Vegfrecine, an inhibitor of VEGF receptor tyrosine kinases isolated from the culture broth of Streptomyces sp.

Article abstract of DOI:10.1021/np300535c

A new inhibitor of VEGF receptor tyrosine kinases, vegfrecine (1), was isolated from the culture broth of Streptomyces sp. MK931-CF8. The molecular structure of 1 was determined by NMR and MS analysis combined with synthesis. Compound 1 showed potent inhi

Full text of DOI:10.1021/np300535c

Note
pubs.acs.org/jnp
Vegfrecine, an Inhibitor of VEGF Receptor Tyrosine Kinases Isolated
from the Culture Broth of Streptomyces sp.
Chisato Nosaka,† Hayamitsu Adachi,*,† Ryuichi Sawa,† Koichi Nakae,† Sonoko Atsumi,†
Naoko Kinoshita,† Yumiko Kubota,† Masayuki Igarashi,† Yoshihisa Sei,‡ Kentaro Yamaguchi,§
Masabumi Shibuya,⊥ Yoshio Nishimura,† and Yuzuru Akamatsu†
†Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
‡Center for Advanced Materials Analysis, Technical Department, Tokyo Institute of Technology, 4259 Nagatsuda-cho, Midori-ku,
Yokohama, Kanagawa 226-8503, Japan
§Tokushima Bunri University, 1314-1 Shido, Sanuki-city, Kagawa 769-2193, Japan
⊥Jobu University, 270-1 Shinmachi, Takasaki, Gunma 370-1393, Japan
S
* Supporting Information
ABSTRACT: A new inhibitor of VEGF receptor tyrosine kinases,
vegfrecine (1), was isolated from the culture broth of Streptomyces sp.
MK931-CF8. The molecular structure of 1 was determined by NMR and
MS analysis combined with synthesis. Compound 1 showed potent
inhibitory activity against vascular endothelial growth factor receptor
(VEGFR) tyrosine kinases in in vitro enzyme assays, but platelet-derived
growth factor receptors (PDGFRs), fibroblast growth factor receptor
(FGFR), and epidermal growth factor receptor (EGFR) responded only
weakly. Compound 1 is a promising new selective VEGFR inhibitor for
investigating new treatments of cancer and inflammatory diseases.
ascular endothelial growth factors (VEGFs) are important
cancer, atherosclerosis, arthritis, ocular neovascular disease, and
Vregulators of angiogenesis, both in normal physiological metastasis formation.22−24 In the course of our screening of
processes such as embryonic development1,2 and in abnormal
microbial metabolites to identify novel inhibitors of Flt-1
tyrosine kinase, compound 1 was isolated from the culture
broth of Streptomyces sp. Compound 1 exhibited potent
inhibitory activity against VEGFR tyrosine kinases in in vitro
enzyme assays, but only weak inhibitory activity against
PDGFRs, FGFR, and EGFR.
disease states. Abnormal angiogenesis is associated with various
pathological processes such as inflammation, rheumatoid
arthritis, ocular neovascularization, psoriasis, and tumor
development. It has been shown that VEGFs are important
for tumor growth and metastasis formation.3−7 VEGF
expression is markedly upregulated in tumors,8 and inhibition
of VEGF-induced angiogenesis significantly inhibits tumor
growth in vivo.9−13 Avastin, the monoclonal antibody against
VEGF-A, has been used as a therapeutic drug for cancer.14
VEGF-A interacts with two potent receptor tyrosine kinases,
Flt-1 (VEGFR-1) and KDR (VEGFR-2). Both Flt-1 and KDR
exist almost exclusively in endothelial cells, and VEGF-A
stimulates vascular endothelial cell proliferation, migration, and
protease production. Several studies showed that KDR is
strongly autophosphorylated in endothelial cells upon VEGF
stimulation and mediates a mitogenic response.15 On the other
hand, tyrosine kinase activity of Flt-1 is weak, leading to no
significant mitogenic response in endothelial cells. Flt-1
signaling does not appear to be involved in physiological
angiogenesis, but in a recent study, activation of Flt-1 was
found to be important for angiogenesis in pathological
conditions. Furthermore, Flt-1 is functionally expressed in
monocytes and macrophages, which play a crucial role in
promoting inflammation.16−21 Indeed, blocking the Flt-1 signal
is sufficient to inhibit strongly pathological angiogenesis such as
In the screening program for microbial metabolites with
