Design, synthesis, and evaluation of indeno[2,1-c]pyrazolones for use as inhibitors against hypoxia-inducible factor (HIF)-1 transcriptional activity

Article abstract of DOI:10.1016/j.bmc.2019.115207

HIF-1 is regarded as a promising target for the drugs used in cancer chemotherapy, and creating readily accessible templates for the development of synthetic drug candidates that could inhibit HIF-1 transcriptional activity is an important pursuit. In this study, indeno[2,1-c]pyrazolones were designed as readily available synthetic inhibitors of HIF-1 transcriptional activity. Nine compounds were synthesized in 4–5 steps from commercially available starting materials. In evaluations of the ability to inhibit the hypoxia-induced transcriptional activity of HIF-1, compound 3c showed a higher level compared with that of known inhibitor, YC-1. The compound 3c suppressed HIF-1α protein accumulation without affecting the levels of HIF-1α mRNA.

Full text of DOI:10.1016/j.bmc.2019.115207

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and evaluation of indeno[2,1-c]pyrazolones for use as
inhibitors against hypoxia-inducible factor (HIF)-1 transcriptional activity
Shinichiro Fusea, Kensuke Suzukia,b, Takahiro Kuchimarub, Tetsuya Kadonosonob, Hiroki Uedaa,b
,
⁎
Shinichi Satoa, Shinae Kizaka-Kondohb, Hiroyuki Nakamuraa,
a Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan
b School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
HIF-1 is regarded as a promising target for the drugs used in cancer chemotherapy, and creating readily ac-
cessible templates for the development of synthetic drug candidates that could inhibit HIF-1 transcriptional
activity is an important pursuit. In this study, indeno[2,1-c]pyrazolones were designed as readily available
synthetic inhibitors of HIF-1 transcriptional activity. Nine compounds were synthesized in 4–5 steps from
commercially available starting materials. In evaluations of the ability to inhibit the hypoxia-induced tran-
scriptional activity of HIF-1, compound 3c showed a higher level compared with that of known inhibitor, YC-1.
The compound 3c suppressed HIF-1α protein accumulation without affecting the levels of HIF-1α mRNA.
Hypoxia-inducible factor-1
Cancer chemotherapy
Indenopyrazolone
Pyrazole
Fused-ring
1. Introduction
for creating synthetic drug candidates that will inhibit HIF-1 transcrip-
tional activity remains an important pursuit.
Hypoxia-inducible factor (HIF)-1 is a member of the basic helix-loop-
helix proteins of the PER-ARNT-single-minded protein family of tran-
scription factors.1 HIF-1 regulates the expression of genes involved in
angiogenesis, cellular energy metabolism, and cell survival during cancer
development. HIF-1 forms a heterodimer with its oxygen-sensitive HIF-
1α and a constitutively expressed HIF-1β subunits.2 Under aerobic con-
ditions (normoxia), proline residues in HIF-1α are hydroxylated by prolyl
hydroxylase (PHD),3 then ubiquitinated by von Hippel-Lindau proteins,4
and degraded by 26S proteasomes. HIF-1α is also regulated by factor
inhibiting HIF (FIH), which is a HIF asparagine hydroxylase.5 Hydro-
xylation of the transactivation domains of HIF-1α by FIH promotes
avoidance of HIF-1α interactions with p300/CBP coactivator that leads
to the repression of HIF-mediated transcription activity.6 Under low
oxygen conditions (hypoxia), the ability of PHD is suppressed and HIF-1α
is stabilized. The resultant HIF-1α forms a heterodimer with its β subunit
and translocates into the nucleus. HIF-1 binds to hypoxia response ele-
ments (HREs) and activates several hundred genes involved in angio-
genesis (VEGF), glucose transport (GLUT1), glycolytic pathways (LDH-
A), ROS signals (iNOS), erythropoiesis (EPO), and other processes. HIF-1
is, therefore, regarded as a promising target for drugs used in cancer
chemotherapy7 and a variety of HIF-1 inhibitors has been reported.4i,8
We have also reported diphenylureas9 and phenoxyacetanilides10 as HIF-
1α inhibitors. Nevertheless, development of a readily accessible template
Fused-ring systems containing heterocycles are frequently found in
the frameworks of marketed drugs.11 We are interested in 6-5-5 ring
systems with a branched 6-membered ring. Indenopyrazole12 and in-
denopyrazolone13 have 6-5-5+6 ring systems that are known to exert
potent inhibitory activity against Chk1 and CDK, respectively. Our
group also reported the EGFR and VEFGR-2 inhibitor indenopyrazolone
114 (Fig. 1) and the structurally similar 6-5-5+6 ring system com-
pounds GN027079,15 and GN4402815,16 (structures are not shown) as
inhibitors against HIF-1 transcriptional activity and tubulin poly-
merization. However, synthetic difficulties encountered with the 6-5-
5+6 ring system compounds have slowed their speed of development
as viable drug candidates. Therefore, we designed pyrazolofuropyrazine
2 and demonstrated its synthesis, but it did not exert potent biological
activity.17 Herein, we wish to report the design and the 4-step synthesis
for indeno[2,1-c]pyrazolones 3. Nine compounds were synthesized, and
compound 3c exerted potent activity against HIF-1 transcriptional ac-
tivity during biological evaluation.
