Development of hypoxia-inducible factor (HIF)-1α inhibitors: Effect of ortho-carborane substituents on HIF transcriptional activity under hypoxia

Article abstract of DOI:10.1016/j.bmcl.2012.11.081

A series of substituted ortho-carboranylphenoxyacetanilides were synthesized and evaluated for their ability to inhibit hypoxia-induced HIF-1 transcriptional activity using a cell-based reporter assay in HeLa cells expressing the HRE-dependent firefly luciferase reporter construct (HRE-Luc) and constitutively expressing CMV-driven Renilla luciferase reporter, and their ability to inhibit cell growth (GI₅₀) using the MTT assay. Among the compounds synthesized, 1g and 1l showed significant inhibition of hypoxia-induced HIF-1 transcriptional activity (IC₅₀: 1.9 ± 0.4 and 1.4 ± 0.2 μM, respectively). Both compounds suppressed HIF-1α accumulation in a concentration-dependent manner. The porcine heart malate dehydrogenase (MDH) refolding assay revealed that compound 1l inhibited human Hsp60 chaperone activity (IC₅₀: 6.80 ± 0.25 μM) and this inhibition activity was higher than that of ETB (IC₅₀: 10.9 ± 0.63 μM).

Full text of DOI:10.1016/j.bmcl.2012.11.081

Bioorganic & Medicinal Chemistry Letters 23 (2013) 806–810
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of hypoxia-inducible factor (HIF)-1
a
inhibitors: Effect of
ortho-carborane substituents on HIF transcriptional activity under hypoxia
⇑
Hiroyuki Nakamura , Yuka Yasui, Minako Maruyama, Hidemitsu Minegishi, Hyun Seung Ban,
Shinichi Sato
Department of Chemistry, Faculty of Science, Gakushuin University, Mejiro, Tokyo 171-8588, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted ortho-carboranylphenoxyacetanilides were synthesized and evaluated for their
ability to inhibit hypoxia-induced HIF-1 transcriptional activity using a cell-based reporter assay in HeLa
cells expressing the HRE-dependent firefly luciferase reporter construct (HRE-Luc) and constitutively
expressing CMV-driven Renilla luciferase reporter, and their ability to inhibit cell growth (GI50) using
the MTT assay. Among the compounds synthesized, 1g and 1l showed significant inhibition of
Received 14 September 2012
Revised 19 November 2012
Accepted 20 November 2012
Available online 8 December 2012
hypoxia-induced HIF-1 transcriptional activity (IC50: 1.9 0.4 and 1.4 0.2
compounds suppressed HIF-1 accumulation in a concentration-dependent manner. The porcine heart
malate dehydrogenase (MDH) refolding assay revealed that compound 1l inhibited human Hsp60
lM, respectively). Both
Keywords:
a
Hypoxia-inducible factor (HIF)-1
ortho-Carborane
Angiogenesis
a
chaperone activity (IC50: 6.80 0.25
(IC50: 10.9 0.63 M).
l
M) and this inhibition activity was higher than that of ETB
l
Inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
Hsp60
Hypoxia-inducible factor 1 (HIF-1) is a key regulator of cellular
transcriptional responses to hypoxia in normal and tumor cells.1,2
HIF-1 is a heterodimeric complex consisting of a hypoxically induc-
increased the binding of receptor of activated C kinase-1 (RACK1),
which recruited elongin C and its E3 ligase complex to HIF-1
a,
resulting in degradation through the ubiquitin–proteasome
ible subunit HIF-1
Under normoxia, HIF-1
a
and a constitutively expressed subunit HIF-1b.
protein undergoes hydroxylation of pro-
pathway.16
a
line residues by prolyl hydroxylase (PHD).3,4 This modification is
recognized by the von Hippel–Lindau tumor suppressor protein
for ubiquitination and rapid degradation.5 Under hypoxia, the
hydroxylation by PHD is inactivated through oxygen-sensing
O
NMe2
H
N
HO
O
O
N
N
N
mechanisms and the unmodified HIF-1a is sufficiently stabilized
H
O
O
HCl
to form a heterodimeric complex with HIF-1b. This complex binds
to the hypoxia response element (HRE) DNA sequence with co-
activators to activate various genes, including VEGF and EPO.6
These growth factors play important roles in tumor angiogenesis.
