Discovery of 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives as non-peptidic selective SUMO-sentrin specific protease (SENP)1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.06.084

We developed 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivative 4 (GN6958) as a non-peptidic selective SUMO-sentrin specific protease (SENP)1 protease inhibitor based on the hypoxia inducible factor (HIF)-1α inhibitor 1 (GN6767). The direct interaction of compound 1 with SENP1 protein in cells was observed by the pull-down experiments using the biotin-tagged compound 2 coated on the streptavidin affinity column. Among the various 1-[4-(N-benzylamino) phenyl]-3-phenylurea derivatives tested, compounds 3 and 4 suppressed HIF-1α accumulation in a concentration-dependent manner without affecting the expression level of tubulin protein in HeLa cells. Both compounds inhibited SENP1 protease activity in a concentration-dependent manner, and compound 4 exhibited more potent inhibition than compound 3. Compound 4 exhibited selective inhibition against SENP1 protease activity without inhibiting other protease enzyme activities in vitro.

Full text of DOI:10.1016/j.bmcl.2012.06.084

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5169–5173
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives as
non-peptidic selective SUMO-sentrin specific protease (SENP)1 inhibitors
⇑
Masaharu Uno, Yosuke Koma, Hyun Seung Ban, Hiroyuki Nakamura
Department of Chemistry, Faculty of Science, Gakushuin University, Mejiro, Toshima-ku, 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:
We developed 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivative 4 (GN6958) as a non-peptidic
Received 27 May 2012
Revised 22 June 2012
Accepted 26 June 2012
Available online 3 July 2012
selective SUMO-sentrin specific protease (SENP)1 protease inhibitor based on the hypoxia inducible fac-
tor (HIF)-1a inhibitor 1 (GN6767). The direct interaction of compound 1 with SENP1 protein in cells was
observed by the pull-down experiments using the biotin-tagged compound 2 coated on the streptavidin
affinity column. Among the various 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives tested, com-
pounds 3 and 4 suppressed HIF-1a accumulation in a concentration-dependent manner without affecting
Keywords:
SENP1
Non-peptidic inhibitors
HIF-1
deSUMOylation
Selective inhibition
the expression level of tubulin protein in HeLa cells. Both compounds inhibited SENP1 protease activity in
a concentration-dependent manner, and compound 4 exhibited more potent inhibition than compound 3.
Compound 4 exhibited selective inhibition against SENP1 protease activity without inhibiting other pro-
tease enzyme activities in vitro.
Ó 2012 Elsevier Ltd. All rights reserved.
Small ubiquitin-like modifier (SUMO), a protein that shares
about 18% sequence identity with ubiquitin, modulates many bio-
logical processes including nuclear transport, transcription, repli-
E2, and E3.9,10 On the contrary, the ‘deSUMOylation’ is promoted
by a family of SUMO/sentrin specific proteases (SENPs).11 In the
mammalian system, six SENPs (SENPs 1–3 and 5–7) have been re-
ported and, in particular, SENP1, a nuclear protease, deconjugates a
large number of SUMOylated proteins.12 For example, SENP1 has
been shown to regulate androgen receptor transactivation by tar-
geting histone deacetylase 1 and induce c-Jun activity through
deSUMOylation of p300.13,14 Moreover, SENP1 is overexpressed
in human prostate cancer specimens.9
cation,
recombination,
and
chromosome
segregation.1,2
Modification of proteins by SUMO is a dynamic and reversible pro-
cess and controlled by a series of on/off enzymes. ‘SUMOylation’,
the covalent interaction between the C-terminus of SUMO and
the e-amino group of a lysine residue in the target protein, is med-
iated by activating (E1),3,4 conjugating (E2),3,5 and ligating (E3) en-
zymes;6–8 however these are entirely distinct from ubiquitin E1,
H
O
HN
S
O
R2O
R1
N
O
O
N
H
H
N
O
N
H
H
HN
H3CO
O
H
N
H
N
N
H
O
O
1 (R1 = H, R2 = CH3; GN6767)
3 (R1 = CH3, R2 = CH3; GN6860)
N
H
N
H
1
2
2 (biotinated probe)
4
(R = CH3, R = C2H5; GN6958)
Figure 1. Structures of 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives 1–4.
