screening and the target
identification method, we recently identified a new natural
angiogenesis inhibitor terpestacin and its cognate target protein,
ubiquinol−cytochrome c reductase binding protein (UQCRB)
of mitochondrial complex III.13,14 Our chemical genetics
Received: June 7, 2014
Published: September 22, 2014
© 2014 American Chemical Society
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a
Scheme 1. General Procedure for the Synthesis of HDNT Derivatives
a
Reagents and conditions: (a) pyridine, acetone, microwave reactor, 100 °C, 40 min.
angiogenesis in vivo.14 In addition, recent research has
demonstrated that UQCRB-mediated mitochondrial ROS
play a central role in hypoxic signaling in tumor cells as well
as vascular endothelial growth factor (VEGF) signaling in
endothelial cells (ECs).20 These results demonstrate that
UQCRB could be a promising therapeutic target for
antiangiogenic and antitumor drug development.
On the basis of a target-based screen with structural
information on the binding mode of terpestacin and
UQCRB, a novel synthetic small molecule targeting UQCRB,
6-((1-hydroxynaphthalen-4-ylamino)dioxysulfone)-2H-
naphtho[1,8-bc]thiophen-2-one (HDNT), was successfully
identified and exhibited potent antiangiogenic activity by
modulating the oxygen-sensing function of UQCRB.21 Thus,
HDNT can serve as a new synthetic small molecule probe to
explore the role of UQCRB in angiogenesis and as a potential
lead compound for medical applications.
presence of potassium hydroxide in ethanol to obtain 2 in high
yields (Scheme 2).
SAR Study of HDNT Derivatives. Initially, HDNT
derivatives were designed based on the docking study of
HDNT and UQCRB. HDNT comprises the sulfonamide core
and two major interacting parts with UQCRB: the 1-
hydroxynaphthalene moiety for hydrogen bonding and hydro-
phobic interaction and the 2H-naphtho[1,8-bc]thiophen-2-one
moiety for π−π interaction with the active site of UQCRB.21
To maintain the major interaction with UQCRB, HDNT
derivatives have retained the sulfonamide core and introduced
diverse functional groups to both sides of the sulfonamide core
(Figure 1). All 17 HDNT derivatives were synthesized and
In the present study, we designed and synthesized a series of
HDNT derivatives with a sulfonylamide backbone and their
biological activities were evaluated both in vitro and in vivo to
further develop novel antiangiogenic agents targeting UQCRB.
On the basis of structure−activity relationship (SAR) studies of
the derivatives, we identified a novel class of mitochondrial
UQCRB modulators with enhanced pharmacological potential.
In addition, a salt form of the most promising HDNT derivative
was synthesized to increase its aqueous solubility, and its
antiangiogenic and antitumor effects were demonstrated in a
glioblastoma mouse xenograft model.
Figure 1. Structures of HDNT and derivatives.
biologically evaluated (Table 1). As the stability of HIF-1α
represents a functional response to hypoxia triggered by the
cellular oxygen sensing system, the bioactivity of the new
synthetic compounds was evaluated by measuring HIF-1α
protein levels using Western blot.16,17 Various functional
groups such as substituted phenyl (methyl, ethyl, tert-butyl,
and biphenyl) and heterocycles (naphthyl and quinolinyl) were
introduced on the R1. Compounds containing small functional
groups such as methyl and ethyl (1a and 1b) showed lower
HIF-1α inhibitory activities than compounds with the bulky
functional groups such as tert-butyl and phenyl group (1c and
1f). Especially 1f with biphenyl group showed the best HIF-1α
inhibitory activities (IC50 = 2.5 μM). The HDNT derivatives
with heterocycles on R1 showed no HIF-1α inhibitory activities
(1n and 1o). We introduced diverse functional groups
including substituted phenyl, naphthyl, and quinolinyl on R2.
Hydroxy group was retained on R2 for the hydrogen bonding
interaction with UQCRB. 1f with 4-hydroxyphenyl group on R2
showed better HIF-1α inhibitory activity (IC50 = 2.5 μM) than
substituted phenyl or heterocycles with hydroxy groups (1g−
m). In addition, the compound with 4-hydroxyphenyl group on
R2 (1c) showed slightly better HIF-1α inhibitory activity (IC50
= 5 μM) than compounds with 2- or 3-hydroxyphenyl group
(1p and 1q, 8 and 8 μM, respectively). Docking study of
RESULTS AND DISCUSSION
■
Chemistry. The general procedure for the synthesis of
HDNT derivatives is illustrated in Schemes 1 and 2.
Sulfonamide-based HDNT derivatives were produced from
commercially available sulfonyl chloride and amine. The
sulfonamide derivatives 1a−q were synthesized by reacting
commercially available sulfonyl chloride with diverse amines in
acetone in the presence of pyridine in a microwave reactor
(Scheme 1). The potassium salt of 1f was prepared in the
a
Scheme 2. Preparation of 2
a
Reagents and conditions: (a) KOH, EtOH, 0 °C.
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subtracting resonance values of UQCRB binding to control
biotin from those of UQCRB binding to BT. For competitive
binding analyses, terpestacin, 1c, and 1f were each incubated
with UQCRB protein and the complexes were subsequently
injected into flow cells of the sensor chip. The competitive
binding of terpestacin to UQCRB inhibited binding between
BT and UQCRB, resulting in a significant decrease in
resonance units (Figure 2). Similar to the preincubation of
terpestacin, the binding efficiency of UQCRB to BT decreased
by 40% by preincubation of either 1c or 1f with UQCRB,
which confirmed that the two small molecules directly bind to
UQCRB.