The growth of tumor blood vessels increases the supply of oxygen
and nutrients to small tumors, which results in tumor growth and
metastasis.7,8 Therefore, HIF-1 is considered to be one of the poten-
tial targets for the development of anticancer agents.2,9
H3CO
O
MeO
Me
O
OH
O
topotecan
NH2
tanespimycin
NH2
OH
O
O
Cl
+
N
O
-O
Cl
O
Although various small molecules have been developed as HIF-
1 inhibitors, their inhibition mechanisms are still not clear in many
cases. Clinical trials were carried out on several HIF-1 inhibitors,
including topotecan,10 tanespimycin,11 PX-866,12 PX-12,13 and
PX-478,14 in the last decade (Fig. 1). In particular, tanespimycin,
a geldanamycin analogue, was widely examined in a clinical
MeO
PX-478
O
H
O
OH
O
N
S
N
S
N
study.15 Tanespimycin suppressed Hsp90 binding to HIF-1
a
and
H
PX-866
Figure 1. Structures of HIF-1 inhibitors under clinical studies.
PX-12
⇑
Corresponding author.
E-mail address: hiroyuki.nakamura@gakushuin.ac.jp (H. Nakamura).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.081

H. Nakamura et al. / Bioorg. Med. Chem. Lett. 23 (2013) 806–810
807
R1
OMe
OMe
B(OH)2
OMe
H
a,b
c
O
O
I
O
O
H
H
N
H
N
H
6
7
8
OH
OH
OMe
OH
LW6 (R1 = CO2Me)
GN26192 (R1 = B(OH)2)
R3
R3
GN26361
e
d
Figure 2. Structures of LW6, GN26192, and their carborane analogues 1.
9a:
b:
c:
10a:
R
R
R
R
1 = Me
1 = Et
R
R
R
R
1 = Me
1 = Et
We recently reported that ortho-carboranylphenoxyacetanilide
b:
c:
d:
(GN26361), an analogue of LW6,17,18 had a potent inhibitory effect
1 = iBu
1 = iBu
on HIF-1
a
activation under hypoxia (Fig. 2).19 Furthermore, we
=
nBu
=
nBu
1
1
d:
synthesized GN26361 multifunctional molecular probes that com-
bined photoaffinity labeling and the click reaction to identify the
target protein, and found that Hsp60 is the primary target of
Scheme 2. Reagents and conditions: (a) ethynyltrimethylsilane, PdCl2(PPh3)2, PPh3,
CuI, diethylamine, THF, reflux, 5 h, 95%; (b) TBAF, THF, rt, 30 min, 86%; (c) B10H14
,
N,N-dimethylaniline, chlorobenzene, microwave, 130 °C, 10 min, 76%; (d) n-BuLi,
GN26361 and affects HIF-1a
accumulation directly or indirectly.20
R1I, THF, ꢀ10 °C, 2 h, 47–93%; (e) BBr3, CH2Cl2, rt, overnight, 90–98%.
In this study, we investigated the effect of the substituents of
ortho-carboranylphenoxyacetanilides (1) on the inhibition of HIF
transcriptional activity under hypoxia. Furthermore, we examined
the inhibition of human Hsp60 chaperone activity by LW6,
GN26192, GN26361, 1l, and 1g and found that the carborane
framework is essential for the inhibition of Hsp60 chaperone
activity.
