Ortho-Carboranylphenoxyacetanilides as inhibitors of hypoxia-inducible factor (HIF)-1 transcriptional activity and heat shock protein (HSP) 60 chaperon activity

Article abstract of DOI:10.1016/j.bmcl.2015.04.088

ortho-Carboranylphenoxy derivatives were synthesized and evaluated for their ability to inhibit hypoxia-induced HIF-1 transcriptional activity using a cell-based reporter gene assay. Among the compounds synthesized, compound 1d showed the most significant

Full text of DOI:10.1016/j.bmcl.2015.04.088

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2624–2628
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
ortho-Carboranylphenoxyacetanilides as inhibitors of hypoxia-
inducible factor (HIF)-1 transcriptional activity and heat shock
protein (HSP) 60 chaperon activity
Guangzhe Li , Soyoko Azuma , Shinichi Sato , Hidemitsu Minegishi a,b, Hiroyuki Nakamura a,
a
b
a
⇑
a
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan
Department of Life Science, Faculty of Science, Gakushuin University, Mejiro, Toshima-ku, Tokyo 171-8588, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
ortho-Carboranylphenoxy derivatives were synthesized and evaluated for their ability to inhibit hypoxia-
induced HIF-1 transcriptional activity using a cell-based reporter gene assay. Among the compounds syn-
thesized, compound 1d showed the most significant inhibition of hypoxia-induced HIF-1 transcriptional
Received 9 March 2015
Revised 25 April 2015
Accepted 27 April 2015
Available online 2 May 2015
activity with the IC50 of 0.53
inhibition of heat shock protein (HSP) 60 chaperon activity among the reported inhibitors: the IC50
toward the porcine heart malate dehydrogenase (MDH) refolding assay was 0.35 M.
lM. Furthermore, compound 1h was found to possess the most significant
l
Keywords:
Hypoxia-inducible factor (HIF)-1
Ó 2015 Elsevier Ltd. All rights reserved.
a
Carborane
Inhibitor
Angiogenesis
Heat shock protein (HSP) 60
Heat shock proteins (HSPs), ubiquitous and evolutionary con-
served proteins in both prokaryotic and eukaryotic cells, constitute
a heterogeneous group of molecules with a wide variety of func-
tions, including assisting the correct folding of nascent polypep-
tides and the refolding of proteins that were partially denatured
as a result of cell stress.1 HSPs are classified into six families
according to their molecular weights: HSP100, HSP90, HSP70,
HSP60, HSP40, and small HSPs (15–30 kDa), with several members
gradually translocate into the nucleus to form a basic helix–
loop–helix heterodimeric complex with a constitutively expressed
HIF-1b. This HIF-1a/b dimer binds to the DNA of hypoxia response
element (HRE) to activate hypoxia-sensitive genes, that encode
proteins including cell immortalization, glucose and energy
14
metabolism, vascularization, invasion and metastasis, and so on.
Therefore, HIF-1 signal targeting is one of the attractive approaches
1
5–17
for development of antitumor agents.
We have developed
2
18
in each family. HSP60 is a family of sequence-related proteins of
various HIF-1 inhibitors, including YC-1 derivatives,
ortho-
60 kDa and typically reside in the mitochondria.3 Hsp60 assem-
carboranylphenoxyacetanilides,
19,20
diaryl-ortho-carboranes,
and indenopyrazoles. We recently identified HSP60 as a primarily
target of ortho-carboranylphenoxyacetanilide (GN26361), a HIF-1
inhibitor (Fig. 1). We clarified that HSP60 plays an important role
for stabilization of HIF-1 protein accumulation under hypoxia and
that inhibition of the HSP60 chaperon activity by GN26361 induces
the degradation of HIF-1 protein through oxygen-independent
21,22
ꢀ
2
3
bles into a tetradecamer that interacts with the co-chaperonin
4
Hsp10 to assist client polypeptides to fold. Although the primary
a
2
4
function of HSP60 is assistance in mitochondrial protein folding,
unfolding, and degradation,5 the expression of HSP60 gene and
HSP60 protein has been investigated in relation to disease progres-
a
6–8
sion, particularly tumor progression.