Regulation of Mitochondrial ROS-Induced Hypoxic
Signal Transduction. UQCRB of mitochondrial complex III
plays a crucial role in the oxygen sensing mechanism that
regulates responses to hypoxia, and thus, the increase in
UQCRB-mediated mitochondrial ROS generation during
hypoxia triggers HIF-1α stabilization and consequently induces
the expression of its target genes, such as VEGF.15 To
determine whether the binding of the new derivatives to
UQCRB regulates the O2 sensing function of UQCRB, we
investigated the effects of 1c and 1f on the mitochondrial ROS-
mediated hypoxic signal transmission procedure. HepG2 cells
were pretreated with 1c or 1f for 30 min and then exposed to
1% O2 for 10 min. Mitochondrial ROS levels were measured
using the MitoSOX Red mitochondrial superoxide indicator.
Compounds 1c and 1f suppressed the hypoxia-induced
mitochondrial ROS generation in HepG2 cells in a dose-
dependent manner (Figure 3A). Moreover, the compounds
significantly inhibited HIF-1α accumulation and VEGF
expression induced by hypoxia (Figures 3B,C). These results
suggest that the binding of the new analogs to UQCRB
modulates the O2 sensing function of UQCRB. Notably, the
derivatives did not affect cell viability at concentrations used in
the present study, indicating that the effects of these
compounds on cellular oxygen sensing do not result from off
target toxicity (Supporting Information Figure S2).
Table 1. HIF-1α Inhibitory Activity of HDNT Derivatives
HIF-1α
a
compd
R1
R2
IC50 (μM)
1a
4-methylphenyl
4-ethylphenyl
4-tert-butylphenyl
4-tert-butylphenyl
4-tert-butylphenyl
4-biphenyl
4-hydroxyphenyl
>20
>20
5
1b
1c
4-hydroxyphenyl
4-hydroxyphenyl
1d
1e
1f
4-hydroxynaphthalen-1-yl
8-hydroxyquinolin-5-yl
4-hydroxyphenyl
>20
20
2.5
>20
>20
>20
>20
>20
>20
20
1g
1h
1i
4-biphenyl
3-fluoro-4-hydroxyphenyl
3-chloro-4-hydroxyphenyl
3-carboxy-4-hydroxyphenyl
4-hydroxynaphthalen-1-yl
5-hydroxynaphthalen-1-yl
7-hydroxynaphthalen-1-yl
8-hydroxyquinolin-5-yl
4-hydroxyphenyl
4-biphenyl
4-biphenyl
1j
4-biphenyl
1k
1l
4-biphenyl
4-biphenyl
1m
1n
1o
1p
1q
2
4-biphenyl
2-naphthyl
>20
>20
8
quinolin-8-yl
4-tert-butylphenyl
4-tert-butylphenyl
4-biphenyl
4-hydroxyphenyl
2-hydroxyphenyl
3-hydroxyphenyl
8
potassium 4-phenolate
5
HDNT
20
a
Experimental details are described in Experimental Section. Each
value represents the mean from three independent experiments.
HDNT derivatives was performed using Discovery Studio
program, and the results are shown in Supporting Information
Figure S1. Hydrogen bond in the sulfonamide core and
hydrophobic interaction between R1 and Leu29 of UQCRB
were observed, but hydrogen bond in hydroxyl group on R2 was
not observed. Among 17 HDNT derivatives, 1c, 1e, 1f, and 1m
with good HIF-1α inhibitory activities (5, 20, 2.5, and 20 μM,
respectively) were evaluated for the thermodynamic solubility
(Table 2). 1f with the best HIF-1α inhibitory activities showed
low thermodynamic solubility (under 2.06 μg/mL in both PBS
and H2O solution). To improve the low solubility of 1f,
potassium salt formation was performed. Potassium salt
compound 2 showed similar inhibitory activity with 1f and
improved solubility (Table 2). 1c, 1f, and 2 with good HIF-1α
inhibitory activity have been selected for additional assays.
Competitive Binding Assays. In our previous reports, we
demonstrated that the in vitro assay based on the competitive
binding of biotin-labeled terpestacin to UQCRB can be
successfully applied to the screening of novel small molecules
binding with UQCRB.14,21 To demonstrate the binding of 1c
and 1f to UQCRB, we performed a surface plasmon resonance
(BIAcore) based competitive binding assay using biotinylated
terpestacin (BT). Controls of biotin and BT were sequentially
immobilized onto the surface of a streptavidin-coated sensor
chip, and the apparent binding affinities were calculated by
In Vitro Angiogenesis Assay. Recently, our study
investigating the role of UQCRB in angiogenesis revealed
that UQCRB enhances VEGF receptor type 2 (VEGFR2)
signaling by increasing the levels of mitochondrial ROS in ECs
and terpestacin blocks mitochondrial ROS-mediated VEGFR2
signaling pathways, thereby inhibiting VEGF-induced angio-
genesis of ECs.20 Therefore, we evaluated the effects of the new
mitochondrial UQCRB modulators on key angiogenic
processes of ECs activated by VEGF. Serum-starved human
umbilical vein endothelial cells (HUVECs) were stimulated by
VEGF with or without 1c or 1f and in vitro angiogenesis assays
were performed. The two compounds dose-dependently
suppressed VEGF-induced invasion and tube formation of
HUVECs (Figure 4). Trypan blue staining was performed in
Table 2. Thermodynamic Solubility of Active HDNT Derivatives
a
thermodynamic solubility (μg/mL)
compd
R1
R2
HIF-1α IC50 (μM)
PBS
H2O
1c
1e
1f
4-tert-butylphenyl
4-tert-butylphenyl
4-biphenyl
4-hydroxyphenyl
5
26.74
12.84
<2.06
7.26
NT
8-hydroxyquinolin-5-yl
4-hydroxyphenyl
20
2.5
20
5
NT
<2.06
NT
1m
2
4-biphenyl
8-hydroxyquinolin-5-yl
potassium 4-phenolate
4-biphenyl
108
>500
a
Thermodynamic solubility was determined by HPLC. Values are the mean from three independent experiments. NT, not tested.