Chemistry: ortho-Carboranylphenoxyacetanilides, in which vari-
ous functional groups (R1 and R2) were introduced to the benzene
ring, were synthesized according to our previously reported proto-
col with modification. As shown in Scheme 1, 4-bromophenol 2
was treated with ethyl chloroacetate and the resulting ethyl ester
was hydrolyzed with LiOH in aqueous THF to afford carboxylic acid
3 in 95% yield. Carboxylic acid 3 was reacted with various anilines,
such as methyl 3-aminobenzoate, methyl 3-amino-4-hydroxyben-
zoate, ethyl 3-amino-4-hydroxybenzoate, and 2-aminobenzophe-
none,
using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDCI) and hydroxybenzotriazole (HOBt) in the presence of diiso-
propylethylamine (DIPEA) in DMF to give corresponding aryloxy-
acetanilides 4a–d, respectively, in high yields. The Sonogashira
coupling reaction was employed for the introduction of an ethynyl
moiety into the phenoxy ring. The reaction of 4a–d with ethynyl-
trimethylsilane proceeded in the presence of PdCl2(PPh3)2 catalyst
in DMF under microwave irradiation condition and the resulting
coupling products were treated with K2CO3 in THF–MeOH solution
to give corresponding acetylenes 5a–d in 21–81% yields. The cou-
pling of acetylenes 5a–d with decaborane (14) was carried out in
the presence of acetonitrile in toluene under reflux condition for
48 h to give ortho-carboranylphenoxyacetanilides 1a–d in moder-
ate yields (17–36%). Ethyl ester 1a was converted into carboxylic
acid 1e by hydrolysis using LiOH in aqueous THF.
O
OH
O
OH
a
b
To introduce substituents (R3) to the carbon of the ortho-carbo-
rane framework, we used the lithiation method in which the lith-
iocarborane generated by treating an ortho-carborane with n-BuLi
was treated as a nucleophile to react with various electrophiles. As
shown in Scheme 2, substituted ortho-carboranylphenols 10 were
synthesized from 4-iodoanisole 6. The Sonogashira coupling of 6
with ethynyltrimethylsilane proceeded in the presence of
PdCl2(PPh3)2 catalyst in THF under reflux condition for 5 h to give
the corresponding coupling product, which was subsequently
treated with tetrabutylammonium fluoride (TBAF) in 5% aqueous
THF solution to remove a trimethylsilyl function, eventually
affording 4-ethynylanisole 7 in 86% yield. The coupling of acety-
lene 7 with decaborane (1.2 equiv) was carried out in the presence
of N,N-dimethylaniline (1.5 equiv) in chlorobenzene as the sol-
vent. The reaction was completed within 10 min at 130 °C in a
sealed vial under microwave irradiation condition and 4-
methoxyphenyl-ortho-carborane 8 was obtained in 76% yield.
When the coupling reaction was carried out under the general
condition (toluene reflux condition), the corresponding ortho-car-
borane product was obtained in low yields (30–50%). Next, various
substituents (R3) were introduced to the ortho-carborane moiety.
Deprotonation of carborane 8 was carried out by using n-BuLi as
the base to generate the lithiocarborane in situ, which was subse-
quently treated with various electrophiles (R1I) to give corre-
sponding substituted ortho-carboranes 9a–d (9a: R1 = methyl,
93%; 9b: R1 = ethyl, 87%; 9c: R1 = i-butyl, 63%; 9d: R1 = n-butyl,
47%). Treatment of 9a–d with BBr3 in CH2Cl2 at room temperature
afforded corresponding ortho-carboranylphenols 10a–d in high
yields.
Br
Br
3
2
R1
R1
O
O
c
O
O
N
H
N
H
R2
R2
Br
R1 = CO2Me, R2 = H
R1 = CO2Me, R2 = OH
R1 = CO2Et, R2 = OH
R1 = COPh, R2 = H
R1 = CO2Me, R2 = H
R1 = CO2Me, R2 = OH
R1 = CO2Et, R2 = OH
R1 = COPh, R2 = H
4a:
b:
5a:
b:
c:
c:
d:
d:
R1
O
d
O
H
N
H
R2
R1 = CO2Me, R2 = H
R1 = CO2Me, R2 = OH
R1 = CO2Et, R2 = OH
R1 = COPh, R2 = H
R1 = CO2H, R2 = H
1a:
b:
c:
d:
e:
e
Scheme 1. Reagents and conditions: (a) (i) ethyl chloroacetate, K2CO3, DMF, rt, 5 h,
(ii) LiOH, THF–H2O, rt, 3 h, 95%; (b) methyl 3-aminobenzoate, methyl 3-amino-4-
hydroxybenzoate, ethyl 3-amino-4-hydroxybenzoate, or 2-aminobenzophenone,
EDCI, HOBt, DIPEA, DMF, rt, 8 h, 88–93%; (c) (i) ethynyltrimethylsilane, PdCl2(PPh3)2,
PPh3, CuI, diethylamine, DMF, microwave, 120 °C, 30 min, (ii) K2CO3, THF–MeOH, rt,
3 h, 21–81%; (d) B10H14, CH3CN, toluene, reflux, 48 h, 17–36%; (e) LiOH, THF/H2O, rt,
38 h, 51%.

