Discovery of indenopyrazoles as a new class of hypoxia inducible factor (HIF)-1 inhibitors

Article abstract of DOI:10.1021/ml3004632

The indenopyrazole framework was investigated as a new class of HIF-1α inhibitors. Indenopyrazole 2l was found to most strongly inhibit the hypoxia-induced HIF-1α transcriptional activity (IC₅₀ = 0.014 μM) among all of the known compounds havin

Full text of DOI:10.1021/ml3004632

Letter
pubs.acs.org/acsmedchemlett
Discovery of Indenopyrazoles as a New Class of Hypoxia Inducible
Factor (HIF)‑1 Inhibitors
Hidemitsu Minegishi, Shinji Fukashiro, Hyun Seung Ban,† and Hiroyuki Nakamura*
Department of Chemistry, Faculty of Science, Gakushuin University, Mejiro, Toshima-ku, Tokyo 171-8588, Japan
S
* Supporting Information
ABSTRACT: The indenopyrazole framework was investi-
gated as a new class of HIF-1α inhibitors. Indenopyrazole 2l
was found to most strongly inhibit the hypoxia-induced HIF-
1α transcriptional activity (IC50 = 0.014 μM) among all of the
known compounds having relatively simple structures, unlike
manassantins. Indenopyrazole 2l suppressed HIF-1α transcrip-
tional activity without affecting both HIF-1α protein
accumulation and HIF-1α/HIF-1β heterodimerization in
nuclei under the hypoxic conditions, suggesting that 2l
probably affected the transcriptional pathway induced by the HIF-1α/HIF-1β heterodimer.
KEYWORDS: HIF-1, indenopyrazole, inhibitor, transcriptional activity
ypoxia inducible factor (HIF)-1 is a transcription factor
Hthat controls the expression of genes influencing
angiogenesis, glucose metabolism, cell proliferation, survival,
and invasion of solid tumors and is thereby considered to be a
central regulator of major adaptive responses to hypoxia in
cancer progression.1 HIF-1 is a heterodimeric complex that
consists of a hypoxia-inducible HIF-1α subunit and a
constitutively expressed HIF-1β subunit.2,3 Under the normoxic
conditions, HIF-1α degradation is facilitated by the hydrox-
ylation of proline residues at the oxygen-dependent degradation
domain (ODDD) by prolyl hydroxylases (PHDs), and the
hydroxylated HIF-1α is degraded by the von Hippel Lindau
(VHL)-mediated ubiquitin proteasome system.4,5 Under the
hypoxic conditions, HIF-1α hydroxylation is impeded due to
the reduced PHD activity. The stabilized HIF-1α is
accumulated in cytosol and translocated to the nucleus where
it dimerizes with HIF-1β. The complex binds to hypoxia
response elements (HREs) together with coactivators to
activate target genes, including vascular endothelial growth
factor (VEGF), erythropoietin (EPO), glucose transporters,
and insulin-like growth factors.6−8 The fact that HIF-1α is
detected at increased levels in many types of human tumors9−14
makes it one of the potential targets of antitumor agents.1,15−17
Various compounds have the potential to inhibit HIF-1
transcriptional activity.17 Such compounds as PX-478,18 PX-
866,19 and EZN-296820 are currently undergoing phase I/II
clinical trials. We recently found that ortho-carboranylphenox-
yacetanilides, which were designed and synthesized based on
the structure of CAY10585,21,22 inhibited HIF-1 transcriptional
activity through hypoxia-induced HIF-1α protein accumula-
tion.23 The identification of heat shock protein (Hsp) 60 as the
primary target of ortho-carboranylphenoxyacetanilides revealed
that Hsp 60 played an important role in the hypoxia-induced
HIF-1α accumulation, both directly and indirectly.24 We also
found that VEGF receptor 2 (VEGFR2) tyrosine kinase
inhibitors, such as AAL993, SU5416, and KRN633, suppressed
HIF-1α transcriptional activity through the inhibition of Akt
and/or ERK phosphorylation signaling pathways.25 From those
findings, it occurred to us that other VEGFR2 tyrosine kinase
inhibitors might also have the potential to inhibit HIF-1α
transcriptional activity. We also found that indenopyrazoles
inhibited VEGFR2 tyrosine kinase activity.26 In this study, we
examined the inhibition of HIF-1α transcriptional activity by
indenopyrazoles under the hypoxic conditions using the dual
luciferase reporter gene assay27 and found that compound 1
(GN2707) significantly inhibited the hypoxia-induced HIF-1α
transcriptional activity, its IC50 being 0.214 μM (Figure 1).
Although compound 1 displayed potent inhibition of HIF-1α
Figure 1. Modification of hit compound 1.