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Figure 2. Effects of the competitors on the interaction between purified UQCRB protein and immobilized terpestacin (Terp) by SPR analysis.
Figure 3. Regulation of mitochondrial ROS-mediated hypoxic signal transduction by new derivatives. (A) Effects of 1c and 1f on mitochondrial ROS
generation. Mitochondrial ROS levels were determined by MitoSOX Red fluorescence. HepG2 cells were pretreated with compounds for 30 min and
then exposed to 1% O2 for 10 min: Nor, normoxia; Hyp, hypoxia. (∗∗) P < 0.01 versus hypoxia control. (B) Effects of 1c and 1f on HIF-1α stability.
HIF-1α protein levels were analyzed by Western blot analysis. HepG2 cells were pretreated with the compounds for 30 min and then exposed to 1%
O2 for 4 h. Western blot data were quantified using densitometry. (∗) P < 0.001 versus hypoxia control. (C) Effect of 1c and 1f on VEGF expression.
HepG2 cells were pretreated with compounds for 30 min and then exposed to 1% O2 for 16 h. The concentration of VEGF protein in the culture
supernatant was determined using an ELISA specific for VEGF. (∗) P < 0.001 versus hypoxia control. Each value represents the mean SE from
three independent experiments.
parallel with in vitro angiogenesis assays, and cytotoxicity was
not observed at the indicated concentrations.
highly resistant to chemotherapy.22−24 Thus, we further
determined the effects of the new UQCRB modulator 1f on
tumor growth and tumor angiogenesis in a glioblastoma mouse
xenograft model. A pilot study was carried out, and lf
administration notably inhibited the tumor growth despite its
poor water solubility (Supporting Information Figure S3). In
particular, a salt form of the compound (2) was synthesized to
increase its aqueous solubility and biological activity was
confirmed by its HIF-1α inhibition activity in HepG2 cells as
well as antiproliferative effect in U87MG cells in vitro
(Supporting Information Figures S4 and S5). Furthermore,
we demonstrated that the bioactivity of 2 is not attributed to
the nonspecific action as a highly active nucleophile by testing
the phenoxide derivatives of other compounds (1a and 1c;
Supporting Information Table S1 and Figure S6). Next, BALB/
c nude mice were inoculated with U87MG cells, and the mice
were intraperitoneally administered with vehicle or 2. As shown
in Figures 6A,B, 2 led to a dramatic reduction in tumor growth
without exhibiting significant body weight loss in mice.
In Vivo Angiogenesis Assay. The antiangiogenic activity
of the new UQCRB targeting small molecules was also
validated in vivo by the chorioallantoic membrane (CAM)
assay. Thermanox coverslips loaded with 1c and 1f were placed
on the surface of CAM from growing chick embryos, and
neovascularized zones were observed under the microscope.
Retinoic acid (RA) was used as a positive control for
antiangiogenic responses. As shown in Figure 5, RA inhibited
angiogenesis by 67% (n = 9), whereas the control coverslips
exhibited 23% (n = 13) inhibition. Compounds 1c and 1f
effectively inhibited the neovascularization of CAM from chick
embryos by 65% (n = 17) and 69% (n = 13), respectively,
without inducing any rupture or toxicity to pre-existing vessels.
These data demonstrate that the synthetic small molecules are a
novel class of antiangiogenic agents targeting UQCRB.
Antitumor Activity in Vivo. Glioblastoma is one of the
most aggressive angiogenic solid tumors and consequently
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complex III, a protein that plays a critical role in tumor
angiogenesis by mediating mitochondrial ROS generation in
tumor cells and ECs.14,20 The development of new small
molecules targeting this protein may offer novel and effective
cancer treatment strategies. By use of a reverse chemical
genetics approach to discover new UQCRB modulators, a new
synthetic small molecule HDNT was identified as a promising
antiangiogenic agent targeting UQCRB.21 In the present study,
a SAR analysis was conducted to improve the pharmacological
potential of HDNT.
In conclusion, this report describes the design, synthesis, and
biological evaluation of a new series of HDNT derivatives with
a sulfonylamide backbone. Notably, of the more potent
derivatives, 1f exhibited 8 times greater inhibitory potency
than the parent small molecule HDNT in a cell-based assay that
measured HIF-1α activity. The novel class of mitochondrial
UQCRB modulators exhibited potent antiangiogenic activity
without inducing cytotoxic effects both in vitro and in vivo by
blocking mitochondrial ROS-mediated angiogenic signaling.
Although the new compounds exhibited the in vitro binding
efficiency to UQCRB to the same extent as terpestacin and
HDNT, their antiangiogenic potential in vitro (IC50 ≤ 2.5 μM)
was 2−10 times stronger than that of the previously reported
small molecules targeting UQCRB (terpestacin, IC50 = 25 μM;
HDNT, IC50 = 5 μM).13,21 These results indicate that the novel
class of UQCRB modulators exhibit greater pharmacological
properties compared to original compounds.