inhibitory activity against Flt-1 tyrosine kinase, we used the
kinase domain of recombinant human Flt-1 expressed in a
baculovirus/insect cell system and 32P-ATP as a tracer to
measure phosphorylation. Strong Flt-1 tyrosine kinase inhib-
itory activity was observed in the ethanol extract of the broth of
Streptomyces sp. MK931-CF8 cultured in solid pressed barley
medium. Ethanol extracts of the solid cultures were loaded onto
Diaion HP-20 and eluted with MeOH. The active fractions
were combined and evaporated to give a brown solid. The solid
was subjected to silica gel column chromatography followed by
Sephadex LH-20 column chromatography to give 1 as a
magenta solid. The molecular formula of 1 was determined to
be C13H11N3O4 by HRESIMS. UV spectra of 1 showed
characteristic absorption maxima at 248, 268, 334, and 524 nm
and bathochromic shifts (297, 340, 592 nm) in alkaline
solution, suggesting the presence of a phenol. In addition,
Received: August 3, 2012
Published: February 15, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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absorption at 1633 cm−1 in the IR spectrum of 1 revealed the
presence of quinone carbonyl groups. The structure of 1 was
the 2-position. Deprotection of the TBS group of 7 produced
the desired compound 1 (see Supporting Information). Thus,
the chemical structure of the active compound 1 was
established as 2-amino-5-((2′-hydroxyphenyl)amino)-3,6-diox-
ocyclohexa-1,4-dienecarboxamide and named vegfrecine. Com-
pound 1 has a unique structure with a p-benzoquinone ring
possessing the same array of amino and carboxamide groups
that are found in the antibiotic G-7063-226 and sarubicin A.27
The quinone skeleton of sarubicin A is derived from 6-
hydroxyanthranilic acid via oxidation of the hydroquinone, 3,6-
dihydroxyanthranylamide. Such oxidation of the hydroquinone
was mimicked in the synthesis of 6 from 5.
We investigated the inhibitory activities of 1 against several
different tyrosine kinases. VEGFRs, PDGFR-α and -β, FGF,
and EGF tyrosine kinases were selected to cover a broad
spectrum of tyrosine kinases. The VEGFRs are predominantly
expressed on endothelial cells. Flt-1 (VEGFR-1) and KDR
(VEGFR-2) are responsible for endothelial cell proliferation
and blood vessel permeability, while Flt-4 (VEGFR-3) seems to
be critical for lymphatic vessel development. These receptor
tyrosine kinases are essential for tumor angiogenesis and
lymphangiogenesis, respectively.28−30 Compound 1 showed
selective inhibition against VEGFRs in vitro and weakly
inhibited PDGFR-α and -β, FGF, and EGF tyrosine kinase.
The inhibitory potency of 1 surpassed that of the positive
control compound, SU5416.31
1
established by H, 13C, HMQC, HMBC, and COSY spectra.
The 13C and DEPT135 spectra revealed the presence of eight
sp2 quaternary carbons including three carbonyls and five sp2
methines, respectively. Uncorrelated proton signals (δ 7.42,
8.66, 8.95, 9.19, 10.09, and 11.00) in the HMQC spectrum
were assigned as exchangeable protons. The COSY spectrum of
1 revealed the connectivities between H-3′ (δ 6.96) and H-6′
(δ 7.26), δ 7.42 and 8.66. The HMBC spectrum of 1 showed
connectivities from H-3′ and H-5′ (δ 6.86) to C-1′ (δ 124.4),
H-4′ (δ 7.12) and H-6′ to C-2′ (δ 151.0), and from δ 7.42 to
167.9, indicating the presence of ortho-disubstituted benzene
and carboxamide moieties. However the structure of 1 could
not be thoroughly clarified because full HMBC correlations
were missing. Therefore, compound 1 was converted into the
1
MOM derivative 2 (Figure 1). The H−13C HMBC spectrum
Compound 1 showed a weak in situ inhibitory activity of over
10 μM against the ligand-induced phosphorylation of Flt-1 in
NIH3T3-Flt-1 cells at two hours after treatment of 1, at which
time cytotoxicity was not observed. The inhibitory activity of 1
in situ is 100-fold less active than that in vitro (Figure 3). The
weak inhibitory activity in situ could originate from poor cell
membrane permeability and nonspecific interaction of 1 with
cell components.
