Synthesis and biological evaluation of kresoxim-methyl analogues as novel inhibitors of hypoxia-inducible factor (HIF)-1 accumulation in cancer cells

Article abstract of DOI:10.1016/j.bmcl.2017.05.024

We designed and synthesized strobilurin analogues as hypoxia-inducible factor (HIF) inhibitors based on the molecular structure of kresoxim-methyl. Biological evaluation in human colorectal cancer HCT116 cells showed that most of the synthesized kresoxim-methyl analogues possessed moderate to potent inhibitory activity against hypoxia-induced HIF-1 transcriptional activation. Three candidates, compounds 11b, 11c, and 11d were identified as potent inhibitors against HIF-1 activation with IC₅₀ values of 0.60–0.94?μM. Under hypoxic condition, compounds 11b, 11c, and 11d increased the intracellular oxygen contents, thereby attenuating the hypoxia-induced accumulation of HIF-1α protein.

Full text of DOI:10.1016/j.bmcl.2017.05.024

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of kresoxim-methyl analogues as
novel inhibitors of hypoxia-inducible factor (HIF)-1 accumulation in
cancer cells
a,
Sanghyuck Lee a, Oh Seok Kwon b, Chang-Soo Lee b, Misun Won c, Hyun Seung Ban d,e, , Choon Sup Ra
⇑
⇑
a School of Chemistry and Biochemistry, Yeungnam University, 280 Daehak-Ro, Gyeongsan-si, Gyeongbuk 38541, Republic of Korea
b Hazards Monitoring Bionano Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
c Personalized Genomic Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
d Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
e Biomolecular Science, University of Science and Technology, Daejeon 34113, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized strobilurin analogues as hypoxia-inducible factor (HIF) inhibitors based on
the molecular structure of kresoxim-methyl. Biological evaluation in human colorectal cancer HCT116
cells showed that most of the synthesized kresoxim-methyl analogues possessed moderate to potent
inhibitory activity against hypoxia-induced HIF-1 transcriptional activation. Three candidates, com-
pounds 11b, 11c, and 11d were identified as potent inhibitors against HIF-1 activation with IC50 values
of 0.60–0.94 mM. Under hypoxic condition, compounds 11b, 11c, and 11d increased the intracellular oxy-
Received 26 March 2017
Revised 6 May 2017
Accepted 8 May 2017
Available online xxxx
Keywords:
Kresoxim analogues
Cancer
gen contents, thereby attenuating the hypoxia-induced accumulation of HIF-1a protein.
Ó 2017 Elsevier Ltd. All rights reserved.
Hypoxia-inducible factor-1 inhibitor
Mitochondrial respiration
Most solid tumors contain hypoxic regions within the tumor
microenvironment and cancer cells adapted to hypoxia are highly
resistant to chemotherapy and radiotherapy.1 Hypoxia-inducible
ing to cytochrome b complex at the Q0 site of complex III in the
electron transport chain (ETC) of mitochondria.8 Of note, in spite
of a broad spectrum of fungicidal activity through the inhibition
of respiration, strobilurins induce low toxicity towards mam-
malian cells.8,9 Various biological activities of strobilurin deriva-
factor (HIF)-1
to hypoxia.2 Under normoxic condition, hydroxylation of HIF-1
by prolyl hydroxylase (PHD) allows the recruitment of ubiquitin
ligase von Hippel Lindau (VHL), and then HIF-1 undergoes ubiq-
uitin and proteasome-dependent degradation.3,4 Under hypoxic
condition, stabilized HIF-1 translocates into the nucleus and
dimerizes with HIF-1b. The HIF-1 /b heterodimer binds to
a is a key regulator of the adaptation of cancer cells
a
tives
have
been
reported
including
antimalarial,10
a
antimicrobial,11 and anticancer activity.12 Until now, several syn-
thetic strobilurins have been developed, and among them, kre-
soxim-methyl is one of the most widely used strobilurin
fungicides.13
a
a
hypoxia-response element (HRE) and induces transcription of a
large number of genes implicated in tumor angiogenesis, glucose
metabolism, cell survival and metastasis.2 Therefore, HIF-1 is an
attractive molecular target for cancer treatment, and various HIF-
1 inhibitors have been developed.5,6
Our research has been focused on the development of a small
molecule-based HIF-1 inhibitor having an inhibitory activity
against mitochondrial respiration. From the results of our previous
studies, the inhibition of mitochondrial respiration could lead to
the degradation of the HIF-1a
protein in cancer cells,14–16 indicat-
Strobilurins, natural products isolated from fungi, are commer-
cial quinone outside inhibitor (Q0I) fungicides.7 The strobilurin
mechanism of action is an inhibition of fungal respiration by bind-
ing that regulation of mitochondria respiration is a promising
approach for treatment of hypoxic cancer. In this study, we
designed and synthesized kresoxim-methyl analogues as inhibitors
of HIF-1a accumulation, and evaluated their HIF inhibitory activity
and anti-proliferative activity in cancer cells under hypoxic
⇑
condition.
Corresponding authors at: Metabolic Regulation Research Center, Korea
Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of
Korea (H.S. Ban) and School of Chemistry and Biochemistry, Yeungnam University,
280 Daehak-Ro, Gyeongsan-si, Gyeongbuk 38541, Republic of Korea (C.S. Ra).
E-mail addresses: banhs@kribb.re.kr (H.S. Ban), csra@yu.ac.kr (C.S. Ra).
Chemical synthesis of a precursor 5, (2-bromomethylphenyl)
methoxyiminoacetic acid methyl ester, leading to kresoxim-
methyl analogues was made using
a well-known literature
http://dx.doi.org/10.1016/j.bmcl.2017.05.024
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lee S., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.05.024