Therefore, HSP60 is used as
a
a biomarker for the diagnosis and prognosis of preneoplastic and
pathway. In this Letter, we studied the further structure–activity
relationship based on the ortho-carboranylphenoxyacetanilides to
develop potent HIF-1a and HSP60 inhibitors.
Chemistry: We first synthesized 3-(ortho-carboranyl)phenols
from 3-iodoanisoles 2 through decaborane coupling with terminal
alkynes as shown in Scheme 1. Sonogashira coupling of 3-iodoani-
sole 2 with ethynyltrimethylsilane proceeded in the presence of
9
–11
neoplastic lesions.
Hypoxia-inducible factor (HIF)-1
that regulates cellular responses to physiological and pathological
a
is a key transcription factor
1
2,13
hypoxia.
degraded via
hypoxic environment, HIF-1
Under the normoxic conditions, HIF-1
proteasome-dependent pathway; whereas in
accumulates in the cytosol and
a is rapidly
a
a
PdCl
with tetrabutylammonium fluoride (TBAF) to remove trimethylsilyl
TMS) group, giving 3-ethynylanisole 4. Decaborane coupling of 4
2 3 2
(PPh ) catalysts in THF and the resulting alkyne 3 were treated
⇑
Corresponding author. Tel.: +81 45 924 5244; fax: +81 45 924 5976.
E-mail address: hiro@res.titech.ac.jp (H. Nakamura).
(
http://dx.doi.org/10.1016/j.bmcl.2015.04.088
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
0

G. Li et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2624–2628
2625
R
R2
B
R1
O
O
A
O
O
N
H
linker
N
H
H
OH
OH
GN26361: R = B(OH)
2
ortho-carboranyl-
phenoxyacetanilides (1)
=
=
BH
C
1
a: R = Bpin (pin = O C Me )
2 2 4
Figure 1. Structure–activity relationship study based on GN26361.
OH
O
c
OMe
H
H
a
b
a
b
O
I
OMe
OMe
R
OMe
TMS
2
3
4
11
1
0
R
O
OH
H
H
c
e
O
O
OMe
OH
O
O
HO
B
1
2
2
H N
1
3
5
5
a: R = H 63%
b: R = Et 68%
6a: R = H 91%
6b: R = Et 88%
d
OH
B
H
N
d, e
OH
B
O
H
N
Scheme 1. Reagents and conditions: (a) ethynyltrimethylsilane, PdCl
CuI, diethylamine, THF, reflux, 5 h; (b) TBAF, THF, rt, 30 min; (c) B10
dimethylaniline, chlorobenzene, microwave, 130 °C, 10 min; (d) (i) n-BuLi, THF,
10 °C, 30 min, (ii) EtI, 1.5 h; (e) BBr , CH Cl , rt, overnight.
2
(PPh
3
)
H
2
, PPh
3
,
O
H
O
14, N,N-
O
O
O
ꢁ
3
2
2
1
d
1e
OH
HO
OH
H
N
was carried out in the presence of N,N-dimethylaniline in
chlorobenzene under microwave conditions to give the 3-(ortho-
carboranyl)anisole 5a in 63% yield. The treatment of 5a with
n-butyllithium generated lithiated ortho-carboranyl intermediate,
which reacted with ethyl iodide to give 5b in 68% yield. The methoxy
groups of 5a and 5b were deprotected by using BBr
-(ortho-carboranyl)phenols 6a and 6b in 91% and 88% yield,
respectively.
H
f
O
1
f
Scheme 3. Reagents and conditions: (a) methyl propiolate, DABCO, CH
overnight, 80%; (b) LiOH, THF–H O, rt, overnight, 60%; (c) (i) oxalyl chloride, CH
0 °C, 2 h, (ii) 13, triisobutylamine, THF, rt, 2 h, 28%; (d) (i) KHF2 (4 M), MeOH, rt,
30 min, (ii) HCl (1 N), 1 h, overnight; (e) H , Pd/C, MeOH–THF, rt, overnight; (f) (i)
KHF (4 M), MeOH, rt, 2 h, (ii) HCl (1 N), rt, overnight, 64%.