808
H. Nakamura et al. / Bioorg. Med. Chem. Lett. 23 (2013) 806–810
R1
CO2Bn
CO2H
CO2Bn
OH
R3
b,c
a
O
O
a, b
Br
O
R3
N
O2N
O2N
N
H
H
OBn
OH
OBn
OH
11
12
13
10a-d
R1 = CO2Et, R3 = Me
1f:
g:
h:
i:
j:
k:
l:
1 = CO2Et, R3 = Et
d
R
R1 = CO2Et, R3 = iBu
a, b
1 = CO2Et, R3
R1 = CO2H, R3
=
=
nBu
nBu
O
O
R
CO2Et
CO2Et
CO2Et
OBn
B
c
1 = B(OH)2, R3 = Me
e
b,c
R
O
O
R1 = B(OH)2, R3 = Et
Br
O
R3
O2N
O2N
N
1 = B(OH)2, R3 = iBu
N
m:
R
H
H
OH
OBn
OH
14
15
16
1k'-m'
Scheme 3. Reagents and conditions: (a) BnBr, K2CO3, DMF, rt, overnight, 64%; (b)
Fe, NH4Cl,EtOH, H2O, reflux, 1 h; (c) bromoacetyl bromide, pyridine, DMAP, rt, 1.5 h;
(d) H2SO4, EtOH, reflux, 12 h, 96%; (e) BnBr, K2CO3,acetone, reflux, overnight, 92%.
Scheme 5. Reagents and conditions: (a) 13, 16 or 20, K2CO3, DMF, rt, overnight, 48–
86%; (b) H2, Pd/C, MeOH, THF, rt, 6 h, 60–85%; (c) (i) KHF2 (4 M), MeOH, rt, 2 h, (ii)
HCl (1 N), rt, overnight, 32–65%.
Benzyl and ethyl ester substituted bromoacetanilides 13 and 16
were also synthesized from 4-hydroxy-3-nitrobenzoic acid 11, as
shown in Scheme 3. Benzoic acid 11 was converted into benzyl es-
ter 12 by treating with benzyl bromide under basic condition, and
reduction of the nitro group of 12 with Fe powder followed by
amide formation with bromoacetyl bromide gave bromoacetani-
lide 13. Furthermore, 4-hydroxy-3-nitrobenzoic acid 11 was con-
verted into ethyl ester 14 in acidic ethanol and the phenol OH
group of 14 was protected with benzyl bromide. Reduction of the
nitro group of resulting benzyl ether 15 with Fe powder followed
by amide formation with bromoacetyl bromide gave bromoacetan-
ilide 16.
Boronic ester substituted bromoacetanilide 20 was synthesized
from 4-bromo-2-nitrophenol 17, as shown in Scheme 4. Compound
17 was converted into benzene boronic ester 18 according to the
procedure reported previously,19 and reduction of the nitro group
of 18 with Fe powder gave aniline 19 in 79% yield. Aniline 19
was treated with bromoacetylbromide to give 20 in 80% yield.
