Boron-containing phenoxyacetanilide derivatives as hypoxia-inducible factor (HIF)-1α inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.12.037

A series of boron-containing phenoxyacetanilide derivatives 8a-f, 9a-f, 15, and 16 were synthesized as hypoxia-inducible factor (HIF)-1α inhibitors. Among the compounds synthesized, carboranylphenoxyacetanilide 16 (GN26361) was found to be a potent inhibitor against HIF-1α accumulation under hypoxic conditions and inhibited the hypoxia-induced HIF-1 transcriptional activity in HeLa cells (IC₅₀ = 0.74 μM). Compound 16 suppressed hypoxia-induced HIF-1α accumulation and vascular endothelial growth factor mRNA expression in a concentration-dependent manner without affecting the expression of HIF-1α mRNA.

Full text of DOI:10.1016/j.bmcl.2009.12.037

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1453–1456
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Boron-containing phenoxyacetanilide derivatives as hypoxia-inducible factor
(HIF)-1a inhibitors
Kazuki Shimizu, Minako Maruyama, Yuka Yasui, Hidemitsu Minegishi, Hyun Seung Ban,
*
Hiroyuki Nakamura
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 boron-containing phenoxyacetanilide derivatives 8a–f, 9a–f, 15, and 16 were synthesized as
Received 31 October 2009
Revised 7 December 2009
Accepted 9 December 2009
Available online 6 January 2010
hypoxia-inducible factor (HIF)-1
acetanilide 16 (GN26361) was found to be a potent inhibitor against HIF-1
conditions and inhibited the hypoxia-induced HIF-1 transcriptional activity in HeLa cells (IC50 = 0.74
Compound 16 suppressed hypoxia-induced HIF-1 accumulation and vascular endothelial growth factor
a
inhibitors. Among the compounds synthesized, carboranylphenoxy-
accumulation under hypoxic
M).
a
l
a
mRNA expression in a concentration-dependent manner without affecting the expression of HIF-1
mRNA.
a
Keywords:
Hypoxia inducible factor (HIF)-1
Inhibitor
a
Ó 2009 Elsevier Ltd. All rights reserved.
Carborane
Boronic acid
Angiogenesis
HeLa cells
The formation of new blood vessels sprouting from existing
host capillaries (angiogenesis), is a necessary process for tumors
to grow beyond a certain critical size.1,2 Specific inhibition of this
tumor-induced angiogenesis prevents growth of many types of so-
lid tumors and provides a novel approach for cancer treatment.
Angiogenesis factors such as vascular endothelial growth factor
(VEGF) and erythropoietin (EPO) are key growth factors in tumor
angiogenesis.3 Various approaches have been studied for inhibition
of their signal transduction to prevent angiogenesis and suppress
tumor growth.4,5
VEGF and EPO.6 HIF-1
a
has been found in a wide variety of human
primary tumors compared with corresponding normal tissue.11–16
HIF-1 is considered to be a potential target for antineoplastic ther-
apy.17,18 Various HIF-1 inhibitors that block HIF-1 activation under
hypoxic conditions have been reported (Fig. 1). Topotecan19 and
PX-47820,21 are undergoing phase I/II clinical trials on patients
expressing high levels of HIF-1a
in their tumors.22 YC-1, originally
developed as a potential therapeutic agent for circulation disor-
ders, is in the lead-optimization stage.23 Recently, AC1-001 was
developed by Lee and co-workers,24 and its benzimidazole analog
(AC-004) has been reported to exhibit antitumor activity in vivo.25
Hypoxia-inducible factors (HIF) are heterodimeric (
scriptional factors and major physiological stimuli for expression
of angiogenesis factors.6–8 The levels of HIF-1
are low under
a/b) tran-
a
NMe2
normal oxygen conditions (normoxia) but increase in response to
hypoxia. HIF-1b, also known as the arylhydrocarbon nuclear trans-
locator,9 is a constitutively expressed nuclear protein. Under nor-
OH
HO
NH2
OH
O
O
N
+
Cl
N
N
N
O
-O
Cl
N
O
moxia, HIF-1a protein undergoes hydroxylation at specific prolyl
HCl
residues by prolyl hydroxylase (PHD). This modification is recog-
nized by the von Hippel–Lindau tumor suppressor protein for rapid
degradation by the ubiquitin–proteasome system.10 Under hypox-
O
Me
topotecan
PX-478
MeO2C
YC-1
CO2H
ia, the hydroxylation by PHD is compromised and HIF-1a is suffi-
H
N
H
N
O
ciently stabilized 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
O
O
N
N
H
H
OH
GN6767
AC1-001
Figure 1. Structure of HIF-1 inhibitors.