2. Results and discussion
2.1. Synthesis
The synthetic plan for 3 is shown in Scheme 1. We planned to
⁎ Corresponding author.
E-mail address: hiro@res.titech.ac.jp (H. Nakamura).
https://doi.org/10.1016/j.bmc.2019.115207
Received 28 September 2019; Received in revised form 31 October 2019; Accepted 4 November 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:ShinichiroFuse,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.115207

S. Fuse, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Scheme 2. Synthesis of 1-phenyl-1H-pyrazole (5a) via Ullmann-type coupling.
Table 1
Synthesis
lithium.-
of
biaryl
ketone
7a
using
5-pyrazolyl
Entry
6
Yielda
1
2
3
4
6a: X1 = COCl
86%
14%
15%
n.r.b
6b: X1 = CN
6c: X1 = CO2Bz
6d: X1 = CO2Me
a
b
Isolated yield.
No reaction.
CD3OD at −78 °C indicated that the lithiation occurred dominantly at
the 5-position of pyrazole. Encouraged by this result, the nucleophilic
acyl substitutions of 5-pyrazolyl lithium against electrophiles 6a-6d
were examined. The reaction of 5-pyrazolyl lithium with benzoyl
chloride 6a afforded the desired ketone 7a in a good yield (entry 1),
whereas the reaction with benzonitrile 6b (entry 2), with benzoic an-
hydride 6c (entry 3), and with benzoic acid methyl ester 6d (entry 4)
did not afford satisfactory results (see Table 2).
Fig. 1. Chemical structures of 6-5-5+6 fused-ring compounds containing het-
erocycles.
prepare N-substituted pyrazole 5 via N-alkylation/arylation of 4. We
expected to obtain ketone 7 from nucleophilic addition/nucleophilic
acyl substitution of 5 against 6. Subsequent cyclization was expected to
afford the desired indeno[2,1-c]pyrazolones 3.
1-Phenyl-1H-pyrazole (5a) was prepared via Ullmann-type coupling
between 4a and 8a in accordance with the reported procedure18
(Scheme 2).
Unfortunately, the palladium-catalyzed dehydrogenative cyclization
conditions reported for the synthesis of fluorenone from benzophe-
none22 did not accomplish the synthesis of 3a from 7a (Scheme 3).
Therefore, 7a was converted to the corresponding iodopyrazole 9a
using ICl (Scheme 3).23 Cyclization via direct CeH arylation of 9a was
examined. Copper (I)-catalyzed conditions that were reported for the
synthesis of fluorenone24 only afforded the undesired 7a. On the other
hand, palladium (II)-catalyzed conditions reported for the synthesis of
substituted fluorenones25 afforded the desired 3a in a 58% yield (2
steps from 7a).