As shown in Scheme 5, substituted ortho-carboranylphenols
10a–d were treated with bromoacetanilide 13 or 16 in DMF under
basic condition and the resulting ethyl esters or a benzyl ester was
hydrogenated to give 1f–j. Meanwhile, ortho-carboranylphenols
10a–c were treated with bromoacetylanilide 20 and deprotection
of resulting pinacol esters 1k0–m0 was carried out using KHF2 in
MeOH followed by HCl to give corresponding boronic acids 1k–
m.21
Table 1
Inhibition of HIF-1 transcriptional activity in HeLa cell-based HRE and CMV dual
luciferase assay and cell growth inhibition
R1
O
O
R3
N
H
R2
1
Compound
R1
R2
R3
IC50
>100
(
l
M)
GI50 (lM)
a
b
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
GN26361
LW6
CO2Me
CO2Me
CO2Et
COPh
H
H
H
H
H
H
Me
Et
iBu
nBu
nBu
Me
Et
>100
>100
18 0.07
53 1.4
>100
2.2 0.1
2.0 0.08
1.9 0.05
6.4 0.02
18 0.3
5.2 0.08
4.2 0.1
2.4 0.06
16 0.7
—
OH
OH
H
31 0.4
19 2.0
69 3.8
>100
2.2 0.2
1.9 0.4
2.1 0.1
CO2H
H
CO2Et
CO2Et
CO2Et
CO2Et
CO2H
OH
OH
OH
OH
OH
OH
OH
OH
OH
25
4
5.7 2.4
1.9 0.2
1.4 0.2
1.8 0.3
2.2 0.2
21 0.9
B(OH)2
B(OH)2
B(OH)2
B(OH)2
iBu
H
a
HeLa cells expressing HRE-dependent firefly luciferase reporter construct (HRE-
Luc) and constitutively expressing CMV-driven Renilla luciferase reporter with
SureFECT Transfection Reagent were established with Cignal™ Lenti Reporter
(SABiosciences, Frederick, MD) according to the manufacturer’s instructions. The
consensus sequence of HRE was 50-TACGTGCT-30 from the erythropoietin gene.
Cells stably expressing the HRE-reporter gene were selected with puromycin. The
cells were incubated for 12 h with or without drugs under the normoxic or hypoxic
condition. After removal of the supernatant, the luciferase assay was performed
using a Luciferase Assay System (Promega, Madison, WI) according to the manu-
facturer’s instructions. The drug concentration required to inhibit the relative light
units by 50% (IC50) was determined from semi-logarithmic dose–response plots,
and the results represent means SD of triplicate samples.
O
O
O
O
Br
B
B
a
O2N
O2N
H2N
OH
OBn
OBn
b
17
18
19
HeLa cells were incubated for 72 h with various concentrations (100 nM to
100 lM) of compounds under the normoxic condition, and viable cells were
determined by the MTT assay. The drug concentration required to inhibit cell
growth by 50% (GI50) was determined from semi-logarithmic dose–response plots,
and results represent means SD of triplicate samples.
O
O
B
b
O
Br
N
H
Biological activity: Synthesized substituted ortho-carboranyl-
phenoxyacetanilide derivatives 1a–m were evaluated for their
ability to inhibit hypoxia-induced HIF-1 transcriptional activity
using a cell-based reporter assay in HeLa cells expressing HRE-
dependent firefly luciferase reporter construct (HRE-Luc) and con-
OBn
20
Scheme 4. Reagents and conditions: (a) Fe, NH4Cl, EtOH, H2O, reflux, 1 h, 79%; (b)
bromoacetylbromide, pyridine, DMAP, rt, 1.5 h, 80%.

H. Nakamura et al. / Bioorg. Med. Chem. Lett. 23 (2013) 806–810
809
stitutively expressing CMV-driven Renilla luciferase reporter, and
their ability to inhibit cell growth (GI50) using the MTT assay.
GN26361 and LW6 were used as positive controls for comparison.
