Diaryl-substituted ortho-carboranes as a new class of hypoxia inducible factor-1α inhibitors

Article abstract of DOI:10.1039/c3dt52828f

Diaryl-substituted ortho-carboranes 1 were synthesized from the corresponding alkynes by decaborane coupling under microwave-irradiated conditions with a combination of N,N-dimethylaniline and chlorobenzene. Among the compounds synthesized, 1a and 1d exhibited significant inhibition of hypoxia-induced HIF-1 transcriptional activity. Both compounds similarly suppressed hypoxia-induced HIF-1α accumulation in a concentration- dependent manner without affecting HIF-1α mRNA expression.

Full text of DOI:10.1039/c3dt52828f

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Diaryl-substituted ortho-carboranes as a new class
of hypoxia inducible factor-1α inhibitors†
Cite this: DOI: 10.1039/c3dt52828f
Hiroyuki Nakamura,*a,b Lisa Tasaki,a Daisuke Kanoh,a Shinichi Satoa and
Received 9th October 2013,
Accepted 18th November 2013
Hyun Seung Ban‡a
DOI: 10.1039/c3dt52828f
www.rsc.org/dalton
Diaryl-substituted ortho-carboranes 1 were synthesized from the inducible factor 1 (HIF-1), a heterodimeric transcription factor
corresponding alkynes by decaborane coupling under microwave- consisting of two subunits, HIF-1α and HIF-1β/ARNT, func-
irradiated conditions with a combination of N,N-dimethylaniline tions to stimulate the physiological expression of those angio-
and chlorobenzene. Among the compounds synthesized, 1a and 1d genesis factors.8 Dachs et al. found that CA4P reduced HIF-1
exhibited significant inhibition of hypoxia-induced HIF-1 transcrip- accumulation in endothelial cells under hypoxia and adversely
tional activity. Both compounds similarly suppressed hypoxia- affected members of the HIF-1 signal transduction pathway.9
induced HIF-1α accumulation in
manner without affecting HIF-1α mRNA expression.
a
concentration-dependent
Recently, much attention has been paid to carborane-based
drug design.10–14 Carboranes (icosahedral dicarba-closo-
dodecaboranes; C2B10H12) possess a stable B–H–B three-center
two-electron bond and show remarkable thermal and catabolic
stability.15 In addition, because of their icosahedral geometry
and exceptional hydrophobicity, they are used as hydrophobic
pharmacophores in biologically active molecules interacting
with their target proteins.16–20 We recently reported that diaryl-
substituted carboranes synthesized as mimics of manassantin
A possess significant inhibitory activity toward HIF-1α protein
accumulation, resulting in suppression of HIF-1 transcrip-
tional activity in human cervical cancer cells, HeLa cells,
under hypoxia.21 In this study, we focused on the cis geometry
of the stilbene framework in CA-4 and designed its ortho-
carborane analogs 1 (Fig. 1).
The synthesis of ortho-carborane analogs 1 is shown in
Scheme 1. The ortho-carborane scaffold can be generated from
the coupling of decaborane (B10H14) with alkynes. Therefore,
we initially synthesized diaryl-substituted alkynes 3 via the
Sonogashira coupling of terminal alkynes 2 with aromatic
iodides (ArI). The reaction of 5-ethynyl-1,2,3-trimethoxy-
benzene 2a and ArI proceeded in the presence of the
Combretastatin A-4 (CA-4) is a mitotic agent isolated from the
bark of the South African bush willow Combretum caffrum. It
exhibits significant cytotoxicity to a wide variety of human
cancer cell lines, including multidrug-resistant cancer cell
lines, and is therefore one of the attractive lead compounds for
the development of anticancer drugs.1,2 Its phosphate prodrug
(CA4P) is a vascular disrupting agent that selectively damages
tumor neovasculature with induction of extensive blood flow
shutdown in metastatic tumors compared to normal tissues.3
A phase II clinical study of CA4P in patients with platinum-
resistant ovarian cancer revealed that the addition of CA4P to
paclitaxel and carboplatin was well tolerated and appeared to
produce a higher response rate in this patient population than
in a patient population that did not receive CA4P.4
Angiogenesis is a necessary process for tumors to grow
beyond a certain critical size. The growth of tumor blood
vessels increases the supply of oxygen and nutrients to small
tumors, resulting in tumor growth and metastasis.5 Vascular
endothelial growth factor (VEGF) and erythropoietin (EPO) are
the key growth factors in tumor angiogenesis.6,7 Hypoxia-
aDepartment of Chemistry, Faculty of Science, Gakushuin University, Mejiro,
Toshima-ku, Tokyo 171-8588, Japan
bChemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta-cho,
Midori-ku, Yokohama, 226-8503, Japan. E-mail: hiro@res.titech.ac.jp;
Fax: +81-45-924-5976; Tel: +81-45-924-5244
†Electronic supplementary information (ESI) available: Spectral data for com-
pounds 1a–g, 6 and 7, Table S1, and experimental details for biology. See DOI:
10.1039/c3dt52828f
‡Present address: Biomedical Genome Research Center, Korea Research Insti-
tute of Bioscience and Biotechnology, Yuseong, Daejeon 305-806, Korea.