Figure 4. Effects of new derivatives on in vitro angiogenesis. Serum-
starved HUVECs were stimulated with VEGF (30 ng/mL) in the
presence or absence of 1c and 1f. (A) Inhibitory activity of the
compounds on endothelial cell invasion. (B) Effects of the new analogs
on the tube-forming ability of HUVECs. Arrows indicate inhibition of
tube formation. The basal levels of invasiveness and capillary tube
formation of HUVECs that were incubated in serum-free medium
were normalized to 100%. (∗) P < 0.001 versus VEGF control. Each
value represents the mean SE from three independent experiments.
Furthermore, 1f/2 significantly suppressed tumor growth
and tumor angiogenesis in a glioblastoma mouse xenograft
model, suggesting that the new UQCRB modulator could be
applied in new therapeutic approaches for human cancer
treatment.
EXPERIMENTAL SECTION
■
Chemistry. All chemicals were obtained from commercial suppliers
and used without further purification. All reactions were monitored by
thin-layer chromatography (TLC) on precoated silica gel 60 F254
(mesh) (Merck, Mumbai, India), and spots were visualized under UV
light (254 nm). Flash column chromatography was performed with
silica (Merck EM9385, 230−400 mesh). 1H and 13C nuclear magnetic
resonance (NMR, Varian Unity Inova) spectra were recorded at 400
and 100 MHz, respectively (Supporting Information Figure S7).
Proton and carbon chemical shifts are expressed in ppm relative to the
internal tetramethylsilane standard, and coupling constants (J) are
expressed in hertz. Splitting patterns are presented as s, singlet; d,
doublet; t, triplet; q, quartet; dd, double of doublets; m, multiplet; and
br, broad. Liquid chromatography−mass spectrometry (LC−MS)
spectra were obtained by using an electrospray ionization (ESI) probe
using a Shimadzu LCMS2010 instrument with an Agilent C18 column
(50 mm × 4.6 mm, 5 μm particle size). The mobile phase consisted of
0.1% formic acid in H2O/0.1% formic acid in CH3CN (1:9) over 10
min at a flow rate of 0.2 mL min−1; the scan mode was ranged from 0
to 500 amu z−1. In the positive mode, the detected ion peaks were
(M+z)/z, where M and z represent the molecular weight of the
compound and charge (number of protons), respectively. High-
resolution ESI-MS measurements were performed on a Micromass
quadrupole-time of flight (Q-TOF) Acquity UPLC−mass system at
Yonsei University in the positive mode. To determine the purity of the
final compounds (1a−q), high performance liquid chromatography
(HPLC) experiments were conducted using the Agilent analytical
column Eclipse-XDB-C18 (150 mm × 4.6 mm, 5 μm) on Shimadzu
HPLC 2010 instruments. All compounds tested in biological assays
were ≥95% pure (Supporting Information Table S2).
Figure 5. Effects of new compounds on the in vivo angiogenesis of
CAM. Fertilized chick eggs were maintained in a humidified incubator
at 37 °C. At the stage of a 4.5-day embryo, 1c-, 1f-, or RA-loaded
Thermanox coverslips were applied to the CAM surface. Two days
later, chorioallantois was observed under a microscope. When CAM
that received treatment showed an avascular zone (arrows), the
response was scored as positive. Calculations were based on the
proportion of positive eggs relative to the total number of eggs tested.
Notably, 2 exhibited significantly increased antitumor activity
compared to lf, indicating that its improved water solubility
may contribute to the increase of clinical availability. We also
evaluated the effects of 2 on tumor angiogenesis by measuring
the tumor blood vessel density (using CD31 as an endothelial
cell marker) and HIF-1α and VEGF expression levels. As
shown in Figure 6C, 2 significantly decreased the tumor
microvessel density and the expression of proangiogenic factors.
These results imply that the new class of UQCRB modulators
could be applied in new cancer therapeutics by blocking tumor
angiogenesis.
CONCLUSION
Recently, we identified a natural antiangiogenic small molecule
probe, terpestacin, that targets UQCRB of mitochondrial
■
General Procedure for the Preparation of Title Compounds
1a−q. Commercially available sulfonyl chloride (1 equiv) and amine
(1.5 equiv) were dissolved in acetone (0.1 M), and then pyridine (3
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Figure 6. Antitumor and antiangiogenic activities of 2 in vivo. Mice bearing glioblastomas consisting of U87MG cells were treated with vehicle or 2
(20 mg/kg). (A) Tumor volumes of two groups (n = 8) after 17 days. (∗∗) P < 0.01 versus vehicle control. (B) Body weight of mice. (C)
Immunohistochemical staining for the detection of CD31, HIF-1α, and VEGF expression. The fluorescence images were originally obtained at 200×
magnification. Quantification of the microvessel density was conducted by counting the number of positive structures in three random fields. The
relative intensity of HIF-1α and VEGF expression was measured by the densitometric analysis of three random fields. (∗∗) P < 0.01 versus vehicle
control.
equiv) was added. The mixture was reacted using microwave reactor at
100 °C for 40 min. The reacted mixture was cooled and quenched
with 10% HCl. The crude was extracted by ethyl acetate. Extracted
organic layer was washed with brine, dried over anhydrous MgSO4,
filtered, and concentrated. The crude product was purified by flash
column chromatography (ethyl acetate/hexane, 1:5 to 1:1).