2
Cl
2
, rt,
3
to afford
2
2
Cl
2
,
3
2
2
Bromoacetylanilide 7 reacted with 3-(ortho-carboranyl)phenols
a and 6b under the basic condition and the resulting 3-(ortho-
6
carboranyl)phenoxyacetanilides 8a and 8b were hydrogenated
using Pd/C under hydrogen atmosphere. Finally the pinacol ester
of 9a and 9b were hydrolyzed to give 1b and 1c as meta-derivatives
of GN26361 (see scheme 2).
ester 11,26 which was hydrolyzed with LiOH in aqueous THF solu-
tion. The resulting carboxylic acid 12 was converted to the acid
chloride, which reacted with 13 to give the corresponding pinaco-
latoboron ester 1d. Deprotection of pinacolatoboron ester followed
by hydrogenesis gave 3-(ortho-carboranyl)phenoxypropananilide
1e. Interestingly, deboronated product 1f was obtained when pina-
We next synthesized ortho-carboranylphenoxy derivatives with
an ethylene linker (Scheme 3).2 4-(ortho-Carboranyl)phenol 10
was treated with methyl propiolate using 1,4-diazabicy-
clo[2.2.2]octane (DABCO) as a base to give the conjugated methyl
5
2
colatoboron ester 1d treated with KHF was reacted with HCl for
overnight.
ortho-Carboranylphenoxyderivative withan propylenelinkerwas
also synthesized as shown in Scheme 4. 4-(ortho-Carboranyl)phenol
1
2 3
0 was treated with ethyl 4-bromobutyrate using K CO as a base
R
OBn
O
O
H
N
B
and the resulting ethyl ester 14 was hydrolyzed to afford the corre-
sponding carboxylic acid 15. The amide bond formation of 15 with
aniline 13 was carried out using 1-[bis(dimethylamino)methylene]-
1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidhexafluorophosphate
(HATU) as a condensation reagent and the resulting pinacolatoboron
O
O
O
a
b
Br
B
N
H
O
O
OBn
O
8a: R = H 71%
b: R = Et 58%
7
8
2
ester 16 was treated with KHF followed by HCl to give 3-(ortho-
carboranyl)phenoxybutananilide 1g.
R
OH
B
R
OH
B
H
H
N
N
c
O
In our previous observation, carborane framework was essential
20
O
O
for inhibition of HSP60 chaperon activity, therefore, we synthe-
sized 3,5-(di-ortho-carboranyl)phenoxyacetanilide derivative as
shown in Scheme 5. Sonogashira coupling of 3,5-dibromoanisole
17 with ethynyltrimethylsilane afforded 18, which was treated
under basic conditions to remove trimethylsilyl (TMS) group, giv-
ing 3,5-diethynylanisole 19. Decaborane coupling of 19 proceeded
under the microwave conditions at 130 °C and the resulting anisole
O
O
HO
OH
9
9
a: R = H 78%
b: R = Et 71%
1b: R = H 35%
1c: R = Et 70%
Scheme 2. Reagents and conditions: (a) 6a or 6b, K
Pd/C, MeOH–THF, rt, overnight; (c) (i) KHF
overnight.
