1-[4-(N-Benzylamino)phenyl]-3-phenylurea derivatives as a new class of hypoxia-inducible factor-1α inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.04.122

A series of 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives 2a-r were synthesized as HIF-1α inhibitors. Among the compounds synthesized, compound 2k was found to be a potent inhibitor against HIF-1α accumulation under hypoxic condition and inhibited the hypoxia-induced HIF-1 transcriptional activity in HEK293 cells (IC₅₀ = 7.2 μM). Furthermore, compound 2k suppressed the hypoxia-induced secretion of VEGF in HeLa cells (IC₅₀ = 15 μM).

Full text of DOI:10.1016/j.bmcl.2009.04.122

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3166–3169
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1-[4-(N-Benzylamino)phenyl]-3-phenylurea derivatives as a new class
of hypoxia-inducible factor-1
a
inhibitors
*
Masaharu Uno, 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 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives 2a–r were synthesized as HIF-1
a
Received 3 April 2009
Revised 23 April 2009
Accepted 25 April 2009
Available online 3 May 2009
inhibitors. Among the compounds synthesized, compound 2k was found to be a potent inhibitor against
HIF-1 accumulation under hypoxic condition and inhibited the hypoxia-induced HIF-1 transcriptional
activity in HEK293 cells (IC50 = 7.2 M). Furthermore, compound 2k suppressed the hypoxia-induced
secretion of VEGF in HeLa cells (IC50 = 15 M).
a
l
l
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Hypoxia
1-[4-(N-Benzylamino)phenyl-3-phenylurea
derivatives
Hypoxia-inducible factor (HIF)-1a inhibitor
Angiogenesis
VEGF
Angiogenesis is a necessary process for tumors to grow beyond
a certain size.1 The growth of tumoral blood vessels supplies oxy-
gen and nutrients to small tumors, and promotes metastasis. Angi-
ogenesis factors, such as vascular endothelial growth factor (VEGF)
and erythropoietin (EPO), are key growth factors in tumor angio-
genesis,2 therefore, various approaches have been studied for inhi-
bition of these signal transduction to prevent angiogenesis and
suppress tumor growth.3 The major physiological stimulus for
VEGF expression is hypoxia, which induces transcription of the
VEGF gene by hypoxia-inducible factor (HIF)-1. HIF-1 is known
as a heterodimeric complex consisting of a hypoxically inducible
sponding normal tissue,7a–f therefore, HIF-1 has been considered
as an important target for development of anticancer agents.8 As
shown in Figure 1, various HIF-1 inhibitors that block HIF-1 activa-
tion under hypoxic condition has been reported, such as YC-1,9
topotecan,10 PX-478,11 and compound 1.12 YC-1 and topotecan
are currently undergoing phase I clinical trials on patients express-
ing high levels of HIF-1
We have screened a chemical library in our laboratory for com-
pounds that can inhibit HIF-1 accumulation in HeLa cells under
a
in their tumors.13
a
hypoxic condition by Western blotting analysis and found that
the 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivative 2b has
subunit HIF-1
a
and a constitutively expressed subunit HIF-1b.
a potential for block of HIF-1
cation based on the structure of compound 2b, we finally discov-
ered compound 2k as a new class of the HIF-1 inhibitor.
As shown in Scheme 1, 4-nitroaniline was treated with Boc2O,
and the protected aniline 4 was converted the diamine 5 by hydro-
genation. Urea formation of 5 with phenylisocyanate, which was
prepared from aniline and triphosgene in situ, followed by depro-
tection of the Boc group with trifluoroacetic acid afforded 1-(4-
aminophenyl)-3-phenylurea 7.14
a accumulation. With further modifi-
HIF-1b is also known as the arylhydrocarbon nuclear translocator
(ARNT), which was originally identified as a binding partner of
the aryl hydrocarbon receptor.4 Under normoxic conditions, HIF-
a
1a protein is subject to oxygen-dependent prolyl hydroxylation,
which leads to rapidly degradation by von Hippel–Lindau tumor
suppressor protein (pVHL)-mediated ubiquitin-proteasome system
(UPS).5 Under hypoxic conditions, oxygen becomes limiting for
prolyl hydroxylase (PHD) activity and HIF-1
a is not degradated
by UPS. As a result, stabilized HIF-1 binds HIF-1b to form a hete-
rodimeric complex, which binds to the hypoxia response element
a
Reductive amination of 7 with various aldehydes 8 was carried
out by using NaCNBH3 in MeOH to give 1-[4-(N-benzyl-
(HRE) DNA sequence with co-activators to activate various genes
amino)phenyl]-3-phenylurea derivatives
2 in 14–99% yields
including VEGF and EPO.6 HIF-1
a
is found at increased levels in a
(Scheme 2 and Table 1). The aldehydes 8j, 8q and 8r were prepared
from 8j, 3-formyl-4-hydroxybenzoic acid and trimesic acid, respec-
tively.15,16 Although esterification of 2h and 2i in methanol gave
the corresponding methyl esters 2k and 2l, respectively, 2g under-
went cyclization under this condition to give 9 in 99% yield.
wide variety of human primary tumors compared with corre-
* Corresponding author. Tel.: +81 339860221; fax: +81 359921029.
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.2009.04.122