1H, J = 7.6 Hz), 7.36−7.29 (m, 2H), 6.87 (d, 1H, J = 8.4 Hz), 6.50 (d,
1H, J = 8.0 Hz), 5.91 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ
155.53, 152.42, 137.35, 131.48, 126.61, 125.95, 125.76, 124.83, 124.62,
123.16, 122.05, 107.26, 34.79, 30.80; ESI (m/z) 354 (MH−); HRMS
(ESI) calcd for C20H21NO3S [MNa+] 378.1134, found 378.1127.
4-tert-Butyl-N-(8-hydroxyquinolin-5-yl)benzenesulfonamide
N-(4-Hydroxyphenyl)-4-methylbenzenesulfonamide (1a). 1H
NMR (400 MHz, DMSO-d6) δ 7.67 (d, 2H, J = 8.4 Hz), 7.44 (d, 2H, J
= 8.4 Hz), 6.60 (d, 2H, J = 8.8 Hz), 6.43 (d, 2H, J = 8.8 Hz), 2.41 (s,
3H); 13C NMR (100 MHz, DMSO-d6) δ 147.88, 145.31, 138.97,
131.70, 130.01, 128.24, 122.48, 113.89, 21.15; ESI (m/z) 262 (MH−);
HRMS (ESI) calcd for C13H13NO3S [MH+] 264.0689, found
264.0675.
1
(1e). H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 9.86 (s, 1H),
8.74 (dd, 1H, JA = 4.0 Hz, JB = 1.4 Hz), 8.11 (dd, 1H, JA = 8.4 Hz, JB =
1.4 Hz), 7.48−7.42 (m, 4H), 7.37−7.34 (m, 1H), 7.00 (d, 1H, J = 8.4
Hz), 6.93 (d, 1H, J = 8.0 Hz), 1.20 (s, 9H); 13C NMR (100 MHz,
DMSO-d6) δ 156.2, 153.0, 148.5, 137.0, 127.0, 126.7, 126.3, 123.0,
121.9, 110.9, 35.2, 31.2; ESI (m/z) 357 (MH+), 355 (MH−); HRMS
(ESI) calcd for C19H20N2O3S [MNa+] 379.1087, found 379.1088.
N-(4-Hydroxyphenyl)biphenyl-4-sulfonamide (1f). 1H NMR
(400 MHz, DMSO-d6) δ 9.76 (s, 1H), 9.29 (s, 1H), 7.79 (d, 2H, J =
8.4 Hz), 7.69 (t, 4H, J = 8.6 Hz), 7.47−7.37 (m, 3H), 6.85 (d, 2H, J =
8.8 Hz), 6.58 (d, 2H, J = 8.8 Hz); 13C NMR (100 MHz, DMSO-d6) δ
155.28, 144.35, 138.82, 138.74, 129.52, 128.94, 128.89, 127.80, 128.59,
127.45, 124.39, 115.89; ESI (m/z) 324 (MH−); HRMS (ESI) calcd for
C18H15NO3S [MH−] 324.0700, found 324.0705.
N-(3-Fluoro-4-hydroxyphenyl)biphenyl-4-sulfonamide (1g).
1H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 9.72 (s, 1H), 7.81
(d, 2H, J = 8.8 Hz), 7.74 (d, 2H, J = 8.4 Hz), 7.67 (d, 2H, J = 7.2 Hz),
7.47−7.38 (m, 3H), 6.85 (dd, 1H, JA = 6.4 Hz, JB = 2.4 Hz), 6.78 (t,
1H, J = 9.2 Hz), 6.69 (dd, 1H, JA = 4.4 Hz, JB = 1.6 Hz); 13C NMR
(100 MHz, DMSO-d6) δ 152.05, 149.65, 144.61, 142.60, 142.48,
138.67, 138.45, 129.52, 127.79, 127.46, 118.50, 118.34, 110.68, 110.47;
ESI (m/z) 342 (MH−); HRMS (ESI) calcd for C18H14FNO3S [MH+]
344.0751, found 344.0733.
4-Ethyl-N-(4-hydroxyphenyl)benzenesulfonamide (1b). 1H
NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 9.25 (s, 1H), 7.53 (d,
2H, J = 8.0 Hz), 7.31 (d, 2H, J = 8.4 Hz), 6.81 (d, 2H, J = 8.8 Hz),
6.56 (d, 2H, J = 8.8 Hz), 2.59 (q, 2H, J = 7.2 Hz), 1.11 (t, 3H, J = 7.6
Hz); 13C NMR (100 MHz, DMSO-d6) δ 155.1, 149.2, 137.4, 129.0,
128.7, 127.2, 124.2, 115.9, 28.3, 15.4; ESI (m/z) 276 (MH−); HRMS
(ESI) calcd for C14H15NO3S [MH+] 278.0845, found 278.0823.
4-tert-Butyl-N-(4-hydroxyphenyl)benzenesulfonamide (1c).
1H NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H), 9.26 (s, 1H), 7.57
(d, 2H, J = 8.4 Hz), 7.50 (d, 2H, J = 8.4 Hz), 6.84 (d, 2H, J = 8.8 Hz),
6.57 (d, 2H, J = 8.8 Hz), 1.22 (s, 9H); 13C NMR (100 MHz, DMSO-
d6) δ 155.51, 154.64, 136.93, 128.69, 126.57, 125.90, 123.50, 115.33,
34.84, 30.78; ESI (m/z) 304 (MH−); HRMS (ESI) calcd for
C16H19NO3S [MH+] 306.1158, found 306.1133.