* Corresponding author.
E-mail address: hiroyuki.nakamura@gakushuin.ac.jp (H. Nakamura).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.037

1454
K. Shimizu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1453–1456
X
X
I
We also found that 1-[4-(N-benzylamino)phenyl]-3-phenylurea
derivatives (e.g., GN6767) inhibit the hypoxia-induced HIF-1 tran-
scriptional activity and suppress the secretion of VEGF without
a
d
O2
N
O2N
H2N
affecting the level of HIF-1a
mRNA.26
OH
OBn
OBn
We studied a boron-based medicinal drug design.27–30 A boron
atom has a vacant orbital and readily interconverts between the
neutral sp2 and anionic sp3 hybridization states. This generates a
new stable interaction between a boron atom and a donor mole-
cule through a covalent bond. The X-ray crystal structure of 20S
proteasome in a complex with bortezomib revealed that the boro-
nic acid of bortezomib covalently interacts with the Thr-1 hydroxyl
group in the active site of the 20S proteasome, forming a hybrid-
ized borate.31 A B–H–B three-center two-electron bond results in
stable formation of various boron clusters. In particular, carboranes
(dicarba-closo-dodecaboranes: C2B10H12) exhibit remarkable ther-
mal stability, and their icosahedral geometry and exceptional
hydrophobicity may allow their use as hydrophobic pharmaco-
phores in biologically active molecules that interact with target
proteins.32–36 In this study, we focused on the structure of AC1-
001 and synthesized boron-containing phenoxyacetanilide deriva-
4a X = Br
:
7
b
X = I
:
b
O
O
O
O
B
B
c
H2N
O2N
OBn
OH
5
6
Scheme 2. Reagents and conditions: (a) BnBr, K2CO3, acetone, reflux, 4a: 85%, 4b:
99%; (b) pinacolatodiboron, PdCl2dppf, AcOK, dioxane, 80 °C, 93% from 4a; (c) Pd/C,
H2, EtOH, 87%; (d) Fe, HCl, EtOH–H2O, reflux, 74% from 4b.
HO
OH
O
O
tives as HIF-1a inhibitors.
B
B
b
a
Chemistry: We first examined introduction of boronic acid into
the benzene ring instead of carboxylic acid. As shown in Scheme 1,
phenols 1a–g having various substituents at the para position were
treated with ethyl chloroacetate. The resulting esters 2a–g were
hydrolyzed with lithium hydroxide to give the corresponding aryl-
oxyacetic acids 3a–g in high yields.
3a e
-
O
O
O
O
N
N
H
H
OH
OH
R = CH3
R
R
8a:
b:
9a: R = CH3
R = CH2CH3
b: R = CH2CH3
c: R = Ph
c: R = Ph
d: R = Cy
R = Cy
d:
e:
e: R = t-Bu
R = t-Bu
Benzyl protection of 4-bromo-2-nitrophenol and 4-iodo-2-
nitrophenol was carried out using benzyl bromide to give the
benzyl ethers 4a and 4b (Scheme 2). The Suzuki–Miyaura-type
diboron coupling reaction of 4a proceeded in the presence of
dichloro(diphenylphosphino-ferrocene)palladium(II) (PdCl2dppf)
catalyst to afford 5 in 93% yield. Hydrogenation of 5 gave anilin-
oboronic acid pinacol ester 6 in 87% yield. Anilines 7 was also de-
rived from 4b by treatment with iron under acidic conditions.