On the other hand, the cytotoxicity of 1 against NIH3T3-Flt-
1 and NIH3T3-KDR cells at 48 h after treatment of 1 was 3
and 12 μM, respectively. The structure−activity relationship
study is ongoing to improve the profile of activity in situ.
Figure 1. Structures of vegfrecine (1) and its MOM derivative (2).
of 2 revealed the following connections: from the MOM1
methylene at δ 5.13 to C-2′ (δ 150.6); from the MOM2
methylene at δ 5.01 to C-1′ (δ 135.8) of ortho-disubstituted
benzene and C-5 (δ 154.7); from H-4 (δ 5.89) to C-2 (δ 154.5)
and C-6 (δ 178.7); from the carboxamide protons (δ 7.17,
8.18) to C-1 (δ 97.2); and from NH2-2 (δ 8.56, 10.50) to C-1
and C-3 (δ 178.2). These data suggested a partial structure of
the 2-amino-3,6-dioxocyclohexa-1,4-dienecarboxamide moiety.
Furthermore, 1H−15N HMBC experiments with 2 demon-
strated the couplings of H-4, H-6′, and MOM2 methylene to a
tertiary amine, resulting in the connectivity between C-1′ of
ortho-disubstituted benzene and C-5 of the quinone ring with
an imino linkage. The protective groups of MOM1 and MOM2
were attached to oxygen and nitrogen, respectively. All of the
carbon connections in structure 2 were established except that
between C-1 and C-6 (Figure 2). Therefore we planned to
synthesize 1 based on the reasonable assumption that a
quinone is present. Scheme 1 illustrates the synthetic plan.
Oxidative amination of 2,5-dihydroxybenzamide 5 with the
TBS derivative 425 afforded the amino benzoquinone 6.
Ammonolysis of 6 gave compound 7, with an amino group at
EXPERIMENTAL SECTION
■
General Experimental Procedures. UV spectra were obtained
on a U-2800 spectrophotometer (Hitachi High-Tech, Tokyo, Japan).
The IR spectrum was obtained on a FT-210 Fourier transform infrared
spectrometer (Horiba, Kyoto, Japan). NMR spectra were recorded
using a JNM-ECA600 (JEOL RESONANCE Inc., Tokyo, Japan) and
an AVANCE 700 spectrometer (Bruker BioSpin, Rheinstetten,
Germany) with TMS as an internal standard. HRESIMS spectra
were measured using an LTQ Orbitrap mass spectrometer (Thermo
Fisher Scientific, San Jose, CA, USA).
Taxonomy. Strain MK931-CF8 was isolated from a soil sample
collected at Sendai-shi, Miyagi Prefecture, Japan, in April 1997. Strain
MK931-CF8 formed well-branched vegetative mycelia and aerial
hyphae that bore straight spore chains. The aerial hyphae of the strain
were light brownish-gray. The vegetative mycelia were pale yellow to
pale yellowish-brown. These characteristics were observed on yeast
extract-starch agar. The diaminopimelic acid isomers in whole-cell
hydrolysates of strain MK931-CF8 were determined to be the LL-form
by the method of Staneck and Roberts.33 The 16S rRNA gene
sequence (1478 bp, positions 28−1524, Escherichia coli numbering
system34 of the strain) was determined and deposited in Genbank
under the accession number AB688982. The sequence of the strain
showed high identity with those of the genus Streptomyces such as
Streptomyces tanashiensis (NBRC 12919T, T: Type strain, Genbank/
EMBL/DDBJ database accession no. AB184245, 1467/1475 bp, 99%),
S. nashvillensis (NBRC 13064T, AB184286, 1467/1477 bp, 99%), and
Figure 2. HMBC correlations of 2.