2
CO
3 2
, DMF, rt, overnight; (b) H ,
(4 M), MeOH, rt, 2 h, (ii) HCl (1 N), rt,
2

2
626
G. Li et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2624–2628
OH
O
Table 1
H
a
O
b
Inhibition of HIF-1 transcriptional activity in HeLa cell-based HRE and CMV dual
luciferase assay and cell growth inhibition
H
OEt
Compound
GN26361
IC50a
(
l
M)
GI50b
(lM)
1
0
14
O
O
2.2 ± 0.2
1.3 ± 0.1
4.2 ± 0.8
1.1 ± 0.3
0.53 ± 0.07
2.4 ± 0.6
1.3 ± 0.2
14.6 ± 2.7
16.4 ± 0.6
13.5 ± 0.6
6.9 ± 0.5
30
5.2 ± 0.8
5.3 ± 5.2
8.7 ± 0.7
7.7 ± 2.3
23.6 ± 0.6
15.6 ± 1.3
B
1
1
1
1
a
b
c
O
O
O
H
OH
O
c
H
B
N
H
d
OH
1e
1f
15
16
1g
1h
HO
OH
a
HeLa cells expressing HRE-dependent firefly luciferase reporter construct (HRE-
O
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 5 -TACGTGCT-3 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
O
d
H
N
H
OH
0
0
1
g
Scheme 4. Reagents and conditions: (a) ethyl 4-bromobutyrate, K
overnight, 57%; (b) LiOH, THF–H
overnight, 48%; (d) (i) KHF (4 M), MeOH, rt, 2 h, (ii) HCl (1 N), rt, overnight, 58%.
2
3
CO , DMF, rt,
using a Luciferase Assay System (Promega, Madison, WI) according to the
2
O, rt, overnight, 93%; (c) 13, HATU, DIPEA, DMF, rt,
manufacturer’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.
HeLa cells were incubated for 72 h with various concentrations (100 nM–
00 lM) of compounds under the normoxic condition, and viable cells were tested
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.
2
b
2
0 was converted to the corresponding phenol 21 by treated with
BBr . Alkylation of 21 with 7 was carried out using NaH as a base
3
and hydrogenesis of the resulting 22 afforded 3,5-(di-ortho-carbo-
ranyl)phenoxyacetanilide derivative 1h in 54% yield.
1
Biological activity: Synthesized substituted ortho-carboranylphe-
noxyacetanilide derivatives 1a–h 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 constitutively express-
ing CMV-driven Renilla luciferase reporter. Cell growth inhibition
compared with GN26361 is probably due to the lipophilicity of the
pinacol ester functional group, which would affect the transmem-
brane property. The meta derivative 1b of GN26361 decreased
the inhibition of HIF-1 transcriptional activity with IC50 of
4
.2 ± 0.8 lM. Previously, we demonstrated the effects of sub-
(
GI50) by the synthesized compounds was also determined by MTT
1
stituents at ortho-carborane (R ) in GN26361 on HIF-1 transcrip-
assay. GN26361 was used as a positive control for comparison. The
results are summarized in Table 1. The IC50 of GN26361 toward
tional activity and found that ethyl group was the most suitable
2
0
among other substituted derivatives (H, Me, and i-Bu). In the case
of the meta derivatives, the ethyl substituent (1c) increased the inhi-
HIF-1 transcriptional activity was 2.2 ± 0.2
ester 1a was more potent than GN26361 (IC50 = 1.3 ± 0.1
higher inhibitory activity of 1a toward HIF-1 transcription
l
M, whereas the pinacol
lM). The
bition of HIF-1 transcriptional activity with IC50 of 1.1 ± 0.3
IC50 of compound 1e having an ethylene linker was slightly
decreased to 2.4 ± 0.6 M, whereas that of compound 1g having a
propylene linker dropped to 14.6 ± 2.7 M, indicating that the
lM. The
l
OMe
l
OMe
OMe
length such as methylene and ethylene linkers is appropriate for
the potency of HIF-1 inhibition. The highest inhibition was achieved
a
b
by compound 1d with the IC50 of 0.53 ± 0.07 lM, suggesting that the
Br
Br
TMS
OMe
TMS
rigid conformation by introducing an unsaturated ethyne linker in
the molecule is attributed to the potency of HIF-1 inhibition.
Interestingly, deboronated derivative 1f also inhibited HIF-1
1
7
18
19
OH
H
H
H
H
transcriptional activity with IC50 of 1.3 ± 0.2
ortho-carboranyl) substituent was not effective; the IC50 of com-
pounds 1h dropped to 16.4 ± 0.6 M. Although the cytotoxicity of
lM. However, 3,5-(di-
c
d
e
l
the compounds was excluded from the HIF-1 transcriptional activity
by correcting the measurement of constitutively expressing CMV-
driven Renilla luciferase activity, cell growth inhibition by the com-
pounds (GI50) was related to the inhibition of the hypoxia-induced
HIF-1 transcriptional activity except compound 1b.