Carboxylic acids 3a–e were reacted with 6 using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI) and hyroxybenzotriaz-
ole (HOBT) to give the corresponding aryloxyacetoanilides 8a–e
in 62–78% yields. Diisopropylethylamine (DIPEA) was an effective
base for these amide formations. Finally, boronic acids 9a–e were
obtained in 22–63% yields from the corresponding pinacol esters
8a–e by treatment with BBr3 in dichloromethane at 0 °C (Scheme
3).
In the case of 4-adamantylphenoxyacetic acid 3f, we first intro-
duced the aniline moiety 7b into 3f using ethyl chloroformate
(ECF) and N-methylmorphorine (NMM). The resulting anilide 10
underwent a Suzuki–Miyaura-type diboron coupling reaction fol-
lowed by hydrogenation to give the boron ester 8f in 63% yield
in two steps. Deprotection of pinacol ester was carried out using
BBr3 to give the corresponding boronic acid 9f in 72% yield
(Scheme 4).
Scheme 3. Reagents and conditions: (a) 6, EDCI, HOBT, DIPEA, 62–78%; (b) BBr3,
CH2Cl2, 0 °C, 22–63%.
O
O
B
I
b, c
a
O
O
3f
O
O
N
H
N
H
OBn
OH
OH
8f
10
HO
B
d
O
O
N
H
OH
9f
Scheme 4. Reagents and condition: (a) 7, ECF, NMM, TEA, THF, À20 oC, 72%; (b)
pinacolatodiboron, PdCl2(dppf), AcOK, DMF, 80 °C, 63%; (c) Pd/C, H2,EtOH, 99%; (d)
BBr3, CH2Cl2, 0 °C, 72%.
We next synthesized a carborane-containing phenoxyacetani-
lide derivative. The Sonogashira coupling reaction of 2g with ethy-
nyltrimethylsilane proceeded in the presence of PdCl2(PPh3)2 and
CuI catalysts at 120 °C in a sealed vial under microwave irradiation
conditions to give the corresponding coupling product in 93% yield.
Removal of the tetramethylsilyl group and ester hydrolysis pro-
ceeded by treatment of LiOH in aqueous THF solution. The result-
ing carboxylic acid 11 was converted to benzyl ester 12. The
decaborane coupling reaction of 12 proceeded in the presence of
CH3CN as a Lewis base under toluene refluxed condition to afford
the corresponding carborane 13 in 31% yield. Hydrogenation of
13 gave 4-ortho-carboranylphenoxyacetic acid 14 in 96% yield.
Amide bond formation of 14 with 6 proceeded using isobutyl chlo-
roformate (IBCF) and the resulting borate 15 was treated with KHF2
in methanol to give the boronic acid 16 (GN25361) in 83% yield
(Scheme 5).
O
O
OH
b
O
a
O
OEt
OH
R
R
R
1a: R = CH3
2
3
b:
R = CH2CH3
c: R = Ph
d: R = Cy
e:
R = t-Bu
f: R = adamantyl
g: R = I
Scheme 1. Reagents and conditions: (a) ClCH2CO2Et, K2CO3, DMF, rt, 90–99%; (b)
LiOH, THF/H2O, 95–97%.