M. Uno et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3166–3169
3167
The synthesized 1-[4-(N-benzylamino)phenyl]-3-phenylurea
derivatives 3a–r were tested for inhibitory effect on HIF-1 accu-
mulation in HeLa and HCT116 cells at a 30 M concentration of
compounds under hypoxic conditions (1% O2, 94% N2, and 5%
CO2) by Western blot analysis. YC-17 was used as a positive control
for comparison. Among compounds 2a–f which have either hydro-
xy or methoxy groups on the benzyl moiety of molecules, com-
H
N
H
N
R
R
a
a
7
+
l
O
CHO
N
H
8
2
j
8a : o-OH
b : m-OH
c : p-OH
j
: o
2a : o-OH
b : m-OH
c : p-OH
d : o-OMe
-COOMe
: o-COOMe
: m-COOMe
: p-COOMe
k
l
m : 2,6-di-OMe
n : 3,5-di-OMe
o : 2,3-di-OMe
d : o-OMe
m : 2,6-di-OMe
n : 3,5-di-OMe
o : 2,3-di-OMe
pounds 2d and 2e exhibited relatively potent HIF-1
a inhibition
e
p
q
r
e
: m-OMe
: 3,5-di-OMe
: m-OMe
b
: p-OMe
: 2-
: p-OMe
OMe, 5-COOMe
f
f
and the inhibitory potency of them was almost the same as that
observed by YC-1 in HeLa and HCT116 cells under hypoxic condi-
tion (Fig. 2a and b). Among the carboxylic acids and their methyl
g
g
p
q
: 3,5-di-COOMe
: 3,5-di-OMe
: o-COOH
: o-COOH
b
: 2-OMe, 5-COOMe
h : m-COOH
h : m-COOH
: p-COOH
: p-COOH
r : 3,5-di-COOMe
i
i
H
N
H
N
esters (2g–l), compound 2k showed the most potent HIF-1a inhi-
bition under hypoxic condition; the corresponding ortho- and para-
isomers (2j and 2l) and carboxylic acids 2g–i were less potent than
compound 2k (Fig. 2c and d). Disubstituted derivatives 2m–r were
O
N
O
9 (99%)
also tested for inhibitory effect on HIF-1a accumulation under hyp-
Scheme 2. Reagents and conditions: (a) NaCNBH3, MeOH, reflux; (b) MeOH, cat.
H2SO4, reflux.
oxic condition. Compound 2o, which has 2,3-dimethoxy groups on
the benzyl moiety, showed a similar inhibition to compounds 2d
and 2k in HeLa cells, although the other disubstituted compounds
showed less potent (Fig. 2e and f). From these results, compound
Table 1
Chemical yields and in vitro inhibition of HIF-1 transcriptional activity in cell-based
HRE reporter assay
2k possesses the highest HIF-1
synthesized, and the inhibitory potency is higher than that of YC-1
[2k: density ratio (HIF-1 b) = 0.39 (HeLa) and 0.39 (HCT116) ver-
sus YC-1: density ratio (HIF-1 b) = 0.47 (HeLa) and 0.52 (HCT116)].
a inhibition among the compounds
a
H
N
H
N
R
a
To confirm the inhibition of transcriptional activity under hyp-
oxic condition, we have investigated the effect of synthesized com-
pounds on hypoxia-induced transcriptional activation of HIF-1
using cell-based reporter assay in recombinant Human Embryonic
Kidney (HEK) 293 cells. YC-1 was used as a positive compound for
O
N
H
2a-r
a
R
Compd
Yield (%)
IC50/lM
a comparison and the IC50 value was 9.24 lM in HEK293 cells.
Compounds 2a–h, 2j, 2m, 2n, and 2p–r showed moderate inhibi-
tion of hypoxia-induced transcriptional activation by HIF-1 (15–
o-OH
m-OH
p-OH
o-OMe
m-OMe
p-OMe
o-COOH
m-COOH
p-COOH
o-COOMe
m-COOMe
p-COOMe
2, 6-OMe
3,5-OMe
2,3-OMe
2,5-OMe
2-OMe, 5-COOMe
3,5-COOMe
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
3r
53
14
14
40
36
51
25
57
32
46
53b
97b
30
38
99
99
45
78
32.7 5.72
69.9 11.9
35.5 8.06
17.4 4.74
52.1 3.29
23.0 9.03
51.7 1.96
41.6 3.22
>100
32.5 20.9
7.21 0.67
>100
39.1 5.72
18.7 0.57
8.62 2.06
29.6 1.85
22.1 7.52
15.1 2.32
9.21 3.24
70
not exhibit inhibitory activity at a 100
pounds. Compounds 2k and 2o, which showed potent HIF-1
l
M), and para-carboxylic acid 2i and its methyl ester 2l did
M concentration of com-
inhi-
l
a
bition in HeLa cells under hypoxic condition, also exhibited
significant inhibition of hypoxia-induced transcriptional activation
NMe2
OH
HO
O
O
N
N
N
N
O
HCl
O
YC-1
Me
a
topotecan
YC-1
The cells were incubated for 16 h with or without drugs under normoxic or
hypoxic condition. After removal of supernatant, the luciferase assay was per-
formed using Luciferase Assay System (Promega, Madison, WI) according to the
manufacturer’s instructions. The drug concentration required to inhibit RLU (Rel-
ative Light Unit) by 50% (IC50) was determined from semilogarithmic concentra-
tion–response plots and results represent the mean SD of triplicate samples.
COOMe
OH
NH2
OH
O
Cl
+
O
N
O
-O
Cl
N
H
b
Yields of esterification from 2h or 2i.
1
PX-478
by HIF-1, and their IC50 values were 7.21 and 8.62
(Table 1).
lM, respectively
Figure 1. Structures of HIF-1 inhibitors.
We next examined concentration-dependent inhibition against
hypoxia-induced HIF-1 accumulation by western blot analysis
and mRNA expression level of HIF-1 by RT-PCR analysis in HeLa
cells.10 As shown in Figure 3a, compound 2k suppressed HIF-1
a
NO2
NO2
NH2
a
b
c
a
a
H2N
BocHN
BocHN
accumulation in a concentration-dependent manner without affect-
3
4
5
ing the expression level of HIF-1b protein. However, 2k did not sup-
press the level of HIF-1a mRNA (Fig. 3b). The effect of compound 2k
on hypoxia-induced VEGF secretion was also measured using ELISA.
As shown in Figure 4, compound 2k significantly inhibited the hy-
poxia-induced secretion of VEGF at a concentration-dependent
H
N
H
H
N
H
d
N
N
O
O
H2N
BocHN
6
7
Scheme 1. Reagents and conditions: (a) Boc2O, CH2Cl2, reflux, 99%; (b) H2, Pd/C,
MeOH, 58%; (c) phenylisocyanate, toluene, reflux, 99%; (d) TFA, CH2Cl2, reflux, 91%.
manner, and the IC50 value was calculated as 15 lM.