We next examined the effects of compounds 1c, 1d, 1f, which
showed significant inhibition of HIF-1 transcriptional activity, and
di-carboranyl compound 1h, on human HSP60 chaperone activity
using the porcine heart malate dehydrogenase (MDH) refolding
2
0
21
O
O
O
O
B
B
H
H
O
O
f
O
H
O
H
N
H
N
H
OBn
OH
2
7,28
assay.
The results are summarized in Figure 2. Inhibition ratio of each com-
pound at 2 M concentration against HSP60 chaperon activity is
indicated by vertical axis. Although epolactaene tert-butyl ester
GN26361 was used as a positive control for comparison.
2
2
1h
l
Scheme 5. Reagents and conditions: (a) ethynyltrimethylsilane, PdCl
CuI, diethylamine, THF, reflux, 5 h, quant.; (b) KOH, MeOH, rt, 6 h, 78%; (c) B10
N,N-dimethylaniline, chlorobenzene, microwave, 130 °C, 37%; (d) BBr , CH Cl
overnight, 100%; (e) 7, NaH, THF, rt, 2 h, 72%; (f) H , Pd/C, MeOH–THF, rt, overnight,
4%.
2
(PPh
3
)
2
, PPh
H
2
, rt,
3
,
,
14
27
(
ETB) exhibited significant inhibition (ꢀ50%) at 6
inhibition was observed at 2 M. Moderate inhibition was observed
in the cases of compounds 1c and 1d, whereas inhibition ratio of
lM, a weak
3
2
l
2
5

G. Li et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2624–2628
2627
however that inhibited HSP60 chaperone activity with the IC50 of
.35 M. It is known that the stability and activity of HIF-1 are reg-
ulated not only by HSP60 but also by HSP90, which is main chap-
eron of HIF-1 . Therefore HIF-1 inhibition may be involved to some
0
l
a
3
2
a
degree in HSP60 inhibition and each inhibitory effect of the com-
pounds might be attributed to different mechanism respectively.
So far, compound 1h possesses the highest inhibitory activity among
compounds reported. Since the expression of HSP60 in relation to
disease progression, particularly tumor progression, has been inves-
tigated, we believe that the current findings of HSP60-binding small
molecules would contribute as a tool for investigation of further
unknown HSP60 functions and HSP60-related diseases.
Acknowledgments
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.
Figure 2. 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 M) and human
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) , and 4 mM ATP). Denatured
MDH was diluted to a concentration of 0.15 M with a buffer (0.1 M Tris/HCl, pH
.6, 7 mM KCl, 7 mM MgCl , 1 mM DTT, and 2 mM ATP) containing HSP60/10 and
each concentrated compound at 30 °C (final concentration of HSP60: 0.2 M,
HSP10: 0.4 M, compound: 2 M). After the reaction mixture was treated for
0 min, the reactivation of MDH by the HSP60/10 chaperon activity was determined
l
l
2
l
7
2
References and notes
l
l
l
1.
Heilman-Miller, S. L.; Pan, J.; Thirumalai, D.; Woodson, S. A. J. Mol. Biol. 2001,
309, 57.
3
+
by measuring the absorbance of NADH at 340 nm, which was converted into NAD
by the refolded MDH with absorption reduction. All compounds were confirmed to
2.
Schlesinger, M. J. J. Biol. Chem. 1990, 265, 12111.
2
5
3. Jindal, S.; Dudani, A. K.; Singh, B.; Harley, C. B.; Gupta, R. S. Mol. Cell. Biol. 1989,
, 2279.
have no inhibitory activity of MDH at 2 lM.
9
4
5
.
.
Bukau, B.; Horwich, A. L. Cell 1998, 92, 351.