Received: December 19, 2012
Accepted: January 27, 2013
Published: January 27, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
transcriptional activity, it was difficult to synthesize derivatives
for structure−activity relationship studies due to the low
chemical yields at the triketone formation step from
corresponding phthalate esters and acetophenones.28 Thus, in
this study, we focused on the alternative indenopyrazole
framework 2 and synthesized a series of indenopyrazole
derivatives.29 We evaluated the synthesized compounds for
potential biological activity and found that they were potent
inhibitors of the hypoxia-induced HIF-1 transcriptional activity
with IC50 of nanomolar order. So far, manassantins isolated
from the aquatic plant Saururus cernuus are known to most
strongly inhibit the hypoxia-induced HIF-1 transcriptional
activity in a T47D-cell-based dual luciferase reporter assay
(IC50 = 3−10 nM).30 Although manassantins are attractive
inhibitors for HIF-1-related biological studies, they have
complex structures with several chiral centers and thus require
a considerable number of synthetic steps.31,32 In this study, we
present an alternative inhibitor that is readily available for HIF-
1-related biological studies.
to their HIF-1 transcription inhibitory activities; the IC50 values
for the former were lower than 30 μM. On the other hand,
compounds 2h−l, which have a substituent on aniline ring B,
more strongly inhibited the hypoxia-induced HIF-1 transcrip-
tional activity than compounds 2a−g. Indenopyrazoles having
the monomethoxy group (2h and 2i), and the dimethoxy
groups (2j) attached to aniline ring B inhibited the hypoxia-
induced HIF-1 transcriptional activity with IC50 = 1.4, 6.1, and
3.2 μM, respectively. The antiproliferative activities of 2h−j
toward human cancer cell lines were similar to their HIF-1
transcriptional activities except for PC3 cells. Interestingly, a
cyclic ether skeleton on aniline ring B dramatically enhanced
the inhibition of the hypoxia-induced HIF-1 transcriptional
activity (2k−m). Compound 2k having a 3′,4′-methylenedioxy
group attached to aniline ring B inhibited the HIF-1
transcriptional activity with IC50 of 0.27 μM. The best result
was obtained for compound 2l, which has a 3′,4′-ethylenedioxy
group attached to aniline ring B: its IC50 was 0.014 μM, which
was 20 times higher than that of compound 2k and 100 times
higher than that of YC-1. The methoxy group substitution at 5-
position of aromatic ring A of 2l decreased the inhibitory effect
on HIF-1 transcriptional activity (2m), revealing that the 3′,4′-
ethylenedioxy substituent on aniline ring B is essential for the
strong inhibition of HIF-1 transcriptional activity. The
antiproliferative activities of indenopyrazoles 2k−m were
weaker than their HIF-1α transcription inhibitory activities by
2 orders of magnitude, revealing that indenopyrazoles 2k−m
suppressed HIF-1 transcriptional activity without affecting cell
viability.
The synthesis of indenopyrazoles is shown in Scheme 1.
Indenopyrazoles 2 were prepared from 2,3-dihydro-1H-inden-
a
Scheme 1. Synthesis of Indenopyrazoles 2a−m
Among the synthesized indenopyrazoles, 2l most strongly
inhibited the hypoxia-induced HIF-1 transcriptional activity.
Then, we examined the effects of 2l on the hypoxia-induced
HIF-1α protein accumulation by Western blot analysis and the
expression of HIF-1α and VEGFR mRNA by RT-PCR analysis
in HeLa cells. The results are shown in Figure 2. Interestingly,
2l did not suppress HIF-1α protein accumulation up to the
concentration of 1 μM. Furthermore, RT-PCR analysis revealed
that 2l inhibited the hypoxia-induced VEGF mRNA expression
in a concentration-dependent manner in the range of 0.001−
1 μM. However, the HIF-1α mRNA expression levels were not
affected by 2l. These results clearly indicate that 2l inhibits the
hypoxia-induced VEGF expression without suppressing HIF-1α
mRNA expression as well as HIF-1α protein accumulation.
As indenopyrazole 2l was found to inhibit the hypoxia-
induced HIF-1 transcriptional activity without suppressing
HIF-1α protein accumulation, we next examined the effect of 2l
on the localization of HIF-1α protein in HeLa cells under the
hypoxic conditions. Immunofluorescence analysis showed that
the basal level of HIF-1α protein was low under the normoxic
conditions, but the accumulated HIF-1α protein was trans-
located into nuclei under the hypoxic conditions (Figure 3a).