Two synthetic approaches for pyrazole-containing biaryl ketone 7
have been reported. One of the two approaches used a nucleophilic
addition of phenyl Grignard reagent against 5-cyano pyrazole and
subsequent hydrolysis of a diaryl imine.19 Another approach used a
nucleophilic acyl substitution of 5-pyrazolyl anion against benzoyl
chloride with a pyrazolyl anion prepared from magnesium, zinc
chloride, and 5-chloro pyrazole.20 Unfortunately, synthesis of the sub-
strates used in these known approaches requires multiple steps, which
prompted us to examine the direct generation of 5-pyrazolyl lithium
from 5a and its nucleophilic addition/nucleophilic substitution against
6 (Table 1). Alley and Shirley reported that the dropwise addition of
1.02 equiv. of n-BuLi at 0 °C in Et2O led to the lithiation of 5a at both
the 5-position of pyrazole and the ortho-position of a phenyl ring.21 We
preliminarily examined lithiation by the dropwise addition of n-BuLi
into a THF solution of 5a at −78 °C. The subsequent deuteration using
The chemical structure of 3a was confirmed by 1H, 13C NMR, IR,
and HRMS analysis. In addition, single crystal of 3e (synthesis is shown
later) was obtained and its structure was unambiguously confirmed by
X-ray analyses (Fig. 2).26
We synthesized indeno[2,1-c]pyrazolones 3b-3i via our developed
procedure (Scheme 4). Pyrazole (4a) or 3-methyl pyrazole (4b) was
coupled with aryl halides 8a-8d to afford N-aryl pyrazoles 5a-5d. The
subsequent nucleophilic acyl substitution of pyrazolyl lithium gener-
ated by 5a-5d and commercially available 5e and 5f against 6a af-
forded the desired ketones 7b-7 g. The following iodination and in-
tramolecular direct CeH arylation of the ketones 7b-7 g afforded the
desired N-substituted indeno[2,1-c]pyrazolones 3b-3 g. In the synthesis
of 3e, an intermediate 7e was not isolated. Removal of either the me-
thyl group or the methoxy phenyl group from 3d was performed to
afford 3 h and 3i.27
2.2. Biological evaluation
The synthesized indeno[2,1-c]pyrazolones 3a-3i were tested via a
dual luciferase assay for their ability to inhibit HIF-1 transcriptional
activity in HeLa cells under hypoxic conditions, and an MTT assay was
used to gauge their antiproliferative activity toward HeLa cells under
normoxic conditions (Table 1, entries 1–9). YC-128 was used as the
positive control (entry 10). Under normoxic conditions, HIF-1α is
Scheme 1. Synthetic plan for indeno[2,1-c]pyrazolones 3.
2

S. Fuse, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Table 2
3d, and 3h contained electron-donating groups (-Me, -OMe, eOH) at
the para-position of the N-aryl ring, and exerted potent HIF-1α in-
hibitory activity (entries 3, 4, and 8). With the exception of 3i (entry 9),
the other compounds, 3a, 3b, 3e, 3f, and 3g (entries 1, 2, 5, 6, and 7),
exerted medium levels of inhibitory activity. Although the compound
3h showed the highest level of HIF-1α inhibitory activity (entry 8), it
also displayed cytotoxicity under normoxic conditions. These results
indicated that compound 3h inhibited not only HIF-1α transcriptional
activity but other biological processes, as well. On the other hand,
compounds 3c and 3d showed no obvious cytotoxicity under normoxic
conditions (entries 3 and 4). A comparison with the reporter gene assay
results between compounds 3a and 3i (entry 1 vs. 9) clearly indicated
the importance of the substituent R2 for inhibiting HIF-1α transcrip-
tional activity. Introduction of a nitrogen atom as a Y (entry 1 vs. 2),
and an electron-withdrawing CF3 group on the N-aryl ring (entry 1 vs.
5) all resulted in detrimental effects against inhibitory activity.
If a compound directly inhibits the luciferase activity, the observed
inhibitory activity is not related to HIF-1 transcriptional activity. To
verify that possibility, compounds 3c and 3d were mixed with luci-
ferase and their luciferase inhibitory activity was investigated. A de-
crease in the intensity of an emission indicates the inhibitory effect of a
compound against luciferase. Compound 3d had a somewhat strong
level of inhibitory activity against luciferase, but no inhibitory activity
was observed in the case of compound 3c (Fig. 3). Thus, the observed
inhibitory activity of 3c against HIF-1α transcriptional activity, as
shown in the reporter gene assay, was reliable. We selected compound
3c as a hit compound.