Hansen, J. J.; Bross, P.; Westergaard, M.; Nielsen, M. N.; Eiberg, H.; Borglum, A.
D.; Mogensen, J.; Kristiansen, K.; Bolund, L.; Gregersen, N. Hum. Genet. 2003,
compound 1f was similar to that of GN26361. The best result was
observed in the case of compound 1h; 100% inhibition was observed
at 2 lM, indicating that the sterically bulky bis-ortho-carborane
moietyis more effectiveon inhibitionof theHSP60 chaperon activity
than the mono-ortho-carborane moiety. Therefore the dose-depen-
dent inhibition of HSP60 chaperon activity by compound 1h was
next examined. As shown in Figure 3, compound 1h inhibited
1
12, 71.
6
7
.
.
Cappello, F. J. Cancer Biol. Ther. 2008, 7, 801.
Bajramovic, J. J.; Geutskens, S. B.; Bsibsi, M.; Boot, M.; Hassankhan, R.; Verhulst,
K. C.; van Noort, J. M. Cell Stress Chaperones 2000, 5, 30.
Khalil, A. A. Cancer Sci. 2007, 98, 201.
Ciocca, D. R.; Calderwood, S. K. Cell Stress Chaperones 2005, 10, 86.
0. Nakamura, H.; Minegishi, H. Curr. Pharm. Des. 2013, 19, 441.
8
9
1
.
.
11. Cappello, F. J. Expert Opin. Ther. Targets 2014, 18, 185.
1
1
1
2. Semenza, G. L. Nat. Rev. Cancer 2003, 3, 721.
3. Ke, Q.; Costa, M. Mol. Pharmacol. 2006, 70, 1469.
4. Semenza, G. L. Curr. Opin. Genet. Dev. 2010, 20, 51.
15. Semenza, G. L. Oncogene 2010, 29, 625.
6. Wu, D.; Zhang, R.; Zhao, R.; Chen, G.; Cai, Y.; Jin, J. PLoS One 2013, 8, e62014.
17. Ban, H. S.; Uto, Y.; Nakamura, H. Expert Opin. Ther. Pat. 2011, 21, 131.
ꢀ
70% of HSP60 chaperon activity at 0.6
IC50 was calculated to be 0.35 ± 0.08 M. So far, compound 1h is
the highest inhibitor of HSP60 chaperon activity among the reported
lM concentration and the
l
2
8
29
30
24
1
four compounds, ETB, mizoribine, EC3016, and GN26361.
In conclusion, we developed inhibitors31 of HIF transcriptional
1
8. Takeuchi, A.; Hori, M.; Sato, S.; Ban, H. S.; Kuchimaru, T.; Kizaka-Kondoh, S.;
activity and HSP60 chaperon activity based on GN26361 as a lead
compound. Among the compounds synthesized, 3-(ortho-carbo-
ranyl)phenoxypropenanilide 1d suppressed HIF-1 transcriptional
activity with the IC50 of 0.53 lM, although the inhibition of HSP60
chaperone activity was not as high as that of GN26361. In contrast,
the inhibition of HIF-1 transcriptional activity of 3,5-(di-ortho-
carboranyl)phenoxyacetanilide 1h with IC50 of 16.4 ± 0.6 lM;
Yamori, T.; Nakamura, H. MedChemComm 2012, 3, 1455.
19. Shimizu, K.; Maruyama, M.; Yasui, Y.; Minegishi, H.; Ban, H. S.; Nakamura, H.
Bioorg. Med. Chem. Lett. 2010, 20, 1453.
0. Nakamura, H.; Yasui, Y.; Maruyama, M.; Minegishi, H.; Ban, H. S.; Sato, S.
Bioorg. Med. Chem. Lett. 2013, 23, 806.
21. Minegishi, H.; Matsukawa, T.; Nakamura, H. ChemMedChem 2013, 8, 265.
2
2
2
2
2
2. Nakamura, H.; Tasaki, L.; Kanoh, D.; Sato, S.; Ban, H. S. Dalton Trans. 2014, 43,
941.