Treatment with CAY10585 at 30 μM potentially suppressed
HIF-1α protein accumulation and nuclear translocation under
the hypoxic conditions. Interestingly, the treatment with 2l at 1
μM did not affect the localization of HIF-1α protein, which was
translocated into nuclei under the hypoxic conditions. To
confirm whether indenopyrazole 2l inhibited HIF-1α/HIF-1β
heterodimerization, we performed immunoprecipitation (IP)
analysis. As shown in Figure 3b, HIF-1α/HIF-1β heterodime-
rization was detected by immunoprecipitation using HIF-1α
antibody. Whole cell lysates and immunoprecipitation products
were immunoblotted with HIF-1α, HIF-1β, and tubulin
antibodies. Although HIF-1α and HIF-1β proteins were
a
Reagents and conditions: (a) LiHMDS, THF, 12 h. (b)
H2NNH2·H2O, AcOH, reflux, 24 h, 43−85%.
1-ones 3 and phenyl isothiocyanates 4 using the procedure in
the literature with modification.29 Compound 2a was identified
by comparison with the reported H NMR spectra in the
1
literature.29 Indenopyrazoles 2b−g, which have a substituent on
aromatic ring A, and indenopyrazoles 2h−l, which have a
substituent on aniline ring B, were synthesized from
corresponding substituted 1-indanones 3b−g and 4a and
substituted phenyl isocyanates 4b−f in a similar manner to
the synthesis of 2a, respectively. Furthermore, indenopyrazole
2m, which has substituents on both aromatic rings A and B, was
also synthesized by the reaction of 3b and 4f in 62% yield.
Synthesized indenopyrazoles 2a−m were tested for their
ability to inhibit HIF-1 transcriptional activity in HeLa cells
under the hypoxic conditions (1% oxygen) using the dual
luciferase reporter gene assay and their antiproliferative
activities toward four human cancer cell lines, HCT116
(human colon cancer), HepG2 (human hepatoma), PC3
(human prostate cancer), and HeLa (human cervical
carcinoma) using the MTT assay. 3-(5′-Hydroxymethyl-2′-
furyl)-1-benzyl indazole (YC-1) and CAY1058521 were used as
positive controls for comparison. The results are summarized in
Table 1. Indenopyrazole 2a at 30 μM, which has no substituent
on aromatic rings A and B, failed to inhibit HIF-1 transcrip-
tional activity. Indenopyrazoles 2b−g, which have a substituent
on aromatic ring A, exhibited weak or no inhibition of HIF-1
transcriptional activity (0−41% inhibition at 30 μM).
Compounds 2a−g had high antiproliferative activities relative
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ACS Medicinal Chemistry Letters
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a
Table 1. Effects of Indenopyrazoles 2 on Proliferative Activities of HCT116, HepG2, PC3, and HeLa Cell Lines and Hypoxia-
b
Induced HIF-1 Transcriptional Activity in HeLa Cells
a
b
Cells were incubated with various concentrations (0.1−30 μM) of compounds for 72 h, and cell viability was determined by the MTT assay. HeLa
cells stably transfected with HRE-firefly luciferase and cytomegalovirus (CMV) promoter-renilla luciferase were incubated for 12 h with compounds
under the hypoxic conditions. After the supernatant was removed, the luciferase assay was performed using the dual luciferase assay system. Average
values standard deviations of triplicate samples (n = 3). Percentage (%) inhibition at 30 μM is indicated in parentheses.
c
d
detected in whole cell lysates, those proteins were also detected
in immunoprecipitation products, and the inhibition of HIF-
1α/HIF-1β heterodimerization by 2l was not observed in HeLa
cells under the hypoxic conditions. Together, the results
indicate that indenopyrazole 2l inhibits the hypoxia-induced
HIF-1 transcriptional activity without affecting HIF-1α/HIF-1β
heterodimerization in nuclei. Furthermore, we examined
immunoprecipitation by anti-HIF-1α antibody to confirm
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ACS Medicinal Chemistry Letters
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Figure 2. Effects of compound 2l on HIF-1α protein and mRNA expression under the hypoxic conditions. HeLa cells were incubated for 4 h with
compound 2l at different concentrations under the hypoxic conditions. (a) HIF-1α protein expression was detected by immunoblot analysis with the
specific antibody. CAY10585 was used as a positive control for the inhibition of HIF-1α protein expression. Tubulin was used as an internal control.
(b) mRNA levels of HIF-1α, VEGF, and GAPDH were detected by RT-PCR. GAPDH was used as an internal control.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and references. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Fax: +81-3-5992-1029. E-mail: hiroyuki.nakamura@
gakushuin.ac.jp.
Present Address
†Biomedical Genome Research Center, Korea Research
Institute of Bioscience and Biotechnology, Yuseong, Daejeon
305-806, Korea.
Funding
This work was partially supported by a Grant-in-Aid for
Scientific Research on Innovative Areas “Chemical Biology of
Natural Products” from The Ministry of Education, Culture,
Sports, Science and Technology, Japan.
Notes
The authors declare no competing financial interest.
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