4-tert-Butyl-N-(4-hydroxynaphthalen-1-yl)benzene-
sulfonamide (1d). 1H NMR (400 MHz, DMSO-d6) δ 8.02 (d, 1H, J
= 8.0 Hz), 7.74 (d, 2H, J = 8.4 Hz), 7.58 (d, 2H, J = 8.4 Hz), 7.53 (d,
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N-(3-Chloro-4-hydroxyphenyl)biphenyl-4-sulfonamide (1h).
1H NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H), 10.03 (s, 1H), 7.81
(d, 2H, J = 8.4 Hz), 7.74 (d, 2H, J = 8.4 Hz), 7.66 (d, 2H, J = 7.2 Hz),
7.44 (t, 2H, J = 7.4 Hz), 7.39 (d, 1H, J = 7.2 Hz), 7.02 (d, 1H, J = 2.4
Hz), 6.88 (dd, 1H, JA = 4.4 Hz, JB = 2.4 Hz), 6.82(d, 1H, J = 8.8 Hz);
13C NMR (100 MHz, DMSO-d6) δ 150.93, 144.63, 138.66, 138.42,
129.82, 129.52, 128.99, 127.80, 127.74, 127.45, 123.80, 122.37, 119.94,
117.30; ESI (m/z) 358 (MH−); HRMS (ESI) calcd for
C18H14ClNO3S [MH+] 360.0456, found 360.0451.
5-(Biphenyl-4-ylsulfonamido)-2-hydroxybenzoic Acid (1i).
1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 7.80 (d, 2H, J =
8.4 Hz), 7.72 (d, 2H, J = 8.4 Hz), 7.66 (d, 2H, J = 7.6 Hz), 7.49 (d,
1H, J = 2.8 Hz), 7.44 (t, 2H, J = 7.4 Hz), 7.38 (d, 1H, J = 7.6 Hz), 7.23
(dd, 1H, JA = 4.5 Hz, JB = 2.6 Hz), 6.84 (d, 1H, J = 8.8 Hz); 13C NMR
(100 MHz, DMSO-d6) δ 171.58, 158.75, 144.62, 138.64, 138.38,
130.43, 129.52, 129.19, 128.99, 127.79, 127.74, 127.45, 123.79, 118.35,
113.44; ESI (m/z) 368 (MH−); HRMS (ESI) calcd for C19H15NO5S
[MH−] 368.0598, found 368.0596.
N-(4-Hydroxynaphthalen-1-yl)biphenyl-4-sulfonamide (1j).
1H NMR (400 MHz, DMSO-d6) δ 8.09 (d, 1H, J = 8.4 Hz), 7.94
(q, 4H, J = 8.4 Hz), 7.75−7.70 (m, 3H), 7.54−7.39 (m, 5H), 6.94 (d,
1H, J = 8.4 Hz), 6.56 (d, 1H, J = 8.0 Hz), 5.98 (s, 2H); 13C NMR (100
MHz, DMSO-d6) δ 146.42, 144.92, 138.33, 135.07, 134.08, 129.63,
129.38, 129.31, 128.11, 127.61, 126.91, 124.85, 123.14, 121.48, 120.18,
105.90; ESI (m/z) 374 (MH−); HRMS (ESI) calcd for C22H17NO3S
[MH+] 376.1002, found 376.1002.
N-(5-Hydroxynaphthalen-1-yl)biphenyl-4-sulfonamide (1k).
1H NMR (400 MHz, DMSO-d6) δ 8.03 (d, 1H, J = 8.4 Hz), 7.97 (d,
2H, J = 8.4 Hz), 7.90 (d, 2H, J = 8.8 Hz), 7.70 (d, 2H, J = 7.2 Hz),
7.50−7.43 (m, 3H), 7.30 (t, 1H, J = 8.0 Hz), 7.18−7.14 (m, 2H), 7.01
(d, 1H, J = 8.4 Hz), 6.66 (d, 1H, J = 7.6 Hz), 5.88 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 146.59, 145.59, 145.49, 138.27, 134.09,
129.64, 129.43, 129.22, 128.35, 128.23, 128.21, 127.64, 124.39, 123.15,
122.43, 118.32, 108.65, 108.53; ESI (m/z) 376 (MH+), 374 (MH−);
HRMS (ESI) calcd for C22H17NO3S [MH+] 376.1001, found
376.1002.
ESI (m/z) 299 (MH−); HRMS (ESI) calcd for C15H12N2O3S [MH+]
301.0641, found 301.0643.
4-tert-Butyl-N-(2-hydroxyphenyl)benzenesulfonamide (1p).
1H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 2H), 7.68 (d, 2H, J = 8.4
Hz), 7.50 (d, 2H, J = 8.4 Hz), 7.12 (d, 1H, J = 8.0 Hz), 6.88 (t, 1H, J =
7.8 Hz), 6.71 (d, 1H, J = 8.0 Hz), 6.65 (t, 1H, J = 7.6 Hz), 1.23 (s,
1H); 13C NMR (100 MHz, DMSO-d6) δ 155.87, 150.17, 138.38,
126.99, 126.19, 126.16, 124.80, 124.04, 119.36, 115.93, 35.24, 31.21;
ESI (m/z) 306 (MH+), 304 (MH−); HRMS (ESI) calcd for
C16H19NO3S [MH−] 304.1013, found 304.1008.
4-tert-Butyl-N-(3-hydroxyphenyl)benzenesulfonamide (1q).