⇑
Corresponding author. Tel.: +81 3 3986 0221; fax: +81 3 5992 1029.
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.2012.06.084

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M. Uno et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5169–5173
H
N
H
CH3
NH
NH2
N
NH2
a
b,c
a,b
O
O2N
O2N
H2N
NH2
O2N
9
5
CH3
N
O
H
N
d
3 or 4
H3CO
H
N
H
N
O
d
c
H2N
2
10
O
N
H
NH2
Scheme 2. Synthesis of the compounds
3 and 4. Reagents: (a) (i) 1-hydrox-
6
ymethylbenzotriazole, EtOH; (ii) NaBH4, THF; (b) (i) triphosgene, toluene; (ii)
aniline, toluene, reflux; (c) H2, Pd/C, MeOH, 2 steps 76%; (d) methyl 3-formylbenzo-
ate or ethyl 3-formylbenzoate, NaCNBH3, MeOH.
H
HN
S
O
of a hypoxically inducible subunit, HIF-1
pressed subunit, HIF-1b. Under normoxic conditions, HIF-1
a
, and a constitutively ex-
O
a
pro-
N
H
N
H
tein is subject to oxygen-dependent prolyl hydroxylation, leading
to rapid degradation by von Hippel–Lindau tumor suppressor pro-
tein (pVHL)-mediated ubiquitin-proteasome system (UPS).15 Un-
der hypoxic conditions, HIF-1
limited oxygen supply for prolyl hydroxylase (PHD) activity. The
stabilized HIF-1 binds to HIF-1b to form a heterodimeric complex,
H
O
O
NH
7:
R = H
O
RO
a is not degraded by UPS due to the
8: R =
N
O
a
which binds to the hypoxia response element (HRE) DNA sequence
with co-activators to activate various genes including angiogenesis
factors, such as vascular endothelial growth factor (VEGF) and
Scheme 1. Synthesis of the biotin-conjugated probe 2. Reagents: (a) (i) triphosgene,
toluene; (ii) 4-nitroaniline, toluene, reflux; (b) H2, Pd/C, MeOH, 2 steps 63%. (c)
methyl benzaldehyde-3-carboxylate, NaCNBH3, MeOH, 10%; (d) 8, cat. DMAP,
CHCl3, 43%.
erythropoietin (EPO).16 HIF-1
a is found at increased levels in a
wide variety of human primary cancers compared with corre-
sponding normal tissue. Therefore, HIF-1 has been considered an
important target for the development of anticancer agents.17–21
We recently reported 1-[4-(N-benzylamino)phenyl]-3-phenylu-
We have focused our efforts on the development of hypoxia-
inducible factor (HIF)-1 inhibitors as pathological angiogenesis
inhibitors. HIF-1 is known as a heterodimeric complex consisting
rea derivative GN6767 as a new class of HIF-1
a inhibitor (com-
Figure 2. Inhibition of SENP1 catalytic domain (SENP1-CD) endopeptidase activity by compounds 3 and 4. Compounds were titrated in HEPES buffer (50 mM HEPES, 0.1 mM
EDTA, pH 7.9), combined with SENP1-CD (3 nM), and incubated for 10 min in 96-well plates before assaying with the SUMO-1-AMC (300 nM). Fluorescence intensity was
plotted on a fluorescence plate reader (excitation/emission wavelengths 380/460 nm; Infinite F200; Tecan) for 30 min after adding the SUMO-1-AMC (a and b). Enzymatic
activity was determined as the relative protease activity 5 min after adding the SUMO-1-AMC (c and d). Statistical significance: ⁄⁄P <0.01, compared with control (À).

M. Uno et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5169–5173
5171
Figure 3. Titration of various proteases assayed in 96-well plates using the SUMO-1-AMC or FITC-casein substrates. (a) Recombinant human SENP2 catalytic domain (SENP2-
CD) was serially diluted in HEPES buffer (50 mM HEPES, 0.1 mM EDTA, pH 7.9), or (b) papain (from papaya latex), (c) trypsin (from bovine pancreas), (d) chymotrypsin (from
bovine pancreas), and (e) thermolysin (from bacillus thermoproteolyticus rokko) were serially diluted in TBS buffer (50 mM TBS, pH 7.2). Enzymatic activity was determined
as the relative protease activity 5 min after adding SUMO-1-AMC substrate (a) or 10 min after adding FITC-casein substrate (b–e) on a fluorescence plate reader. Statistical
significance: ⁄P <0.05 and ⁄⁄P <0.01, compared with control (À).