3. Minegishi, H.; Fukashiro, S.; Ban, H. S.; Nakamura, H. ACS Med. Chem. Lett. 2013,
, 297.
4. Ban, H. S.; Shimizu, K.; Minegishi, H.; Nakamura, H. J. Am. Chem. Soc. 2010, 132,
1870.
4
4
1
5. Nail, R.; Won, M.; Kim, B. K.; Xia, Y.; Choi, H. K.; Jin, G.; Jung, Y.; Kim, H. M.; Lee,
K. J. Med. Chem. 2012, 55, 10564.
2
2
2
6. Cong, Z.-Q.; Nishino, H. Synthesis 2008, 2686.
7. Nielsen, K. L.; Cowan, N. J. Mol. Cell 1998, 2, 93.
8. Nagumo, Y.; Kakeya, H.; Shoji, M.; Hayashi, Y.; Dohmae, N.; Osada, H. Biochem.
J. 2005, 387, 835.
2
3
3
9. Itoh, H.; Komatsuda, A.; Wakui, H.; Miura, A. B.; Tashima, Y. J. Biol. Chem. 1999,
2
74, 35147.
0. Chapman, E.; Farr, G. W.; Furtak, K.; Horwich, A. L. Bioorg. Med. Chem. Lett.
009, 19, 811.
1. Spectral data for representative compounds: 3-[(3-(1-Ethyl-1,2-dicarba-closo-
carboranyl)phenoxyacetylamino)]-4-hydroxy benzene boronic acid (1c): 1
NMR (500 MHz; CD OD): d 8.23–8.30 (m, 1H), 7.31–7.43 (m, 4H), 7.20 (dd,
J = 7.0 Hz, 1.5 Hz, 1H), 6.81–6.87 (m, 1H), 4.85 (s, 2H), 1.87 (q, J = 7.5 Hz, 2H),
2
H
3
1
1
1
1
4
.00 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl
3
, 125 MHz): d 168.6, 159.1, 150.6, 133.5,
32.9, 131.5, 128.5, 125.9, 125.8, 119.3, 118.2, 115.6, 85.0, 84.6, 68.8, 29.7,
4.1. IR (NaCl) 3382, 3285, 2925, 2856, 2588, 2359, 1779, 1604, 1431, 1339,
ꢁ
1
+
200, 757 cm
;
HRMS (ESI) m/z Calcd for
C
18
H
28
B
11NO
5
Na [M+Na] :
80.2966. Found: 480.2965. HPLC purity (CH
2
Cl /MeOH = 20:1): 98.2%
2
(
254 nm), retention time: 7.56 min. (E)-3-[(4-(1,2-Dicarba-closo-carboranyl)-
1
phenoxyacryloylamino]-4-hydroxybenzene boronic acid pinacol ester (1d):
H
Figure 3. Dose-dependent inhibition of human Hsp60 chaperone activity by
compound 1h. IC50 value was calculated to be 0.35 ± 0.08 M.