1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.41 (s, 1H), 7.67
(d, 2H, J = 8.4 Hz), 7.54 (d, 2H, J = 8.8 Hz), 6.94 (t, 1H, J = 7.8 Hz),
6.57 (t, 1H, J = 2.0 Hz), 6.50 (d, 1H, J = 8.0 Hz), 6.35 (dd, 1H, JA =
4.0 Hz, JB = 2.0 Hz), 1.23 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ
158.26, 156.18, 139.36, 137.42, 130.25, 126.92, 126.52, 111.22, 110.33,
106.72, 35.29, 31.16; ESI (m/z) 306 (MH+), 304 (MH−); HRMS
(ESI) calcd for C16H19NO3S [MH−] 304.1013, found 304.1006.
General Procedure for the Preparation of Title Compound
2. To a solution of 1f in EtOH, 1 M solution of KOH in EtOH (1.1
equiv) was added at 0 °C. The reaction mixture was warmed to room
temperature and filtered after salt formed. The precipitate was washed
by EtOH three times and dried in vacuo.
Potassium 4-(Biphenyl-4-ylsulfonamido)phenolate (2). 1H
NMR (400 MHz, DMSO-d6) δ 7.77−7.68 (m, 6H), 7.48 (t, 2H, J =
7.6 Hz), 7.40 (t, 1H, J = 7.2 Hz), 6.81 (d, 2H, J = 8.8 Hz), 6.55 (d, 2H,
J = 8.8 Hz); 13C NMR (100 MHz, DMSO-d6) δ 155.29, 144.36,
138.81, 138.74, 129.52, 127.81, 127.59, 127.44, 124.40, 116.00; ESI
(m/z) 324 (M−).
HIF-1α Stability Analysis. HepG2 cells were pretreated with the
indicated concentrations of the synthetic derivatives for 30 min and
then exposed to 1% O2 for 4 h. HIF-1α and tubulin protein levels were
analyzed by Western blot analysis and quantified using densitometry.
Competitive Binding Assays by SPR. Biotin and biotinylated
terpestacin were sequentially immobilized onto the surface of a
streptavidin-coated sensor chip. Competitors (25 μM) were incubated
with purified UQCRB protein (25 μM) in pH 7.4 running buffer (10
mM HEPES, 150 mM NaCl, and 3 mM EDTA), and then UQCRB−
competitor complexes were injected onto the sensor chip at a flow rate
of 30 μL/min. The surface of the sensor chip was regenerated by
injecting 5 μL of regeneration buffer (50 mM NaOH). Molecular
interaction analysis was performed using the BIAcore 2000 system
(BIAcoreAB), and apparent binding affinities were calculated by
subtracting resonance values of UQCRB binding to control biotin
from those of UQCRB binding to biotinylated terpestacin.
Measurement of Mitochondrial ROS. Mitochondrial ROS levels
were measured using a MitoSOX Red mitochondrial superoxide
indicator (Invitrogen). Once in the mitochondria, the MitoSOX Red
reagents oxidized by superoxide emit red fluorescence. HepG2 cells
were pretreated with indicated concentrations of synthetic derivatives
for 30 min and then exposed to 1% O2 for 10 min. After incubation
with MitoSOX Red (5 μM) for 10 min, cells were lysed and
centrifuged to remove debris. The fluorescence in the protein-
normalized supernatant was determined using a FL600 microplate
fluorescence reader (Bio-Tek) at the excitation and emission
wavelengths of 510 and 580 nm, respectively.
In Vitro Angiogenesis Assays. The invasiveness of HUVECs was
examined using a Transwell chamber system with 8.0 μm pore-sized
polycarbonate filter inserts (Corning Costar, Acton, MA, USA) as
previously described.25,26 The total number of invaded cells was
counted using an optical microscope at 100× magnification. The tube
formation assay was performed using HUVECs grown on Matrigel
(BD Biosciences, San Jose, CA, USA) as previously described.25 Tube
formation was quantified by counting the number of connected cells in
randomly selected fields at 100× magnification and dividing that
number by the total number of cells in the same field.
CAM Assay. Fertilized chick eggs were maintained in a humidified
incubator at 37 °C for 3 days. Approximately 2 mL of egg albumin was
removed with a hypodermic needle allowing the CAM and yolk sac to
drop away from the shell membrane. On day 3.5 the shell was punched
N-(7-Hydroxynaphthalen-1-yl)biphenyl-4-sulfonamide (1l).
1H NMR (400 MHz, DMSO-d6) δ 8.04 (d, 1H, J = 9.2 Hz), 7.92
(s, 4H), 7.72 (d, 2H, J = 7.6 Hz), 7.50−7.43 (m, 4H), 7.19 (t, 1H, J =
7.8 Hz), 7.00−6.95 (m, 2H), 6.65 (d, 1H, J = 7.8 Hz), 5.81 (s, 2H);
13C NMR (100 MHz, DMSO-d6) δ 146.98, 146.55, 145.46, 138.23,
134.95, 133.51, 129.65, 129.45, 129.38, 128.74, 128.19, 127.64, 125.35,
121.46, 119.88, 118.40, 115.57, 108.48; ESI (m/z) 376 (MH+), 374
(MH−); HRMS (ESI) calcd for C22H17NO3S [MH+] 376.1002, found
376.1004.