pound 1 in Fig. 1).20 Although GN6767 exhibited potent inhibition
against HIF-1 accumulation under hypoxic condition and inhib-
ited the hypoxia-induced HIF-1 transcriptional activity in KEK293
We thought that SENP1 inhibitor has potential for development
as an anti-tumor agent. No inhibitor of SENP1 was reported when
we started the current study.23,24 We actually synthesized biotin-
conjugated chemical probe of compound 1 and tried to identify
the target molecules of compound 1. Fortunately, SENP1 protein
was detected from the affinity column coated with a biotin mole-
cule 2 (Scheme 1). Thus we screened compounds, synthesized as
a
cells (IC50 = 7.2
lM), the inhibition mechanism has not been ob-
served yet.21
In 2007, Yeh and co-workers investigated the way in which
SENP1 controls EPO production by regulating the stability of HIF-
1
a
under hypoxia. SUMOylation of HIF-1
motes its binding to pVHL, which results in degradation of HIF-1
through a proline hydroxylation-independent UPS. Thus, SENP1
plays a key role in regulation of HIF-1
stability under hypoxia.22
a
induced by hypoxia pro-
HIF-1a
inhibitors in our laboratory,20,21 for the inhibition of SENP1
a
activity using in vitro SENP protease assay, and discovered that
compound 1 displayed SENP1 protease inhibition (Fig. 1). We
tested various 1-[4-(N-benzylamino)phenyl]-3-phenylurea deriva-
a

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M. Uno et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5169–5173
of compounds 3 and 4 were calculated to be 39.5 0.8
lM and
29.6 0.5 M, respectively. During our study of SENP1 inhibitors,
l
first SENP1 inhibitors were reported by Bogyo and the co-work-
ers.23 They developed the peptide-based inhibitors, however, those
compounds also inhibited SENP2 activity.
Since compound 4 showed significant inhibition toward SENP1
endopeptidase activity, we further investigated the selectivity of
compound 4 against other proteases.26 We chose SENP2 and pa-
pain as cysteine protease, trypsin and chymotrypsin as serine pro-
tease, and thermolysin as metallo protease, and examined
inhibition of compound 4 toward these protease activities. The
fluorogenic substrate SUMO-1-AMC was employed for measure-
ment of the enzymatic activities of SENP2. N-Ethylmaleimide
(NEM), known as a cysteine protease inhibitor, was used as a posi-
Figure 4. Effect of compounds 3 and 4 on hypoxia-induced accumulation of HIF-1
protein in HeLa cells. HeLa cells were incubated for 6 h with the indicated
a
concentrations of compounds under hypoxic condition. The levels of HIF-1 protein
a
were detected by immunoblot analysis with the protein specific antibody. Tubulin
was used as load control.
tive control.27 As shown in Figure 3a, NEM (10-100
lM) inhibited
SENP2 endopeptidase activity markedly. However, compound 4
did not exhibit significant inhibition of SENP2 at a range of 10–
tives including the compounds reported previously,20 and found
that the methyl- (compound 3, R1 = CH3, R2 = CH3) and the ethyl-
(compound 4, R1 = CH3, R2 = C2H5) substituents at the urea moiety
possess more efficient for the SENP1 protease inhibition. (See
Scheme 2)
Synthesis of the biotin-conjugated chemical probe of compound
1 is shown in Scheme 1. The reaction of 4-nitroaniline with tri-
phosgene in toluene afforded the corresponding isocyanate, which
reacted with 4-nitroaniline and the resulting 1,3-bis(4-nitro-
phenyl)urea was hydrogenated to give 1,3-bis(4-aminophe-
nyl)urea 5 in 63% yield in two steps. Reductive amination of 5
with methyl 3-formylbenzoate gave compound 6, which reacted
with activated biotin-conjugated carboxylic acid 8 to afford the
biotinylated probe 2.