Effects of indeno[2,1-c]pyrazolones 3b-3i on the proliferative activity of HeLa
cells
and
on
the
HIF-1α
transcriptional
activity
in
HeLa
cells.
Entry
Compound
Cytotoxicitya
HRE-Lucb
IC50 (μM)
3
Y
R1
R2
IC50 (μM)
> 10c
1
3a
3b
3c
C
N
C
C
C
C
C
C
C
H
H
H
H
H
Me
H
H
H
Ph
1.4
0.83
0.46
0.16
0.10
2
Ph
52
5.7
10
3
4-MePh
4-MeOPh
4-CF3Ph
Ph
> 10c
> 10c
0.79
0.61
23
4
3d
3e
3f
5
> 100
> 30c
0.92
6
4.8
0.86
7
3g
3h
3i
Me
49
4.3
8.8
0.079
0.088
8
4-HOPh
H
2.2
0.067
0.93
0.39
> 30c
2.4
9
> 30c
56
10
YC-1
0.51
a
HeLa cells were incubated at 37 °C for 72 h in medium containing various
concentrations of compounds under normoxic conditions, and cell viability was
determined via MTT assay.
b
HeLa cells transiently transfected with HRE-firefly luciferase and cytome-
galovirus promoter-Renilla luciferase were incubated at 37 °C for 12 h with
compounds under hypoxic conditions. After the supernatant was removed, a
luciferase assay was performed using the dual luciferase assay system.
In order to further elucidate the mechanism of action of compound
3c, the effects of 3c on the hypoxia-induced HIF-1α protein accumu-
lation were evaluated by Western blot analysis and the expression of
HIF-1α mRNA was evaluated by RT-PCR analysis in HeLa cells (Fig. 4).
The compound 3c suppressed HIF-1α protein accumulation at con-
centrations higher than 10 μM (Fig. 4a). However, the levels of HIF-1α
mRNA were not affected by compound 3c (Fig. 4b).
c
Solubility of these compounds was insufficient, and, their maximum con-
centrations were set at 10 or 30 μM.
2.3. Conclusions
In summary, we designed indeno[2,1-c]pyrazolones to develop
readily available synthetic inhibitors against HIF-1 transcriptional ac-
tivity. Nine compounds were synthesized in 4–5 steps from commer-
cially available starting materials. Evaluation of the ability to inhibit
the hypoxia-induced transcriptional activity of HIF-1 revealed that the
compound 3c had a higher level of inhibitory activity compared with
that of YC-1. The compound 3c suppressed HIF-1α protein accumula-
tion without affecting the levels of HIF-1α mRNA. The obtained results
showed the usefulness of indeno[2,1-c]pyrazolones as a readily avail-
able scaffold for future synthetic drug development targeting HIF-1
transcriptional activity or degradation inducing activity.
Scheme 3. Synthesis of 1-phenylindeno[2,1-c]pyrazol-8(1H)-one (3a) via in-
tramolecular CeH direct arylation.
3. Experimental section
3.1. General methods
NMR spectra were recorded on either using a BRUKER BIOSPIN
AVANCE II 400 (400 MHz for 1H, 100 MHz for 13C) or using a BRUKER
BIOSPIN AVANCE III HD500 (500 MHz for 1H, 125 MHz for 13C) in the
indicated solvent. Chemical shifts are reported in units of parts per
million (ppm) relative to the signal (0.00 ppm) for internal tetra-
methylsilane for solutions in CDCl3 (7.26 ppm for 1H, 77.2 ppm for 13C)
or methanol‑d4 (3.31 ppm for 1H, 49.0 ppm for 13C). Multiplicities are
reported by using the following abbreviations: s, singlet; d, doublet; dd,
doublet of doublets; t, triplet; m, multiplet; and, J, coupling constants in
Herts (Hz). The IR spectra were recorded on a JASCO FT/IR-4100 with
KBr pellets. Only the strongest and/or structurally important peaks are
reported as IR data given in cm−1. HRMS (EI-MS) were measured using
Fig. 2. ORTEP drawing of 3e.
continuously degraded, and their concentration was expected to be low.