Fig. 1 Molecular design of ortho-carborane analogs 1.
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Table 1 Chemical yieldsa and inhibition of HIF-1 transcriptional activity
in the cell-based HRE reporter assayb
Compound
Yield of 3c (%)
IC50 (µM)
1a
1b
1c
1d
1e
1f
61 (20)
50 (6)
71 (12)
53 (9)
78 (20)
53 (14)
63 (22)
0.6
1.2
5.1
0.8
6.4
5.5
4.6
1.5
1g
YC-1
a Reactions were carried out in the presence of three equivalents of
N,N-dimethylaniline as Lewis base in chlorobenzene under
a
microwave-irradiated conditions in a sealed tube for 15 min. b HeLa
cells expressing HRE-dependent firefly luciferase reporter construct
(HRE–Luc) and constitutively expressing CMV-driven Renilla luciferase
reporter with SureFECT Transfection Reagent were incubated for 12 h
with or without drugs under normoxic or hypoxic conditions. After
removal of the supernatant, the luciferase assay was performed using a
Luciferase Assay System (Promega, Madison, WI, USA) according to the
manufacturer’s instructions. The fluorescence intensity of HRE–Luc
was normalized to that of constitutively expressing CMV-driven Renilla
luciferase control. The drug concentration required to inhibit relative
light units by 50% (IC50) was determined from semi-logarithmic dose–
response plots. c Yields of 3 under the toluene-refluxed conditions
using acetonitrile as a Lewis base are shown in parentheses.
Scheme 1 Reagents and conditions: (a) ArI, Pd(PPh3)4, CuI, TEA–THF,
reflux, 4–6 h; (b) (i) B10H14, CH3CN–toluene, 110 °C, 24 h or (ii) B10H14
,
N,N-dimethylaniline–chlorobenzene, microwave, 150 °C, 15 min.
Pd(PPh3)4 catalyst (10 mol%) in THF to give the corresponding
diaryl-substituted alkynes 3a–d in 42–90% yields. In a similar expressing CMV-driven Renilla luciferase reporter.25 The com-
manner, alkynes 3e–g were obtained from 4-ethynyl-1,2- pound concentrations required to inhibit relative light units by
dimethoxybenzene 2b in 46–87% yields.22 The substituted 50% (IC50) are summarized in Table 1. YC-1 (3-(5′-hydroxy-
decaborane coupling of alkyne 3a with decaborane was carried methyl-2′-furyl)-1-benzylindazole) was used as the positive
out in the presence of acetonitrile as the Lewis base under control.26,27 Significant inhibition of the hypoxia-induced
toluene reflux conditions.23 However, 1a was obtained only in HIF-1 transcriptional activity was observed in all synthesized
20% yield. Furthermore, in the cases of 3b–d, the corres- ortho-carborane analogs 1a–g with IC50 values between 0.6 and
ponding diaryl-substituted ortho-carboranes 1b–d were 6.4 µM. Notable was that compound 1a, which has similar sub-
obtained in even lower yields (5–12%) under similar con- stituents to CA-4 on both benzene rings, and compounds 1b
ditions. In order to increase the yields of diaryl-substituted and 1d exhibited higher inhibitory activity than YC-1, the
ortho-carboranes, we optimized the reaction conditions of the respective IC50 values being 0.6, 1.2, and 0.8 μM. We recently
decaborane coupling using 1,2-diphenylethyne as the starting reported diaryl-substituted ortho- and meta-carboranes as
material. As shown in Table S1 (see the ESI†), the combination mimics of manassantin A.21 The present diaryl-substituted
of N,N-dimethylaniline and chlorobenzene, a protocol develo- ortho-carborane analogs 1a, 1b, and 1d are more strongly inhi-
ped by Nagasawa et al.,24 worked effectively. Indeed, the coup- biting the hypoxia-induced HIF-1 transcriptional activity than
ling of decaborane with 1,2-diphenylethyne proceeded the previously synthesized carborane-containing mimics of
smoothly in the presence of three equivalents of N,N-dimethyl- manassantin A.
aniline as the Lewis base in chlorobenzene by microwave
irradiation for 15 min, giving 1,2-diphenyl-ortho-carborane in cant inhibition of the hypoxia-induced HIF-1 transcriptional
75% yield (entry 7 in Table S1†).
activity in HeLa cells, we further examined the effects of those
Because ortho-carborane analogs 1a and 1d showed signifi-
With the optimum conditions established, we synthesized two compounds on hypoxia-induced HIF-1α protein accumu-
ortho-carborane analogs 1 again by coupling decaborane with lation by western blot analysis as well as HIF-1α mRNA
3 under microwave irradiation. The results are shown in expression by RT-PCR analysis. As shown in Fig. 2, both 1a
Table 1. Diaryl-substituted alkynes 3a–g underwent coupling and 1d similarly suppressed the hypoxia-induced HIF-1α
with decaborane by microwave irradiation in the presence of protein accumulation in a concentration-dependent manner
N,N-dimethylaniline in chlorobenzene to give the corres- without affecting the expression of tubulin protein (Fig. 2a).
ponding ortho-carborane analogs 1a–g in 50–78% yields.