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Scheme 1. Synthesis of 1
Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden)
and eluted with MeOH to yield 1 (43 mg).
Vegfrecine (1): magenta, amorphous solid; UV (MeOH) λmax (log
ε) 248 (4.02), 268 (4.06), 334 (4.04), 524 (3.30) nm, (MeOH-HCl)
λmax (log ε) 248 (4.02), 268 (4.06), 334 (4.04), 523 (3.29) nm,
(MeOH/NaOH) λmax (log ε) 297 (3.94), 340 (3.95), 592 (3.72) nm;
IR (KBr) νmax 3417, 3311, 1633, 1581, 1519, 1459, 1375, 1349 cm−1;
1H and 13C NMR data are listed in Table 1; HRESIMS (positive) m/z
274.0822 (M + H)+ (calcd for C13H12N3O4, 274.0822).
Synthesis of 2. To a solution of 1 (4 mg, 0.0146 mmol) in
dimethyl formamide (0.2 mL) were added methoxymethyl chloride
(11 μL, 0.146 mmol) and N,N-diisopropylethylamine (51 μL, 0.292
mmol), and the reaction mixture was stirred overnight. The reaction
mixture was diluted with ethyl acetate, washed with water, dried over
MgSO4, and filtered. The filtrate was evaporated to give a crude solid,
which was subjected to preparative TLC on silica gel developed with a
mixture of chloroform and methanol (50:1) to give 2 (4.2 mg, 80%
yield). 2: H and 13C NMR data are listed in Table 1; HRESIMS
1
Figure 3. Effect of 1 on the autophosphorylation of Flt-1 (VEGFR-1)
(a) and KDR (VEGFR-2) (b) in NIH3T3 cells.
(positive) m/z 384.1158 (M + Na)+ (calcd for C17H19N3O6Na,
384.1166).
Synthesis of 1. To a solution of 5 (3.13 g, 20.4 mmol) in a mixture
of MeOH and water (1:2, 664 mL) were added compound 425 (10.5 g,
47 mmol) in MeOH (66 mL) and NaIO4 (15.27 g, 71.4 mmol) in
water (132 mL), and the reaction mixture was stirred overnight at
room temperature. The reaction mixture was diluted with ethyl
acetate, washed with water, dried over MgSO4, and filtered. The filtrate
was evaporated to give a crude solid, which was subjected to column
chromatography on silica gel. Elution with a mixture of toluene and
acetone (20:1) gave 6 (8.4 g, 70% yield).
S. polychromogenes (NBRC 13072T, AB184292, 1454/1470 bp, 98%).
These morphological characteristics together with genetic analysis of
strain MK931-CF8 suggested that the strain belongs to the genus
Streptomyces. Therefore, the strain was designated as Streptomyces sp.
MK931-CF8
Fermentation and Isolation of 1. A slant culture of Streptomyces
sp. MK931-CF8 was inoculated into 110 mL of medium in 500 mL
baffled flasks and incubated for 2 days at 27 °C on a rotary shaker at
180 rpm. Each flask contained 110 mL of seed medium consisting of
2% galactose, 2% dextrin, 1% soypeptone, 0.5% corn steep liquor, 0.2%
(NH4)2SO4, and 0.2% CaCO3 in deionized water and was adjusted to
pH 7.2 before sterilization. Aliquots (7 mL) of the seed culture were
transferred to fifty 500 mL Erlenmeyer flasks containing sterilized solid
production medium composed of barley (15 g) and deionized water
(25 mL), and the medium was cultivated statically at 30 °C for 17