2
S. Lee et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Scheme 1. Reagents and conditions: (a) NaCN, Aliquat 336, DCM, H2O, rt, 2 h; (b) 85% H2SO4, NaBr, rt, 2 h; MeOH, 65 °C, 2 h; (c) MeONH2ÁHCl, MeOH, 65 °C, 12 h; (d) NBS,
AIBN, CCl4, 80 °C, 12 h, 70%.
Scheme 2. Reagents and conditions: (a) o-methoxyamine hydrochloride, MeOH, 65 °C, 12 h; (b) 5, K2CO3, acetone, 60 °C, 12 h.
Scheme 3. Reagents and conditions: (a) R-Br, K2CO3, acetone, 60 °C, 12 h; (b) 5, K2CO3, acetone, 60 °C, 12 h.
procedure.17 In these sequences, commercially available o-toluoyl
chloride was converted to 5 via a four-step route on a multigram
scale without column chromatography (Scheme 1). Cyanation of
Table 1
Effects of kresoxim-methyl analogues on the hypoxia-induced HIF transcriptional
activation and cancer cell growth.
o-toluoyl chloride 1 with sodium cyanide and aliquat 336 in
dichloromethane and water (1:1 v/v) quantitatively yielded ben-
zoyl cyanide 2, which was converted to keto ester 3 by treatment
with a methanolic solution of sodium bromide and sulfuric acid.
Reaction of 3 with o-methylhydroxylamine hydrochloride gave
the corresponding imine in a mixture of E/Z = 85:15, and following
recrystallization from dichloromethane and hexane to afford (E)-
methyl 2-methoxyimino-2-o-tolylacetate 4. The benzylic radical
bromination of 4 using the Wohl–Ziegler procedure with N-bromo-
succinimide (NBS) yielded 5 in a good yield.
Cmpd
R1
R2
HRE-Luca IC50 (mM)b
11f
8a
H
H
OH
9.16 0.95
3.40 0.20
Reaction of phenolic carbonyl compounds 6 with o-methoxya-
mine hydrochloride in methanol gave their corresponding oximes
7 in good yields and resorcinol was converted into its benzylic
derivatives 10 in modest to good yields. Finally oximes 7, resorci-
nol (9) and its benzylic derivatives 10 were reacted with 5 in the
presence of potassium carbonate in acetone to afford kresoxim-
8b
H
4.52 0.15
14.88 0.19
1.12 0.57
8c
OCH3
H
11a
methyl analogues
(Schemes 2 and 3).
8 and 11, respectively, in modest yields
11b
H
0.60 0.25
We next evaluated the biological activity of the synthesized
kresoxim-methyl analogues. First, we examined the effects on
hypoxia-induced transcriptional activation of HIF-1 using a cell-
based HRE reporter gene assay in human colorectal cancer
HCT116 cells; the results are shown in Table 1. Under hypoxic con-
ditions, 2-hydroxy compound (11f) showed moderate inhibitory
activity against HIF-1 activation. Replacement of the hydroxyl
group to methyl oxime significantly increased the HIF-1 inhibitory
activity (8a and 8b). However, introduction of methoxy group into
R1 position (8c) resulted in reduction of the inhibitory activity.
Furthermore, the addition of benzyloxy substituents on R2 position
(11a–11d) also showed significant inhibitory activity. Among
11c
11d
H
H
0.87 0.28
0.94 0.15
11e
H
>20
a
HIF transcriptional activity was determined using HCT116 cells stably trans-
fected with HRE-Luc.
The drug concentrations required to inhibit HIF activation by 50% (IC50) was
determined from the semilogarithmic dose-response plots, and the results are
means SD of triplicate samples.
b