3168
M. Uno et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3166–3169
Figure 2. Relative ratios of HIF-1
a
and -1b levels by Western blot analyses at 30
lM concentration of compounds. The levels of each protein were detected by immunoblot
analysis with the HIF-1a or HIF-1b specific antibodies after 4 h-incubation of cells with compounds. The density ratios of HIF-1a protein level to HIF-1b were calculated, and
the mean value of density ratio in hypoxia control group was set to 1.0. Values are the means from triplicate experiments with the SEM shown by vertical bars. Statistical
significance: ###: P < 0.001 versus normoxia control; *: P < 0.05, **: P < 0.01, and ***: P < 0.001 versus hypoxia control.
10
###
IC50 = 15 μM
8
**
a
b
6
4
2
HIF-1α
HIF-1α
***
GAPDH
HIF-1β
-
-
-
-
1
3
10 30
3
10 30
2k (μM)
2k (μM)
O2 (%) 20
1
O2 (%) 20
1
0
2k (μM)
-
-
3
10
30
Figure 3. Effect of 2k on the accumulation of the HIF-1
a
protein and mRNA induced
O2 (%)
20
1
by hypoxia. (a) HeLa cells were incubated with compound 2k at different
concentrations under hypoxic condition for 4 h. HIF-1b was used as the loading
control and ‘–’ is DMSO as control. The levels of each protein were detected by
Figure 4. Compound 2k selectively inhibited hypoxia-induced secreted VEGF
protein in HeLa cells. The cells were incubated for 12 h with or without drugs.
Values are the means from triplicate experiments with the SEM shown by vertical
bars. Statistical significance: ###: P < 0.001 versus normoxia control; *: P < 0.05, **:
P < 0.01, and ***: P < 0.001 versus hypoxia control.
immunoblot analysis with the HIF-1
analysis for the effect of compound 2k on the hypoxia-induced mRNA level of HIF-
in HeLa cells for 2 h. GAPDH was used as the loading control and ‘–’ is DMSO as
control.
a or HIF-1b specific antibodies. (b) RT-PCR
1a

M. Uno et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3166–3169
3169
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the significant suppression of the hypoxia-induced transcriptional
activity (IC50 = 7.21
results suggest that 1-[4-(N-benzylamino)phenyl]-3-phenylurea
derivative 2k will be a new class of HIF-1 inhibitor. Further de-
inhibition by 2k is currently
lM) and VEGF secretion (IC50 = 15 lM). These
a
tailed mechanistic studies of HIF-1
a
under investigation in our group.
Acknowledgments
We thank Professor Hideko Nagasawa, Gifu Pharmaceutical
University, for providing us HEK293 cells stably transfected with
hypoxia response element (HRE)-Luc as well as useful suggestions
regarding cell-based HRE reporter assay. This work is supported by
the Ministry of Education, Science, Sports, Culture and Technology,
Grant-in-Aid for Scientific Research (B) (No. 18350090) from Japan.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.04.122.
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