days. Ethanol was added to the medium, and the combined ethanol
extracts (2000 mL) from the solid cultures were evaporated to give a
brown residue, which was dissolved in 8000 mL of water. The aqueous
solution of the residue was loaded onto HP-20 (600 mL) (Mitsubishi
Chemical Co., Tokyo, Japan), washed with 60% MeOH, and then
eluted with MeOH. The MeOH fractions were combined and
evaporated to give a brown solid (2.27 g). The solid was subjected to
silica gel column chromatography (62 g, Cica silica gel 60N, Kanto
Chemical Co. Inc., Tokyo, Japan) and eluted with 200 mL of CHCl3/
MeOH (50:1) followed by 400 mL of CHCl3/MeOH (40:1). The
active fractions (140 mL) were combined and evaporated to give
solids, which were further subjected to column chromatography over
1
6: H NMR (600 MHz, CDCl3) δ 0.16 (6H, s), 0.23 (6H, s), 0.91
(9H, s), 1.02 (9H, s), 5.54 (1H, brd, J 3.3 Hz), 6.05 (1H, s), 6.85 (1H,
d, J 8.5 Hz), 6.90 (1H, dd, J 1.4 and 7.8 Hz), 6.91−7.0 (2H, m), 7.03
(1H, dd, J 0.6 and 3.1 Hz), 7.08−7.16 (2H, m), 7.32 (1H, dd, J 0.6 and
3.2 Hz), 8.53 (1H, s), 9.36 (1H, brs); 13C NMR (150 MHz, CDCl3) δ
−4.35, 18.1, 18.2, 25.6, 25.7, 98.6, 98.8, 119.4, 119.9, 121.1, 121.5,
121.6, 125.4, 125.6, 127.6, 129.0, 131.7, 144.9, 147.6, 148.8, 157.1,
171.0, 177.5, 178.6; HRESIMS (positive) m/z 616.2620 (M + Na)+
(calcd for C31H43N3O5NaSi2, 616.2633).
To a solution of 6 (10.8 g, 18.1 mmol) was added a 7 M ammonia
in methanol solution (Aldrich) (114 mL), and the reaction mixture
was stirred at room temperature for 2 h. Evaporation of the solvent
gave a crude solid, which was subjected to column chromatography on
silica gel. Elution with a mixture of CHCl3 and MeOH (20:1) gave 7
(6.65 g, 95%).
1
7: H NMR (600 MHz, CDCl3) δ 0.04 (6H, s), 0.81 (9H, s), 5.37
(1H, brs), 5.97 (1H, s), 6.72 (1H, dd, J 1.2 and 8.4 Hz), 6.81 (1H, dt, J
1.2 and 7.8 Hz), 6.90 (1H, dt, J 1.2 and 7.8 Hz), 7.17 (1H, dd, J 1.8
and 7.8 Hz), 7.45 (1H, brs), 8.54 (1H, brs), 8.79 (1H, brs), 10.65 (1H,
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Note
a
1
Table 1. H and 13C NMR Data of 1 and 2
1
2
position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
1
96.5 C
157.3 C
175.7 CO
95.9 CH
148.2 C
176.6 CO
124.4 C
151.0 C
116.2 CH
127.4 CH
119.2 CH
124.2 CH
167.9
97.2 C
154.5 C
178.2 CO
104.8 CH
154.7 C
178.7 CO
135.8 C
150.6 C
115.3 CH
126.7 CH
122.1 CH
127.4 CH
169.4
2
3
4
5.55, s
5.89, s
5
6
1′
2′
3′
4′
5′
6′
6.96, dd (1.0, 7.7)
7.12, dt (1.5, 7.7)
6.86, dt (1.0, 7.7)
7.26, dd (1.5, 7.7)
7.10, dd (1.0, 7.6)
7.21, dt (1.0, 7.6)
7.01, dt (1.0, 7.6)
7.19, dd (1.0, 7.6)
1-CONH2
1-CONH2
7.42, d (3.2)
8.66, d (3.2)
8.95, br
7.17, d (3.5)
8.18, d (3.5)
8.56, br
2-NH2
11.00, br
9.19, s
10.50, br
5-NH
2′-OH
MOM1
10.09, s
94.3 CH2
55.7 CH3
85.0 CH2
55.9 CH3
5.13, s
3.30, s
5.01, s
3.24, s
MOM2
a
NMR spectra were measured at 600 MHz for 1 in DMSO-d6 and 700 MHz for 2 in DMSO-d6. Chemical shifts are expressed in ppm with TMS as
an internal standard.