The results are summarized in Table 1. Among compounds 1a–e
that have no substituent at the R3 group of the ortho-carborane
framework, compounds 1a and 1d that have no hydroxyl group
on the aniline ring minimally inhibited HIF-1 transcriptional activ-
ity even at 100
lM concentration under hypoxia. Whereas com-
pound 1d, which has a phenyl ketone moiety at the R1 group,
showed low inhibitory activity (IC50 = 69 lM), compounds with a
hydroxyl group at the R2 group (1b and 1c) inhibited HIF-1 tran-
scriptional activity similarly to the adamantyl derivative LW6
(IC50 = 19–31 lM). These results indicate that the hydroxyl substi-
tuent at the R2 position is essential for the inhibition of HIF-1 tran-
scriptional activity and ethyl ester 1c is more potent than methyl
ester 1b (19 lM vs 31 lM). Interestingly, the inhibitory activity
was dramatically enhanced when substituents were introduced
at the R3 position of the ortho-carborane framework. Compounds
1f–h, where methyl, ethyl, and i-butyl groups were substituted
at the R3 position, respectively, displayed significant inhibition of
HIF-1 transcriptional activity with IC50 values of 1.9–2.2
substitution with an n-butyl group at the R3 position reduced the
inhibitory activity of 1i (25 M), whereas the introduction of the
same n-butyl group increased the inhibitory activity of carboxylic
acid derivative 1j (5.7 M). Since the carboxylic acid 1j was consid-
lM. The
l
l
ered as the hydrolyzed form of the ester 1i in cells, we prepared the
carboxylic acid derivative of 1g and tested. The carboxylic acid
inhibited hypoxia-induced HIF-1 transcriptional activity (1.1 lM),
revealing that the esters 1g and 1i are considered to be pro-drugs
that could be hydrolyzed to the active carboxylic acids. We previ-
ously reported that boronic acid on the aniline ring of 1 enhanced
the inhibition of the hypoxia-induced HIF-1 transcriptional activ-
ity19 (i.e., 1c vs GN26361 in Table 1). Therefore, these alkyl groups
were introduced at the R3 position of GN26361. The methyl (1k),
ethyl (1l), and i-butyl (1m)-substituted boronic acid analogues
showed slight enhancement of the inhibitory activity with IC50 val-
Figure 4. Hsp60 chaperone activity was analyzed by using MDH as substrate.
Porcine MDH was denatured in 10 mM HCl at rt for 2 h. Hsp60 (4
Hsp10 (8 M) were pre-incubated for 90 min at 30 °C in a buffer (50 mM Tris/HCl,
pH 7.6, 300 mM NaCl, 20 mM KCl, 20 mM Mg(OAc)2, and 4 mM ATP). Denatured
MDH was diluted to a concentration of 0.15 M with a buffer (0.1 M Tris/HCl, pH
7.6, 7 mM KCl, 7 mM MgCl2, 1 mM DTT, and 2 mM ATP) containing Hsp60/10 and
each concentrated compound at 30 °C (final concentration of Hsp 60: 0.2 M, Hsp
10: 0.4 M, compound: 0.6–20 M). After the reaction mixture was treated for
lM) and human
l
l
l
l
l
30 min, MDH reactivation was measured with NADH and oxaloacetic acid. (A)
Time-dependent decreasing ratio of absorbance of NADH at 340 nm. ‘+’, NADH only;
‘h’, NADH with denatured MDH; ‘o’, NADH with MDH refolded by Hsp60/10; ‘ꢁ’,
NADH with native MDH (15 nM; 1/10 amount); ‘x’, NADH with MDH and Hsp60/10
in the presence of compound. The decreasing ratio of absorbance was calculated
based on the absorbance at 0 s. (B) Dose-dependent inhibition of human Hsp60
chaperone activity by compound. IC50 values were calculated for ETB
ues of 1.4–1.9 lM, and significant enhancement was not observed
by these substituents compared to the case of ester derivative 1c. It
should be noted that cell growth inhibition by the compounds
(GI50) was related to the inhibition of the hypoxia-induced HIF-1
transcriptional activity, although the cytotoxicity of the com-
pounds was excluded from the HIF-1 transcriptional activity by
correcting the measurement of constitutively expressing CMV-dri-
ven Renilla luciferase activity.
(10.9 0.63 lM), GN26361 (2.46 0.33 lM), and 1l (6.80 0.25 lM).
As compounds 1g and 1l possess significant inhibitory activity
toward the hypoxia-induced HIF-1 transcriptional activity among
the ethyl ester derivatives and boronic acid analogues, respec-
tively, we next examined the effects of those two compounds on
tubulin protein expression level (Fig. 3). These results indicate that
the inhibition of hypoxia-induced HIF-1 transcriptional activity is
caused by the suppression of HIF-1a accumulation by the
compounds.