K. Shimizu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1453–1456
1455
O
O
O
adamantly group resembles the carborane cage in molecular size
and shape.32 Therefore, we synthesized compounds 15 and 16,
which have an ortho-carboranyl group substituted at the para po-
sition of the benzene ring, and tested for the hypoxia-induced tran-
scriptional activity of HIF-1. The carborane substituent enhanced
the inhibition of the hypoxia-induced transcriptional activity of
HIF-1 (Table 1). The pinacol ester 15 displayed significant inhibi-
O
O
c
a, b
OH
OBn
2g
12
11
O
O
O
H
H
OBn
OH
OH
d
e
tion (1.7
lM), but the best result was obtained in carboranyl boro-
nic acid 16; the IC50 value was 0.74
l
M, which was one-fold lower
13
14
than that of AC1-001. In all cases, the pinacol ester derivatives
exhibited higher inhibition in comparison with their boronic acid
derivatives due to the transmembrane property of the more lipo-
philic pinacol ester functional group (except the carboranyl boro-
nic acid 16). HIF-1 inhibitors do not affect on the cell growth but
affect on suppression of angiogenic factors in cancer cells. There-
fore, cell growth inhibition of synthesized compounds was also
investigated, and the compound concentrations required for 50%
growth inhibition of HeLa cells are shown in Table 1. Although
compounds 8a and 8c displayed relatively higher inhibition of cell
HO
O
O
B
B
f
g
O
O
O
O
H
H
N
N
H
H
OH
OH
15
16 (GN26361)
Scheme 5. Reagents and conditions: (a) ethynyltrimethylsilane, PdCl2(PPh3)2, CuI,
DEA, DMF, microwave, 120 °C, 93%; (b) LiOH, THF–H2O, rt, 82%; (c) BnBr, Na2CO3,
DMF, rt, 93%; (d) B10H14, CH3CN, toluene, reflux, 31%; (e) Pd/C, H2, EtOH, 96%; (f) 6,
IBCF, 30%; (g) KHF2, MeOH, 83%.
growth (GI50 = 8.6 and 5.8
compounds were in the range of 12.1–85.9
We next examined the effects of compound 16 against hypoxia-
induced HIF-1 accumulation by western blot analysis and the
mRNA expression level of HIF-1 and VEGF by RT-PCR analysis in
HeLa cells.20 Compound 16 suppressed HIF-1
accumulation in a
lM, respectively), GI50 values of other
lM.
a
Biological activity: The synthesized boron-containing phenoxy-
acetanilide derivatives 8a–f, 9a–f, 15, and 16 were tested for the
hypoxia-induced transcriptional activity of HIF-1 using a cell-
based reporter assay in HRE (Â5)-Luc transfected HeLa cells and
cell growth inhibition (GI50) using the MTT assay. AC1-001 was
used as a positive compound for comparison. The results are sum-
marized in Table 1. The pinacol esters 8a–c and the boronic acids
9a–c did not show significant inhibition of the hypoxia-induced
a
a
concentration-dependent manner without affecting the expression
level of HIF-1b protein (Fig. 2a). RT-PCR analysis revealed that com-
pound 16 inhibited the hypoxia-induced expression of VEGF mRNA
in a concentration-dependent manner (Fig. 2b); however, the levels
of HIF-1
indicated that compound 16 inhibits the hypoxia-induced expres-
sion of VEGF via suppression of HIF-1 accumulation. We also
a mRNA were not affected by compound 16. These results
transcriptional activation by HIF-1 (IC50 P90 lM). Enhancement
of inhibition was observed along with bulky substituents at the
a
examined the effect of compound 16 on cell growth inhibition in