and 20 mM Tris-HCl, 1 mM MnCl2, 10 mM MgCl2, 50 μg/mL poly
GluTyr, 100 mM NaCl, 1 μM Na3VO4, 1 μM DTT, and 0.5% BSA at
pH 7.4 for KDR, EGFR, and FGFR. Each kinase (55 ng/20 μL buffer)
and the compound dissolved in 2% DMSO buffer solution (10 μL)
were added to a well, and the reaction was started by addition of 4 μM
ATP/10 μCi/mL 32P-ATP buffer solution (10 μL). After incubation
for 20 min at room temperature the reaction was stopped by the
addition of 30% TCA. Filter plates were washed three times with 15%
TCA, and radioactivity was measured on a scintillation counter (Tri-
Carb 2800TR, PerkinElmer, Waltham, MA, USA).
Cell Culture. Flt-1 overexpressing NIH3T3-Flt-1 cells and KDR
overexpressing NIH3T3-KDR cells were grown in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal calf serum, 2
mmol of glutamine, and 200 μg/mL G418. Human umbilical vein
endothelial cells were obtained from LONZA (Walkersville, MD,
USA) and cultured in endothelial growth medium (LONZA).
Cytotoxicity Assay. Cell viability was measured by the MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colori-
metric method.35 The cells were seeded in 96-well microplates at
densities of 3 × 103cells in 100 μL of growth medium per well in the
absence or presence of test samples. After 2 days’ incubation, MTT
(10 μL of 5 mg/mL) was added to each well, and the plate was further
incubated for 2 h. Resultant MTT formazan was solubilized with 10%
sodium dodecyl sulfate overnight, and the absorbance at 570 nm was
measured using an ARBO-SX 1420 multilabeled counter (PerkinElm-
er).
Table 2. Inhibitory Activities of 1 and SU5416
IC50 (μM)
RTK
1
SU5416
Flt-1 (VEGFR-1)
KDR (VEGFR-2)
Flt-4 (VEGFR-3)
PDGF-α
0.018
0.011
0.0018
3.2
3
1
0.5
0.2
0.4
>100
5
PDGF-β
0.48
2.6
EGF
FGF
0.73
brs); 13C NMR (150 MHz, CDCl3) δ −4.30, 18.1, 25.5, 25.6, 96.7,
97.2 (br), 119.5, 121.7, 122.1, 126.6, 127.8, 128.2, 146.8, 148.2, 149.6,
156.6, 170,0, 176.4, 177.1; HRESIMS (positive) m/z 388.1681 (M +
H)+ (calcd for C19H26N3O4Si, 388.1687).
To a solution of 7 (1.04 g, 2.71 mmol) in tetrahydrofuran (303 mL)
were added acetic acid (5.89 mL, 102.9 mmol) and a 1 M
tetrabutylammonium fluoride solution (2.7 mL) in tetrahydrofuran,
and the reaction mixture was stirred at room temperature for 5 min.
The reaction mixture was diluted with ethyl acetate, washed with
water, dried over MgSO4, and filtered. The filtrate was evaporated to
give a solid, which was washed with MeOH to afford synthetic
1
compound 1 (660 mg, 90% yield). The H and 13C NMR spectra of
synthetic compound 1 were identical to those of vegfrecine (see
Supporting Information).