100 lM concentrations. The fluorogenic substrate fluorescein iso-
thiocyanate (FITC)-Casein28 was employed for measurement of
the other enzymatic activities (Fig. 3b–e). Leupeptin hydrochloride
(Leu), known as serine/cysteine inhibitor,29 was used as a positive
control for the experiments of papain and trypsin (Fig. 3b and c),
and N-p-tosyl-L
-phenylalanine chloromethyl ketone (TPCK)30 and
EDTA31 were used as positive controls for the experiments of chy-
motripsin and thermolysin (Fig. 3d and e), respectively. Although
weak inhibitions of compound 4 against papain were observed in
a concentration-dependent manner (Fig. 3b), compound 4 did not
exhibit significant inhibition toward trypsin, chymotripsin, and
thermolysin at a range of 10–100 lM concentrations (Fig. 3c–e).
These results indicate that compound 4 possesses selective inhibi-
Synthesis of compounds 3 and 4 is shown in Scheme 1. N-
Methyl-4-nitroaniline 9, derived from 4-nitroaniline,25 was treated
with triphosgene to afford the corresponding isocyanate, which re-
acted with aniline to give urea derivative. The nitro group was
hydrogenated under palladium catalyzed condition to give com-
pound 10 in 76% yield in two steps. Reductive amination of com-
pound 10 with methyl 3-formylbenzoate or ethyl 3-
formylbenzoate gave compound 3 and 4 in 46% and 61% yields,
respectively.
We first examined the direct interaction of 1-[4-(N-benzyl-
amino)phenyl]-3-phenylurea derivatives with SENP1 protein in
cells. The streptavidin HP SpinTrap affinity column was coated with
the biotin-tagged compound 2 and HeLa cell lysate was passed
through this column. The proteins trapped by the affinity column
were eluted with 2% SDS solution and the eluted materials were
analyzed by western blots using a SENP1 antibody. SENP1 protein
was retained on the affinity column in the presence of biotin con-
jugated 2, whereas no SENP1 protein was detected from the affin-
ity column coated with a biotin molecule 7, revealing that biotin
conjugated 2 interact with SENP1 in the cells (Fig. S1 in the Sup-
plementary data).
tory potency toward SENP1 enzymatic activity.
We also demonstrated the effect of the synthesized compounds
3 and 4 on the hypoxia-induced HIF-1
man cervical cancer cells by western blot analysis. The results are
shown in Figure 4. Both compounds 3 and 4 suppressed HIF-1
accumulation in concentration-dependent manner without
affecting the expression level of tubulin protein and compound 4
displayed higher inhibitory potency against HIF-1 accumulation
than compound 3 in HeLa cells. Actually, significant suppression
of HIF-1 accumulation was observed at a 3 M concentration of
compound 3 under hypoxic condition.
a accumulation in HeLa hu-
a
a
a
a
l
In conclusion, we developed 1-[4-(N-benzylamino)phenyl]-3-
phenylurea derivative 4 as a SENP1 protease inhibitor. Inhibition
of compound 4 against SENP1 protease activity is not as high as
that against HIF-1
suppression of HIF-1
a
accumulation (Fig. 2d vs Fig. 4), suggesting that
accumulation could be independent of
a
SENP1 inhibition by compound 4. Even in these cases, as far as
we know, there are only two reports of SENP1 protease inhibitors;
the peptidic SENPs protease inhibitors23 and benzodiazepine-
based non-peptidic inhibitors.24 Therefore, the current investiga-
tion is a great potential not only as tools for the study of SUMO-re-
lated action mechanisms but also as a new type of anticancer drug
design.
We next examined the effect of compounds on in vitro SENP1
endopeptidase activity. Each compound was combined with a
SENP1 catalytic domain and incubated with fluorogenic SUMO-1-
AMC (7-amino-4-methylcoumarin). The enzymatic activity was
determined by the release of fluorescent AMC. Although GN6767
displayed 40% inhibition of the SENP1 endopeptidase activity at
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.06.084.
100
50
l
M concentration, significant inhibition was not observed at
lM (Fig. S2). A methyl substituent on the urea nitrogen
(R1 = CH3) in the urea derivatives increased inhibitory potency of
SENP1 endopeptidase activity. As shown in Figure 2, both
methyl-substituted urea derivatives, 3 and 4, inhibited SENP1
endopeptidase activity in a concentration-dependent manner.
Compound 3 displayed 74% inhibition of the SENP1 endopeptidase
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