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S. Lee et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Fig. 1. Effects of kresoxim-methyl analogues on intracellular oxygen tension. HCT116 cells were treated with the indicated concentration of compound 11b (A) or 5 mM
compounds 11c, 11d, and 11e (B) for 6 h under hypoxic conditions. The intracellular oxygen tension was detected by hypoxia-sensitive probe MAR (0.5 mM). Nuclei were
stained with NUCLEAR-ID Red DNA stain. The scale bar indicates 100 mm. (C) The fluorescence intensity ratios of MAR probe to NUCLEAR-ID are shown by vertical bars.
Statistical significance: **P < 0.01, compared with hypoxia control.
them, compound 11b containing 4-chloro-benzyloxy exerted the
most potent inhibitory activity with an IC50 value of 0.6 mM. How-
ever, cyanide (11e) did not show any influence on hypoxia-induced
HIF activation.
and 11d at 5 mM potently enhanced the oxygen level, but com-
pound 11e at 5 mM did not (Fig. 1B). The fluorescent intensity ratio
of MAR probe to NUCLEAR-ID is presented in Fig. 1C. Furthermore,
other kresoxim-methyl analogues at 5 mM also regulated the intra-
cellular oxygen levels in parallel with the HIF inhibition (Supple-
mentary Fig. S1). These results indicate that the inhibitory effect
of HIF activation by kresoxim-methyl analogues is mediated by
regulation of intracellular oxygen tension.
To assess ability of the synthesized compounds to regulate
intracellular oxygen, we determined the oxygen level in cancer
cells using a hypoxia-sensitive fluorescence probe, mono azo rho-
damine (MAR).18 The results are shown in Fig. 1. Under normoxia,
the MAR probe is non-fluorescent, but it turns fluorescent due to
the reduction of the azo group when exposed to hypoxic condi-
tions. Effects of kresoxim-methyl analogues on the change of intra-
cellular oxygen levels were determined. As shown in Fig. 1A,
compound 11b at 1–5 mM attenuated the reduction of the MAR
probe, indicating that the intracellular oxygen level increased in
a concentration-dependent manner. In addition, compounds 11c
We next explored whether the increased oxygen by kresoxim-
methyl analogues lead to degradation of the HIF-1
hypoxic cells. As shown in Fig. 2, incubation of HCT116 cells under
hypoxia for 6 h significantly induced accumulation of HIF-1 pro-
tein. Under these conditions, treatment with compounds 11b, 11c,
and 11d at 1 to 5 mM suppressed the hypoxia-induced HIF-1 accu-
mulation in a concentration-dependent manner, while compound
a protein in
a
a
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S. Lee et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.05.
024.
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Fig. 2. Effects of kresoxim-methyl analogues on the hypoxia-induced accumulation
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suppressed the hypoxia-induced accumulation of HIF-1
a protein
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results from this study could provide an impetus to further develop
kresoxim-methyl analogues as anticancer agents.
Acknowledgements
This work was supported by Yeungnam University Research
Grant (216A251117) and the KRIBB Initiative of the Korea Research
Council of Fundamental Science and Technology.
Please cite this article in press as: Lee S., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.05.024