NMR (500 MHz; CDCl ): d 9.92 (s, 1H), 7.87 (d, J = 9.0 Hz, 2H), 7.57 (dd,
3
l

2
628
G. Li et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2624–2628
J = 8.0 Hz, 1.5 Hz, 1H), 7.53 (d, J = 9.0 Hz, 2H), 7.39 (s, 1H), 7.52 (d, J = 1.5 Hz,
H), 7.05 (d, J = 9.0 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 5.80 (d, J = 11.5 Hz, 2H), 3.93
3H), 6.81–6.88 (m, 3H), 4.92 (s, 1H), 4.07 (t, J = 6.0 Hz, 2H), 2.61 (t, J = 7.0 Hz,
2H), 2.13–2.18 (m, 2H); 13C NMR (CDCl
, 125 MHz): d 174.4, 161.5, 151.9,
133.2, 130.5, 130.1, 127.1, 126.3, 116.8, 115.6. IR (NaCl) 3296, 3065, 2935,
2
3
s, 1H), 1.32 (s, 12H); 1 C NMR (CDCl
3
, 125 MHz): d 165.7, 157.5, 156.8, 152.0,
34.2, 130.0, 129.7, 129.0, 125.0, 119.8, 117.8, 104.0, 83.8, 75.6, 60.5, 24.8. IR
(
1
3
ꢁ
1
2596, 1513, 1338, 1256 cm ; HRMS (ESI) m/z Calcd for C18H B11NO Na
28 5
ꢁ
1
+
(
NaCl) 2978, 2924, 2852, 2598, 2359, 2345, 1681, 1544 cm ; HRMS (ESI) m/z
[M+Na] : 480.2966. Found: 480.2964. HPLC purity (AcOEt): 95.5%
+
Calcd for C23
CH Cl /MeOH = 20:1): 100.0% (254 nm), retention time: 7.10 min. 3-[(4-(1,2-
Dicarba-closo-carboranyl)phenoxypropanamido)]-4-hydroxybenzene boronic
acid (1e): 1H NMR (500 MHz; CD
OD): d 7.99 (s, 1H), 7.46 (d, J = 9.0 Hz, 2H),
.31 (dd, J = 8.0 Hz, 1.5 Hz, 1H), 6.85 (d, J = 9.0 Hz,1H), 4.95 (s, 1H), 4.33 (t,
H
34
B
11NO
5
Na [M+Na] : 546.3435. Found: 546.3438. HPLC purity
(254 nm), retention time: 4.83 min. 3-[(3,5-Di(1,2-dicarba-closo-carboranyl)
(
2
2
phenoxyacetylamino]-4-hydroxy benzene boronic acid pinacol ester (1h): 1
H
NMR (500 MHz; CDCl
1H), 7.46 (d, J = 1.5 Hz, 1H), 7.35 (t, J = 2.0 Hz, 1H), 7.17 (d, J = 2.5 Hz, 2H), 7.08
(d, J = 10.0 Hz, 1H), 4.69 (s, 2H), 3.96 (s, 2H), 1.36 (s, 12H); 13C NMR (CDCl
3
): d 9.12 (s, 1H), 8.38 (s, 1H), 7.66 (dd, J = 10.0 Hz, 1.5 Hz,
3
7
3
,
1
3
J = 6.0 Hz, 2H), 2.89 (t, J = 6.0 Hz, 2H); C NMR (CDCl
3
, 125 MHz): d 171.9,
61.2, 151.6, 133.0, 130.1, 129.9, 127.5, 126.3, 116.4, 115.7, 115.6, 78.4, 65.5,
2.5, 62.4, 37.6. IR (NaCl) 3309, 2930, 2594, 1540, 1512, 1338, 1254, 1186,
125 MHz): d 166.1, 156.6, 151.8, 136.3, 135.0, 128.9, 123.7, 121.5, 120.0, 115.8,
1
6
7
4
84.0, 74.4, 67.1, 60.0, 24.9. IR (NaCl) 2979, 2924, 2596, 2359, 2341, 1670, 1594,
ꢁ
1
1506, 1360, 1241, 1141, 1116 cm ; HRMS (ESI) m/z Calcd for C24
H
45
B
21NO
5
ꢁ
1
+
+
55 cm ; HRMS (ESI) m/z Calcd for C17
H
27
B11NO
5
[M+H] : 444.2990. Found:
[M+H] : 654.5407. Found: 654.5409. HPLC purity (CH
(254 nm), retention time: 5.28 min.
2 2
Cl /MeOH = 20:1): 99.7%
44.2992. HPLC purity (AcOEt): 95.6% (254 nm), retention time: 4.80 min. 3-
[
(4-(1,2-Dicarba-closo-carboranyl)phenoxybutanamido)]-4-hydroxybenzene
32. Liu, Y. V.; Baek, J. H.; Zhang, H.; Diez, R.; Cole, R. N.; Semenza, G. L. Mol. Cell
2007, 25, 207.
1
3
boronic acid (1g): H NMR (500 MHz; CD OD): d 7.88 (s, 1H), 7.31–7.45 (m,