Therefore, the compounds that exert low cytotoxicity under normoxic
conditions (MTT assay) and high inhibitory activity under hypoxic
conditions (reporter gene assay) are candidates for use as HIF-1α in-
hibitors. The assay results revealed that the compound 3h exerted po-
tent toxicity under normoxic conditions (entry 8). On the other hand,
none of the other compounds exerted potent toxicity. Compounds 3c,
a
JEOL JMS-700. All reactions were monitored by thin layer
3

S. Fuse, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Scheme 4. Synthesis of indeno[2,1-c]pyrazolones 3b-3i.
chromatography carried out on 0.2 mm E. Merck silica gel plates (60
F
254) with UV light, as visualized by p-anisaldehyde solution, 10%
ethanolic phosphomolybdic acid, and dinitrophenylhydrazine EtOH
solution. Flash column chromatography was performed on silica gel
(Fuji Silysia, CHROMATOREX PSQ 60B, 50–200 µm).
3.2. Cell growth assay (MTT assay)
Human cervical carcinoma HeLa cells were used for the cell viability
assay. These cells (5 × 103 cells per well of a 96-well plate) were in-
cubated at 37 °C for 72 h in RPMI-1640 medium (100 µL) containing at
various concentrations of indenopyrazolones 3a-3i (10 mM DMSO so-
lution). After the incubation, the medium was removed, RPMI-1640
medium (100 µL) and 3′-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) in PBS (5 mg/mL, 10 µL) were added to each
well, and the cells were further incubated at 37 °C for 2 h. After removal
of the medium, DMSO (100 µL) was added and the absorbance at
Fig. 3. Inhibitory activity of compounds 3c and 3d against luciferase.
Luciferase was incubated at 37 °C for 10 min in separate batches of medium that
contained one of each of the compounds (1 μM), luciferase assay reagent was
added to each mixture, and the emission intensity was measured.
4

S. Fuse, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Biotechnology, Santa Cruz, CA). After further incubation with horse-
radish peroxidase (HRP)-conjugated secondary antibody, protein ex-
pression was visualized with a Molecular Imager ChemiDoc XRS System
(Bio-Rad, Hercules, CA).
3.6. Quantitative RT-PCR
HeLa cells (1 × 106 cells per well of a 6-well plate) were incubated
for 12 h with or without drugs under either normoxic or hypoxic con-
ditions. Total RNA was extracted from the cells using a RNeasy Mini Kit
(QIAGEN, German) according to the manufacturer’s instructions. After
extraction, the extracted RNA (1.0 µg) was reverse transcribed at 40 °C
for 50 min by adding 3 μg/μL random R.P. (0.50 μL), 10 mM dNTPs
(5.0 μL), 5 × buffer (2.5 μL) and M-MLV (0.50 μL) (Promega, WI).
Quantitative RT-PCR was carried out with SYBR Thunderbird (Toyobo,
Japan) using the following primers: HIF-1α forward 5′-TTT TCA AGC
AGT AGG AAT TGG AA-3′, HIF-1α reverse 5′-GTG ATG TAG TAG CTG
CAT GAT CG-3′, β2-microglobulin (B2M) forward 5′-TAC ATG TCT
CGA TCC CAC TT-3′, B2M reverse 5′-TAC ACT GAA TTC ACC CCC AC-
3′. The mRNA level of B2M was used to normalize the value of HIF-1α.
Acknowledgements
We thank Mr. Yoshihisa Sei (Technical Department, Tokyo Institute
of Technology) for his technical assistance with X-ray crystallographic
analysis. This study was partially supported by Scientific Research (B)
(No. 18H02685) from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
Fig. 4. Effects of compound 3c on HIF-1α protein and mRNA expression under
hypoxic conditions. (a) HIF-1α protein expression was detected by immunoblot
analysis with the specific antibody. YC-1 was used as a positive control for the
inhibition of HIF-1α protein expression. (b) mRNA level of HIF-1α was detected
by RT-PCR.
Declaration of Competing Interest
595 nm was measured with a microplate reader. The drug concentra-
tion required to reduce cell viability by 50% (IC50) was determined
from semilogarithmic dose–response plots.
The authors declare no competing interests.
Appendix A. Supplementary material
3.3. HIF-1 transcriptional activity assay (Luciferase reporter gene assay)
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.115207.