1
N-(8-Hydroxyquinolin-5-yl)biphenyl-4-sulfonamide (1m). H
NMR (400 MHz, DMSO-d6) δ 8.62 (d, 1H, J = 4.0 Hz), 8.47 (d, 1H, J
= 8.4 Hz), 7.89 (d, 2H, J = 8.4 Hz), 7.81 (d, 2H, J = 8.4 Hz), 7.67 (d,
2H, J = 7.2 Hz), 7.49−7.41 (m, 3H), 7.33−7.30 (m, 1H), 7.21 (d, 1H,
J = 8.4 Hz), 6.58 (d, 1H, J = 8.8 Hz), 6.17 (s, 2H); 13C NMR (100
MHz, DMSO-d6) δ 150.60, 146.02, 145.46, 141.83, 138.55, 134.69,
134.60, 131.55, 129.59, 129.47, 129.24, 127.60, 127.56, 124.00, 119.87,
118.18, 105.93; ESI (m/z) 377 (MH+), 375 (MH−); HRMS (ESI)
calcd for C21H16N2O3S [MH+] 377.0954, found 377.0954.
N-(4-Hydroxyphenyl)naphthalene-2-sulfonamide (1n). 1H
NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.18−8.15 (m, 2H),
8.07 (d, 1H, J = 8.4 Hz), 7.80−7.73 (m, 2H), 7.67 (t, 1H, J = 7.2 Hz),
6.58 (d, 2H, J = 8.8 Hz), 6.35 (d, 2H, J = 9.2 Hz), 5.15 (s, 2H); 13C
NMR (100 MHz, DMSO-d6) δ 147.94, 138.95, 134.93, 131.43, 129.81,
129.57, 127.97, 122.71, 122.49, 113.89; ESI (m/z) 298 (MH−);
HRMS (ESI) calcd for C16H13NO3S [MH+] 300.0689, found
300.0685.
1
N-(4-Hydroxyphenyl)quinoline-8-sulfonamide (1o). H NMR
(400 MHz, DMSO-d6) δ 9.12 (d, 1H, J = 4.4 Hz), 8.50 (d, 1H, J = 8.0
Hz), 8.22−8.17 (m, 2H), 7.72−7.69 (m, 1H), 7.63 (t, 1H, J = 7.8 Hz),
6.70 (d, 2H, J = 8.4 Hz), 6.41 (d, 2H, J = 8.4 Hz); 13C NMR (100
MHz, DMSO-d6) δ 155.06, 151.81, 143.14, 137.44, 135.71, 134.34,
132.21, 128.96, 126.05, 124.00, 123.03, 115.66, 68.68, 27.84, 22.16;
7996
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out and removed, and the shell membrane was peeled away. When the
chick embryos were 4.5 days old, Thermanox coverslips (NUNC,
Rochester, NY, USA) containing the synthetic compounds were air-
dried and then applied to the CAM surface. Two days later, 2 mL of
10% fat emulsion (Greencross Co., Yongin, South Korea) was injected
into the chorioallantois, and the CAM was observed under a
microscope. Because retinoic acid (RA) is a known antiangiogenic
compound, RA was used as a positive control for antiangiogenic
responses. The response was scored as positive when CAM treated
with the sample showed an avascular zone similar to that of RA-treated
CAM with very few vessels compared to that of the control coverslip.
The response was calculated as the percentage of positive eggs relative
to the total number of eggs tested.
2012M3A9D1054520), the Translational Research Center for
Protein Function Control, KRF (Grant 2009-0083522), the
Next-Generation BioGreen 21 Program (Grant
PJ0079772012), Rural Development Administration, National
R&D Program, Ministry of Health &Welfare (Grant 0620360-
1), and the Brain Korea 21 Plus Project, Republic of Korea.
ABBREVIATIONS USED
■
UQCRB, ubiquinol−cytochrome c reductase binding protein;
ROS, reactive oxygen species; HDNT, 6-((1-hydroxynaphtha-
len-4-ylamino)dioxysulfone)-2H-naphtho[1,8-bc]thiophen-2-
one; HIF-1α, hypoxia inducible factor 1α; VEGF, vascular
endothelial growth factor; EC, endothelial cell; SPR, surface
plasmon resonance; BT, biotinylated terpestacin; VEGFR2,
vascular endothelial growth factor receptor type 2; HUVEC,
human umbilical vein endothelial cell; CAM, chorioallantoic
membrane; RA, retinoic acid
In Vivo Mouse Tumor Xenograft Assay. All mice were housed
in the specific pathogen-free (SPF) facility of the Laboratory Animal
Research Center (LARC) in Yonsei University, which is operated in
accordance with the Institutional Animal Care and Use Committee
(IACUC) and international guidelines on the ethical use of animals.
U87MG glioblastoma cells (5 × 106) suspended in 200 μL of PBS/
Matrigel (1:1) were subcutaneously implanted into the dorsal flank of
athymic nude mice (4 week-old female Balb C nu/nu, OrientBio).
Once the tumors became palpable (50−100 mm3), mice were
randomly divided into two groups (n = 8, per group) and
intraperitoneally treated with vehicle or 2 at a dosage of 20 mg/kg
every other day. The tumor volume and body weight of the mice were
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ASSOCIATED CONTENT
* Supporting Information
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Analytical data (HPLC analysis and NMR spectra) for all
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +82-2-2123-5883. Fax: +82-2-362-7265. E-mail:
kwonhj@yonsei.ac.kr.
■
Author Contributions
∥H.J.J. and M.C. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We are grateful to Dr. Chulho Lee for his help with the SAR
study. This study was partly supported by grants from the
National Research Foundation of Korea funded by the Korean
Government (Grants 2009-0092964, 2010-0017984, and
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