the hypoxia-induced HIF-1
sis in HeLa cells. Compounds 1g and 1l suppressed HIF-1
a
accumulation by western blot analy-
accumu-
We previously reported that Hsp60 is the primary target of
GN26361 and affects HIF-1
accumulation directly or indirectly.20
a
a
lation in a concentration-dependent manner without affecting
Therefore, we examined whether compounds 1g and 1l inhibit hu-
man Hsp60 chaperone activity using the porcine heart malate
dehydrogenase (MDH) refolding assay. This assay was performed
according to the literature protocol.22,23 Briefly, denatured porcine
MDH was refolded by Hsp60/10 in the presence or absence of the
compounds, and the reactivation of MDH by the Hsp60/10 chaper-
one activity was determined by measuring the absorbance of
NADH at 340 nm, which was converted into NAD+ by the refolded
MDH with absorption reduction. The time course of NADH absorp-
tion is shown in Figure 4A. In this assay system, it was calculated
that approximately 25% of MDH was reactivated by Hsp60/10 by
comparing the activities between native MDH and refolded MDH
and the compound that inhibited it (Fig. 4A). The Hsp60 inhibitor
ETB23 was tested as positive control and found to inhibit MDH
Figure 3. Effect of compounds 1g and 1l on hypoxia-induced accumulation of HIF-
1a
protein. HeLa cells were incubated for 4 h with or without the indicated
concentrations of compounds under the hypoxic condition. Protein levels were
refolding at the micromolar level (IC50 = 10.9 0.63
lM). In addi-
detected by immunoblot analysis with HIF-1
Tubulin was used as the loading control.
a- or tubulin-specific antibodies.
tion, GN26361 showed more potent activity (IC50 = 2.46 0.33
l
M)

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H. Nakamura et al. / Bioorg. Med. Chem. Lett. 23 (2013) 806–810
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than ETB. Compounds 1g and 1l having potent inhibitory activity
toward the hypoxia-induced HIF-1 transcriptional activity were
tested as well. Compound 1l, which has an ethyl group at the R3
position of the ortho-carborane framework of GN26361, showed
Hsp60/10 inhibitory activity (IC50 = 6.80 0.33 lM), whereas 1g,
which has an ethyl ester at the R1 position of 1l, did not show sig-
nificant activity. We also tested the carboxylic acid derivative of 1g
and observed that the carboxylic acid derivative of 1g inhibited the
Hsp60/10 chaperone activity similar to the boronic acid 1l. These
results suggest that the acid moiety such as carboxylic acid and
boronic acid enhances HIF-1 suppression by inhibiting Hsp60
chaperon activity. In order to evaluate the importance of the
ortho-carborane framework in Hsp60 inhibition, two adamantyl
derivatives, LW6 and GN26192, having a methyl ester and a boro-
nic acid group at the R1 position, respectively (Fig. 2), were tested.
Both of them showed little activity, attesting that the ortho-
carborane framework is important for Hsp60 inhibition (Fig. 4B).
In conclusion, we have developed two novel inhibitors of HIF
transcriptional activity, compounds 1g and 1l, which have a substi-
tuted ortho-carborane framework. The two compounds suppressed
19. Shimizu, K.; Maruyama, M.; Yasui, Y.; Minegishi, H.; Ban, H. S.; Nakamura, H.
Bioorg. Med. Chem. Lett. 2010, 20, 1453.
20. Ban, H. S.; Shimizu, K.; Minegishi, H.; Nakamura, H. J. Am. Chem. Soc. 2010, 132,
11870.
21. Spectral data for representative compounds: 1-Methyl-2-[ethyl-4-hydroxy-3-(2-
phenoxyacetamido)benzoate]-1,2-dicarba-closo-dodecaborane (1f): 1H NMR
(400 MHz; CDCl3): d 9.39 (s, 1H), 8.51 (s, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.80 (s,
1H), 7.67 (d, J = 9.2 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 9.2 Hz, 2H), 4.73
(s, 2H), 4.36 (q, J = 7.1 Hz, 2H), 1.71 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H); 13C NMR
(100 MHz; CDCl3): d 167.5, 165.8, 158.0, 152.8, 133.0, 129.2, 125.5, 124.3,
124.2, 122.9, 119.6, 115.0, 81.5, 67.0, 61.1, 23.1, 14.3; MS (ESI) m/z 470
[MꢀH]ꢀ, 506 [M+Cl]ꢀ; Anal. Calcd for C20H29B10NO5: C, 50.94; H, 6.20; N, 2.97.