HeLa cells transiently transfected with HRE-Luc29 and a cytomega-
lovirus promoter-Renilla luciferase (Promega, Madison, WI, USA) re-
porter genes (2 × 104 cells per well of a 96-well plate) using Lipo-
fectamine 2000 (Thermo Fisher Science, MA, USA) were incubated at
37 °C for 12 h with or without drugs under normoxic or hypoxic (1%
O2) conditions. After removal of supernatant, luciferase assay was
performed using a Dual Luciferase Assay System (Promega, Madison,
WI, USA) according to the manufacturer’s instructions. The drug con-
centration required to inhibit relative light units by 50% (IC50) was
determined from the semilogarithmic dose-response plots, and the re-
References
1. Wang GL, Jiang BH, Rue EA, Semenza GL. Proc Nat Acad Sci USA.
1995;92:5510–5514.
2. Jiang B-H, Rue E, Wang GL, Roe R, Semenza GL. J Biol Chem.
1996;271:17771–17778.
3. (a) Bruick RK, McKnight SL. Science. 2001;294:1337–1340
(b) Epstein ACR, Gleadle JM, McNeill LA, et al. Cell. 2001;107:43–54.
4. (a) Maxwell PH, Wiesener MS, Chang G-W, et al. Nature. 1999;399:271–275
(b) Tanimoto K, Makino Y, Pereira T, Poellinger L. EMBO J. 2000;19:4298–4309
(c) Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, Conaway JW. Proc
Nat Acad Sci USA. 2000;97:10430–10435
sults are means
SD of triplicate samples.
(d) Ohh M, Park CW, Ivan M, et al. Nat Cell Biol. 2000;2:423–427
(e) Cockman ME, Masson N, Mole DR, et al. J Biol Chem. 2000;275:25733–25741
(f) Masson N, Willam C, Maxwell PH, Pugh CW, Ratcliffe PJ. EMBO J.
2001;20:5197–5206
3.4. Luciferase inhibitory activity assay
To a solution of compound in DMSO and PBS buffer (10 μL), a so-
lution of QuantiLum Recombinant Luciferase (Promega, WI) in PBS
(20 ng/μL, 10 μL) was added. After incubation at 37 °C for 10 min, 10 μL
of LAR (Promega) was added to the mixture and measured the emission
intensity by luminometer infinite F200 (TECAN, Switzerland).
(g) Ivan M, Kondo K, Yang H, et al. Science. 2001;292:464–468
(h) Yu F, White SB, Zhao Q, Lee FS. Proc Nat Acad Sci USA. 2001;98:9630–9635
(i) Melillo G. Cancer Metastasis Rev. 2007;26:341–352.
5. (a) Pugh CW, O'Rourke JF, Nagao M, Gleadle JM, Ratcliffe PJ. J Biol Chem.
1997;272:11205–11214
(b) Jiang B-H, Zheng JZ, Leung SW, Roe R, Semenza GL. J Biol Chem.
1997;272:19253–19260
3.5. Western blotting
(c) Mahon PC, Hirota K, Semenza GL. Genes Dev. 2001;15:2675–2686.
6. (a) Freedman SJ, Sun Z-YJ, Poy F, et al. Proc Nat Acad Sci USA.
2002;99:5367–5372
After drug treatment for 12 h, the cells were dipped in 100 μL of
sample buffer (50 mM Tris, pH 7.4, 4% SDS, 10% glycerol, 4% 2-
thioethanol, and 50 μg/ml bromophenol blue) for 5 min and the lysate
was boiled for 5 min. The cell lysates were subjected to SDS-poly-
acrylamide gel electrophoresis (PAGE), transferred to polyvinylidene
difluoride (PVDF) membrane (GE Healthcare Buckinghamshire, UK),
and immunoblotted with anti-HIF-1α antibody (BD Transduction
Laboratories, Lexington, KY) and anti-tubulin antibody (Santa Cruz
(b) Dames SA, Martinez-Yamout M, De Guzman RN, Dyson HJ, Wright PE. Proc Nat
Acad Sci USA. 2002;99:5271–5276.