various cell lines, such as HeLa, colon 26 (mouse colon cancer),
B16 (mouse melanoma), and C6 (rat glioma). Cells were incubated
for 72 h with compound 16 and viable cells were determined by
MTT assay. Selective inhibition of cell growth by compound 16
para position of the benzene ring: cyclohexyl group (8d and 9d,
70.4 and 84.8
and 40.3 M, respectively), and adamantly group (8f and 9f, 3.1
and 4.6 M, respectively). The potency of compounds 8f and 9f
lM, respectively), tert-butyl group (8e and 9e, 35.3
l
l
were similar to that of AC1-001 (3.1 lM). It is considered that an
was observed in colon 26 cells (GI50 = 5.0
lM) whereas significant
inhibition was not observed at 10 M in other cell lines. The effi-
l
Table 1
cacy of the boronic acid group is presumably due to the Lewis acid-
ity. A boronic acid has a vacant p-orbital and can interact with
target protein(s) not only through hydrogen bonding but also cova-
lent bonding, which may cause enhanced biological activity,
although we do not have any evidence for the role of the boronic
acid group in the current study.31,37,38
In conclusion, carboranyl boronic acid 16 was found to be the
most potent inhibitor of hypoxia-induced HIF-1a protein accumu-
lation among the compounds synthesized. We confirmed that
compound 16 showed significant suppression of the hypoxia-in-
Inhibition of HIF-1 transcriptional activity in cell-based HRE reporter assay and cell
growth inhibition
a
b
Compd
IC50
(
l
M)
GI50 (lM)
8a
8b
8c
8d
8e
8f
9a
9b
9c
9d
9e
9f
90.6 2.67
91.8 8.24
>100
70.4 1.88
35.3 1.64
3.1 0.15
>100
17.7 0.66
8.6 0.46
5.8 0.82
13.4 0.53
12.1 0.67
16.0 0.47
25.7 0.21
14.9 0.23
85.9 0.73
29.2 6.73
21.3 0.40
15.2 1.13
52.4 2.49
16.2 0.70
51.6 0.89
>100
>100
duced transcriptional activity (IC50 = 0.74 lM) and expression of
84.8 5.53
40.3 6.77
4.6 0.86
1.7 0.58
0.74 0.24
3.1 0.07
A
B
15
HIF-1α
HIF-1α
16(GN26361)
AC1-001
VEGF
HIF-1β
16(μM)
O2 (%) 20
a
HeLa cells stably transfected with HRE-Luc were incubated for 12 h with or
without drugs under normoxic or hypoxic conditions. After removal of supernatant,
luciferase assay was performed using Luciferase Assay System (Promega,
-
-
0.3
1
1
3 10
GAPDH
16(μM)
-
-
3
1
10
a
a
Madison, WI, USA) according to manufacturer’s instructions. The drug concentra-
tion required to inhibit the relative light unit by 50% (IC50) was determined from
semi-logarithmic dose–response plots, and results represent the mean SD of
triplicate samples.
O2 (%) 20
Figure 2. Effect of compound 16 on hypoxia-induced accumulation of HIF-1
a
protein and expression of VEGF mRNA. HeLa cells were incubated for 4 h with or
without the indicated concentrations of compound 16 under hypoxic condition. (A)
b
HeLa cells were incubated for 48 h with various concentrations (100 nM to
100 lM) of compounds under normoxic condition, and viable cells were deter-
The levels of each protein were detected by immunoblot analysis with HIF-1
a- or
mined by MTT assay. The drug concentration required to inhibit cell growth by 50%
(GI50) was determined from semi-logarithmic dose–response plots, and results
represent the mean SD of triplicate samples.
HIF-1b-specific antibodies. HIF-1b was used as the loading control, and ‘–’ was
DMSO used as the control. (B) HIF-1a, VEGF, and GAPDH mRNA expression was
detected by RT-PCR.