RT-PCR analysis revealed that both 1a and 1d did not suppress
Then, the synthesized ortho-carborane analogs 1a–g were the HIF-1α mRNA expression (Fig. 2b). These results indicate
evaluated for their ability to inhibit hypoxia-induced HIF-1 that the inhibition of the hypoxia-induced HIF-1 transcrip-
transcriptional activity by conducting a cell-based reporter tional activation by compounds 1a and 1d is due to the sup-
gene assay in HeLa cells expressing HRE-dependent firefly pression of HIF-1α accumulation without affecting HIF-1α
luciferase reporter construct (HRE-Luc) and constitutively mRNA expression under hypoxic conditions in HeLa cells.
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Fig. 2 Effect of compounds 1a and 1d on HIF-1α protein and mRNA
expression induced by hypoxia. (a) HeLa cells were incubated in the
presence of compounds 1a and 1d at different concentrations under
hypoxia for 4 h. HIF-1β was used as the loading control and ‘−’ is DMSO
as the control. The levels of each protein were determined by immuno-
blot analysis with HIF-1α or HIF-1β specific antibodies. (b) RT-PCR analy-
sis for the effect of compounds 1a and 1d on the hypoxia-induced
mRNA level of HIF-1α and VEGF in HeLa cells for 4 h. GAPDH was used
as the loading control and ‘−’ is DMSO as the control.
Fig. 3 Effect of 1a, 1d, and 7 on HIF-1α protein accumulation induced
by hypoxia. HeLa cells were incubated with compounds at 3 and 30 μM
under hypoxic conditions for 4 h. Tubulin was used as the loading
control and ‘−’ is DMSO as the control. The levels of each protein were
detected by immunoblot analysis with HIF-1α or tubulin specific
antibodies.
Scheme 2 (a) NaNO2, KI–CH3CN, 0 °C, 5 h; (b) 3a, Pd(PPh3)4, CuI,
TEA–THF, reflux, 4 h; (c) B10H14, N,N-dimethylaniline–chlorobenzene,
microwave, 120 °C, 15 min; (d) Pd/C/EtOH, r.t., overnight; (e) propargyl
bromide, K2CO3–acetone, 60 °C, 1.5 h.
To investigate further the inhibitory mechanism of the
hypoxia-induced HIF-1 transcriptional activity by compounds
1a and 1d, we designed multifunctional probe molecule 9
based on conventional chemical biology techniques, including
photo-affinity labeling, click conjugation, and biotinylation,
which are highly useful tools for the detection of target pro-
teins of biologically active molecules having undefined mecha-
nisms of action.28–30 As shown in Scheme 2, we synthesized a
multifunctional molecular probe of 1a by substituting the
benzophenone moiety that would form covalent bonds with
target proteins by UV irradiation (photo-affinity labeling), and
an acetylene moiety that would conjugate with azide-linked
biotin by the click reaction. Aniline derivative 4, the starting
material, was converted into iodoketone 5. The Sonogashira
coupling of ethynylbenzene 3a with iodoketone 5 followed by
the coupling with decaborane afforded diaryl-substituted
ortho-carborane 6. Reduction of 6 followed by propargylation
of the resulting phenol under basic conditions gave multi-
functional probe molecule 7.
Fig. 4 Pull-down assay of 7-biotin. HeLa cells were plated on ø100
dishes (5 × 106 cells) and incubated at 37 °C for 24 h. After treatment
with compound 7 (10 μM) for 1 h, the cells were suspended in 100 μL of
PBS and then UV irradiated at 360 nm on ice for 20 min. After the click
reaction was carried out with azide-linked biotin in the cell lysates, the
mixture was incubated with avidin resin. Proteins bound to 7-biotin were
separated by SDS-PAGE and tubulin was detected by immunoblot
analysis.
To confirm whether the synthesized probe molecule 7 exhi-
bits HIF inhibitory activity, we first examined the effects of 7
on the hypoxia-induced activation of HIF in HeLa cells by
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immunoblot analysis. As shown in Fig. 3, the reduction of the
HIF-1α protein expression by probe 7 was similar to that by 1a
and 1d.
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Conclusions
We succeeded in the synthesis of diaryl-substituted ortho-
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