Immunoblotting. Subconfluent cells were starved for 16 h and
then incubated for 2 h with samples. Cells were subsequently
stimulated for 5 min with 50 ng/mL recombinant human VEGF-A
(R&D Systems Inc., Minneapolis, MN, USA). Cells were then lysed in
lysis buffer (50 mM HEPES [pH 7.2], 1% Triton X-100, 150 mM
NaCl, 10% glycerol, 1 mM EGTA, 1 mM Na3VO4, 1 mM PMSF, 500
U/mL aprotinin), and the insoluble material was removed by
centrifugation. Equal amounts of protein were separated electro-
phoretically on 7.5% polyacrylamide-SDS gels and transferred to
polyvinylidene difluoride membranes. The membranes were blocked
in blocking agent (5% nonfat milk in Tris-buffered saline containing
In Vitro Tyrosine Kinase Assay. The kinase domain of Flt-1 was
expressed as a GST-fusion protein by infection of Sf9 cells with
engineered baculoviruses. GST-Flt-1 was purified from infected Sf9
cell lysates on glutathione sepharose. The enzymes KDR, Flt-4
(VEGFR-3), PDGFR-α and -β, EGFR, and FGFR were purchased
from Invitrogen Co Ltd. The assays were performed in 96-well filter
plates. The assay buffers were 20 mM Tris-HCl, 3 mM MnCl2, 3 mM
MgCl2, 50 μg/mL poly GluTyr, 100 mM NaCl, 1 μM Na3VO4, 1 μM
DTT, and 0.5% BSA at pH 7.4 for Flt-1, Flt-4, and PDGFR-α and -β,
718
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Note
0.1% Tween 20) and incubated with anti-p-tyr antibody (Cell
Signaling Technology), anti-Flt-1 goat polyclonal antibody (R&D
systems Inc.), anti-KDR rabbit polyclonal antibody (SantaCruz
Biotechnology Inc., Santa Cruz, CA, USA), and anti-actin mouse
monoclonal antibody (Abcam PLC., Cambridge, UK).
To analyze the phosphorylation of Flt-1, cell lysates were
immunoprecipitated with anti-Flt-1 rabbit polyclonal antibody (Santa
Cruz Biotechnology Inc.), and immune complexes were then
separated by SDS-PAGE gel and blotted with the antibodies described
above.
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Manova, K.; Benezra, R.; Rafii, S. Nat. Med. 2001, 7, 1194−1201.
(21) Hiratsuka, S.; Maru, Y.; Okada, A.; Seiki, M.; Noda, T.; Shibuya,
M. Cancer Res. 2001, 61, 1207−1213.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR, 1H−1H COSY, 1H−13C HMQC, and
1H−13C HMBC spectra of 1. H and 13C NMR, H−13C
HMQC, 1H−13C HMBC, and 1H−15N HMBC spectra of 2. 1H
and 13C NMR spectra of synthetic vegfrecine (1). This material
is available free of charge via the Internet at http://pubs.acs.org.
1
1
AUTHOR INFORMATION
Corresponding Author
*Tel: +81-3-3441-4173. Fax: +81-3-3441-7589. E-mail:
adachih@bikaken.or.jp (H.A.).
■
(22) Wu, Y.; Zhong, Z.; Huber, J.; Bassi, R.; Finnerty, B.; Corcoran,
E.; Li, H.; Navarro, E.; Balderes, P.; Jimenez, X.; Koo, H.;
Mangalampalli, V. R.; Ludwig, D. L.; Tonra, J. R.; Hicklin, D. Clin.
Cancer Res. 2006, 12, 6573−6583.
(23) Choi, S. T.; Kim, J. H.; Seok, J. Y.; Park, Y. B.; Lee, S. K. Clin.
Rheumatol. 2009, 28, 333−337.
Author Contributions
The manuscript was written through contributions of H.
Adachi, C. Nosaka, R. Sawa, and N. Kinoshita. All authors have
given approval to the final version of the manuscript.
Notes
(24) De Bandt, M.; Ben Mahdi, M. H.; Ollivier, V.; Grossin, M.;
Dupuis, M.; Gaudry, M.; Bohlen, P.; Lipson, K. E.; Rice, A.; Wu, Y.;
Gougerot-Pocidalo, M. A.; Pasquier, C. J. Immunol. 2003, 171, 4853−
4859.
(25) Daniel, D.; Arkadii, V.; Oscar, M.; Silvana, L.; Stephane, R.,
Sylvie, F.; Giliane, B.; Zhihong, Z., Isabelle, P.; Frederic, G.; Ljubomir,
I. WO2005092899
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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The authors express their thanks to Dr. M. Hatano for the
characterization of Streptomyces sp. MK931-CF8. This research
was supported by the Intramural Research Project of the
Microbial Chemistry Research Foundation (MCRF).
(26) Noble, M.; Noble, D.; Sykes, R. B. J. Antibiot. 1977, 30, 455−
459.
(27) Gould, S. J.; Eisenberg, R. L.; Hills, L. R. Tetrahedron 1991, 47,
7209−7218.
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