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K. Shimizu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1453–1456
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a
a
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Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific
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38. Spectral data for lead compounds: 3-(4-Adamantyl-phenoxyacetylamino)-4-
hydroxybenzene boronic acid pinacol ester (8f): 1H NMR (400 MHz; CDCl3) d
9.56 (s, 1H), 8.57 (s, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.41 (s, 1H), 7.35 (d, J = 8.0,
2H), 7.06 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 2H), 4.66 (s, 2H), 2.10 (s, 3H),
1.90 (s, 6H), 1.73–1.82 (m, 6H), 1.34 (s, 12H); 13C NMR (75 MHz; CDCl3) d
168.4, 154.5, 151.9, 146.0, 134.6, 129.0, 126.3, 124.1, 119.8, 114.5, 83.8, 67.2,
43.3, 36.7, 35.7, 28.9, 24.8; IR(KBr) 3379.1, 2900.7, 2846.7, 2684.7, 2619.2,
2484.1, 1662.5, 1562.2, 1512.1, 1076.2, 1033.8, 1010.6, 856.3, 829.3 cmÀ1; MS
(ESI, positive) m/z 504 ([M+H]+); Anal. Calcd for C30H38BNO5: C, 71.57; H, 7.61;
N, 2.78. Found: C, 71.25; H, 7.77; N, 2.75. 3-(4-Adamantylphenoxyace-
tylamino)-4-hydroxybenzene boronic acid (9f): 1H NMR (400 MHz; CD3OD) d
8.23 (d, J = 11.2 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.19 (d, J = 9.2 Hz, 2H), 6.85 (d,
J = 8.8 Hz, 2H), 6.71-6.76 (m, 1H), 4.50 (s, 2H), 1.93 (br s, 3H), 1.77 (s, 6H), 1.63-
1.71 (m, 6H); 13C NMR (75 MHz; CD3OD) d 169.2, 156.8, 151.0, 146.5, 132.7,
128.3, 127.9, 127.1, 125.8, 115.6, 68.9, 44.5, 37.9, 36.8, 30.5; IR(KBr) 2900.7,
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806.2 cm-1
;
MS (ESI, positive) m/z 436 ([M(OMe)+H]+); Anal. Calcd for
C24H28BNO5: C, 68.42; H, 6.70; N, 3.32. Found: C, 68.14; H, 6.59; N, 3.19. 3-
(4-o-Carboranylphenoxyacetylamino)-4-hydroxybenzene boronic acid pinacol
ester (15): 1H NMR (400 MHz; CDCl3) d 9.28 (s, 1H), 8.43 (s, 1H), 7.63 (d,
J = 8.4 Hz, 1H), 7.52 (d, J = 9.2, 2H), 7.42 (s, 1H), 7.06 (d, J = 7.6 Hz, 1H), 6.95 (d,
J = 8.0 Hz, 2H), 4.66 (s, 2H), 3.91 (br s, 1H), 1.34 (s, 12H); 13C NMR (75 MHz;
CDCl3) d 167.3, 157.7, 151.8, 134.8, 128.9, 127.9, 123.9, 119.9, 114.9, 83.9, 67.1,
60.7, 24.9; IR(KBr) 3375.2, 2738.7, 2572.9, 2360.7, 1651.0, 1512.1, 1072.3,
1026.1, 1002.9, 964.3, 925.8, 879.5, 833.2 cmÀ1; Anal. Calcd for C22H34B11NO5:
C, 51.67; H, 6.70; N, 2.74. Found: C, 51.51; H, 6.56; N, 2.95. 3-(4-o-
Carboranylphenoxyacetylamino)-4-hydroxybenzene boronic acid (16): 1H
NMR (400 MHz; CD3OD) d 8.17 (d, J = 12.4 Hz, 1H), 7.44 (d, J = 9.2 Hz, 2H),
7.20–7.33 (m, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.71–6.77 (m, 1H), 4.92 (br s, 1H),
4.62 (s, 2H); 13C NMR (75 MHz; CD3OD) d 168.4, 160.0, 150.6, 132.9, 130.3,
128.6, 125.8, 116.1, 115.5, 78.1, 68.6, 62.4; IR(KBr) 3066.6, 2927.7, 2866.0,
2603.7, 2341.4, 1882.4, 1670.2, 1512.1, 1188.1, 1122.5, 1072.3, 1022.2, 972.1,
918.1, 879.5, 837.0 cmÀ1; MS (ESI, negative) m/z 428 ([MÀH]À); Anal. Calcd for
C16H24B11NO5: C, 44.77; H, 5.64; N, 3.26. Found: C, 44.57; H, 5.47; N3.23.
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