7. (a) Harris AL. Nat Rev Cancer. 2002;2:38–47
(b) Semenza GL. Nat Rev Cancer. 2003;3:721–732
(c) Giaccia A, Siim BG, Johnson RS. Nat Rev Drug Discov. 2003;2:803–811
(d) Denko NC. Nat Rev Cancer. 2008;8:705–713
(e) Wigerup C, Påhlman S, Bexell D. Pharmacol Ther. 2016;164:152–169.
8. (a) Onnis B, Rapisarda A, Melillo G. J Cell Mol Med. 2009;13:2780–2786
(b) Wang R, Zhou S, Li S. Curr Opin Chem Biol. 2011:3168–3189
5

S. Fuse, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
(c) Ban HS, Uto Y, Nakamura H. Expert Opin Ther Pat. 2011;21:131–146
17. Fuse S, Inaba M, Sato S, Joshi M, Nakamura H. Synthesis. 2018;50:1493–1498.
18. Correa A, Bolm C. Adv Synth Catal. 2007;349:2673–2676.
19. Fliege W, Huisgen R, Kolbeck W, Weberndörfer V. Chem Ber. 1983;116:3438–3460.
20. Piller FM, Metzger A, Schade MA, Haag BA, Gavryushin A, Knochel P. Chem Eur J.
2009;15:7192–7202.
(d) Xia Y, Choi H-K, Lee K. Eur J Med Chem. 2012;49:24–40
(e) Hu Y, Liu J, Huang H. J Cell Biochem. 2013;114:498–509
(f) Kim SY, Yang EG. Molecules. 2015;20:20551–20568
(g) Ban HS, Uto Y, Won M, Nakamura H. Expert Opin Ther Pat. 2016;26:309–322.
9. Uno M, Ban HS, Nakamura H. Bioorg Med Chem Lett. 2009;19:3166–3169.
10. (a) Shimizu K, Maruyama M, Yasui Y, Minegishi H, Ban HS, Nakamura H. Bioorg
Med Chem Lett. 2010;20:1453–1456
21. Alley PW, Shirley DA. J Am Chem Soc. 1958;80:6271–6274.
22. Li H, Zhu R-Y, Shi W-J, He K-H, Shi Z-J. Org Lett. 2012;14:4850–4853.
23. Nayak M, Batra S. RSC Adv. 2012;2:3367–3373.
(b) Ban HS, Shimizu K, Minegishi H, Nakamura H. J Am Chem Soc.
2010;132:11870–11871.
24. Haggam RA. Tetrahedron. 2013;69:6488–6494.
25. Qabaja G, Jones GB. J Org Chem. 2000;65:7187–7194.
11. Bemis GW, Murcko MA. J Med Chem. 1996;39:2887–2893.
12. (a) Tong Y, Przytulinska M, Tao Z-F, et al. Bioorg Med Chem Lett.
2007;17:5665–5670
26. CCDC-1943606 contains the supplementary crystallographic data for this paper. The
data can be obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.jk/getstructures.
(b) Tao Z-F, Li G, Tong Y, et al. Bioorg Med Chem Lett. 2007;17:5944–5951.
13. Nugiel DA, Vidwans A, Etzkorn A-M, et al. J Med Chem. 2002;45:5224–5232.
14. Usui T, Ban HS, Kawada J, Hirokawa T, Nakamura H. Bioorg Med Chem Lett.
2008;18:285–288.
27. A synthesis of compound 1i from 2-ethynylbenzoyl chloride was previously re-
ported, see: Kende AS, Journet M. Tetrahedron Lett. 1995;36:3087–3090.
28. (a) Li SH, Shin DH, Chun Y-S, Lee MK, Kim M-S, Park J-W. Mol Cancer Ther.
2008;7:3729–3738
15. Minegishi H, Fukashiro S, Ban HS, Nakamura H. ACS Med Chem Lett.
2013;4:297–301.
(b) Chun Y-S, Yeo E-J, Park J-W. Cancer Lett. 2004;207:1–7.
29. Kizaka-Kondoh S, Itasaka S, Zeng L, et al. Clin Cancer Res. 2009;15:3433–3441.
16. Minegishi H, Futamura Y, Fukashiro S, et al. J Med Chem. 2015;58:4230–4241.
6