Found: C, 51.08; H, 6.06; N, 2.65. 1-Ethyl-2-[ethyl-4-hydroxy-3-(2-
phenoxyacetamido)benzoate]-1,2-dicarba-closo-dodecaborane (1g): 1H NMR
(400 MHz; CDCl3): d 9.40 (s, 1H), 8.50 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.79 (s,
1H), 7.65 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.6 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H), 4.73
(s, 2H), 4.36 (q, J = 7.1 Hz, 2H), 1.87 (q, J = 7.5 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H),
0.99 (t, J = 7.5 Hz, 3H); 13C NMR (100 MHz; CDCl3) d 167.5, 165.9, 158.0, 152.7,
133.0, 129.2, 125.4, 124.1, 122.9, 119.5, 115.0, 83.4, 82.9, 67.0, 61.1, 29.7, 28.6,
14.3, 13.8 MS (ESI) m/z 484 [MꢀH]ꢀ; Anal. Calcd for C21H31B10NO5: C, 51.94; H,
6.43; N, 2.88. Found: C, 51.92; H, 6.31; N, 2.51. 1-Isobutyl-2-[ethyl-4-hydroxy-
3-(2-phenoxyacetamido)benzoate]-1,2-dicarba-closo-dodecaborane (1h): 1H
NMR (400 MHz; CDCl3): d 9.37 (s, 1H), 8.52 (s, 1H), 7.86 (d, J = 8.4 Hz, 1H),
7.81 (s, 1H), 7.64 (d, J = 10.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 10.4 Hz,
2H), 4.73 (s, 2H), 4.36 (q, J = 7.1 Hz, 2H), 1.79–1.69 (m, 1H), 1.66 (d, J = 5.6 Hz,
2H), 1.39 (t, J = 7.1 Hz, 3H), 0.83 (d, J = 6.4 Hz, 6H); 13C NMR (100 MHz; CDCl3):
d 167.5, 165.8, 157.9, 152.8, 133.2, 129.3, 125.5, 124.3, 124.2, 122.9, 119.7,
114.9, 83.6, 82.6, 66.9, 61.1, 51.6, 43.7, 28.4, 23.4, 14.3; MS (ESI) m/z 512
HIF-1a accumulation as well. We also evaluated the inhibition of
human Hsp60 chaperone activity and found that the carborane
framework is essential for the inhibition of Hsp60 chaperone
activity. Indeed, LW6 and GN26192 did not inhibit Further
structure-activity relationship studies based on Hsp60 inhibitory
activity are in progress.
Acknowledgment
This work was partially supported by a Grant-in-Aid for Scien-
tific Research on Innovative Areas ‘Chemical Biology of Natural
Products’ from The Ministry of Education, Culture, Sports, Science
and Technology, Japan.
References and notes
[MꢀH]ꢀ.
1-Methyl-2-[4-hydroxy-3-(2-phenoxyacetamido)phenylboronic
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acid]-1,2-dicarba-closo-dodecaborane (1k): 1H NMR (400 MHz; CD3OD):
d
8.26 (s, 1H), 7.69 (d, J = 9.2 Hz, 2H), 7.32 (d, J = 8.2 Hz, 1H), 7.12 (d, J = 9.2 Hz,
1H), 6.85 (d, J = 8.2 Hz, 1H), 4.76 (s, 2H), 1.72 (s, 3H); 13C NMR (100 MHz;
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115.6, 83.8, 79.2, 68.5, 23.4; MS (ESI) m/z 480 [M+Na]+, 496 [M+K]+; Anal. Calcd
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1-Isobutyl-2-[4-hydroxy-3-(2-phenoxyacetamido)phenylboronic
acid]-1,2-
dicarba-closo-dodecaborane (1m): 1H NMR (400 MHz; CDCl3): d 8.20 (s, 1H),
7.60 (d, J = 10.3 Hz, 2H), 7.25 (d, J = 8.2 Hz, 1H), 7.06 (d, J = 10.3 Hz, 2H), 6.80 (d,
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