Identification of SENP1 inhibitors through in silico screening and rational drug design

Article abstract of DOI:10.1016/j.ejmech.2016.06.018

The small ubiquitin-related modifier (SUMO)-specific proteases (SENPs) catalyze the deconjugation of SUMO from their substrate proteins. SENP1 which is the most studied isoform is closely related to many cancers such as prostate cancer and colon cancer, t

Full text of DOI:10.1016/j.ejmech.2016.06.018

European Journal of Medicinal Chemistry 122 (2016) 178e184
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Identification of SENP1 inhibitors through in silico screening and
rational drug design
Yaxue Zhao, Zhongli Wang, Jianchen Zhang, Huchen Zhou*
State Key Laboratory of Microbial Metabolism, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
The small ubiquitin-related modifier (SUMO)-specific proteases (SENPs) catalyze the deconjugation of
SUMO from their substrate proteins. SENP1 which is the most studied isoform is closely related to many
cancers such as prostate cancer and colon cancer, thus representing a potential therapeutic target for
cancer treatment. In the present study, we identified eleven SENP1 inhibitors representing a variety of
scaffolds through in silico screening. Based on these scaffolds, a series of new compounds were designed
and synthesized in order to improve their SENP1 inhibitory potency. As a result, compounds with IC50 as
Received 25 March 2016
Received in revised form
11 June 2016
Accepted 11 June 2016
Available online 14 June 2016
low as 3.5
discussed.
m
M (compound 13m) were obtained and a preliminary structure-activity relationship was
Keywords:
SENP1 inhibitor
In silico screening
Rational design
Cancer
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
been shown to be overexpressed in more than 50% of prostate
cancer, and the critical role of SENP1 in tumorigenesis has been
Small ubiquitin-like modifier (SUMO) is a member of the
ubiquitin-like protein family that modulates cellular functions of a
variety of target proteins [1e3]. Sumoylation has emerged as an
important protein post-translational modification that can partic-
ipate in transcriptional regulation, nuclear transport, maintenance
of genome integrity, and signal transduction in human [4,5].
Dysfunction of sumoylation is associated with a broad range of
diseases including cancer, neurodegenerative syndrome, diabetes,
viral infection, and development defects [6]. As a reversible process,
sumoylation is mediated by SUMO-activating enzyme (E1), SUMO-
conjugation enzyme (E2) and SUMO protein ligase (E3), and is
readily reversed by a family of SUMO-specific proteases (SENPs) [7].
SENPs, a family of cysteine proteases are responsible for the
deconjugation of SUMO from substrate proteins [8]. Six isoforms of
SENPs (SENP1, 2, 3, 5, 6, and 7) from human have been identified
and characterized, each of them has a distinct subcellular locali-
zation and substrate specificity [9,10]. Among them, SENP1 has
demonstrated in transgenic mice [11e13]. Furthermore, it has been
found that SENP1 is essential for colon cancer cell growth [14], and
enhances vascular endothelial growth factor (VEGF) production by
regulating the stability of hypoxia-inducible factor-1 (HIF-1)
[15,16]. Thus, SENP1 may serve as an attractive target for devel-
oping new cancer therapeutics.
Since 2011, several SENP1 inhibitors have been reported by
our and other groups [17e20], including the benzodiazepine-
based peptidomimetic covalent inhibitors from our group [17],
the SUMO-derived peptide-based covalent inhibitors from
Bogyo’s group [18], the 2-(4-chlorophenyl)-2-oxoethyl 4-
benzamidobenzoates and 1-[4-(N-benzylamino)phenyl]-3-
phenylureas as non-covalent inhibitors from Zhang [19] and
Chen’s [20] groups, respectively. These inhibitors showed
moderate inhibitory activity which needs major improvement in
order to be useful for in-depth in vivo studies. At the same time,
the availability of a variety of scaffolds would also facilitate the
discovery of potent inhibitors, and eventually lead to the
development of therapeutic agents for SENP1-related patho-
logical conditions and chemical probes to further the under-
standing of the biological functions of SENP1.
Abbreviations: SUMO, small ubiquitin-related modifier; SENP, SUMO-specific
protease; pre-SUMO, SUMO precursor; E1, SUMO-activating enzyme; E2, SUMO-
conjugation enzyme; E3, SUMO protein ligase; VEGF, vascular endothelial growth
factor; HIF1, hypoxia-inducible factor 1; HTVS, high-throughput virtual screening;
SP, standard precision; XP, extra precision.
In this study, we identified a number of new scaffolds as SENP1
inhibitors through in silico screening. Two of the scaffolds were
analyzed and a hybrid structure based on these two scaffolds was
designed and optimized by structure-based rational design. Thus, a
* Corresponding author.
E-mail address: hczhou@sjtu.edu.cn (H. Zhou).
http://dx.doi.org/10.1016/j.ejmech.2016.06.018
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

Y. Zhao et al. / European Journal of Medicinal Chemistry 122 (2016) 178e184
179
series of new SENP1 inhibitors were developed and an IC50 value as
low as 3.5 M (compound 13m) was achieved. This work demon-
criteria: binding pose should closely resemble that of pre-SUMO, at
least four hydrogen bonds should be formed, and scaffold diversity
should be considered. Finally, 30 compounds were selected and
purchased for SENP1 inhibitory assay.
We showed that 11 of the 30 selected compounds showed
SENP1 inhibitory activities with IC50 below 50 mM (Table 1). These
m
strated that in silico screening followed with rational design is an
effective strategy for the discovery of inhibitors against novel tar-
gets such as SENP1. It also provided new lead compounds for
further development of SENP1 inhibitors as potential therapeutic
agents and functional probes.
inhibitors represent a diversity of scaffolds, providing hits for
further optimization and development (see Table 2).
2. Results and discussion
2.2. Representative structures of SENP1 inhibitors
2.1. Inhibitor identification via in silico screening
Among the identified SENP1 inhibitors with IC50 values below
20 mM, the structures of compounds 1, 5, 7 and 9 can be summa-
The SENP1 protein undergoes significant conformational change
upon substrate protein binding and a binding tunnel capped by
residues Trp465 and Val532 is induced. Thus, the co-crystal struc-
ture of SENP1 and pre-SUMO [21] (PDB ID: 2IY1) was used as the
starting structure for in silico screening as it represents the binding
conformation of SENP1 with the presence of the binding tunnel
(Fig. 1A).
rized into a representative structure, A1-L1-A2-L2-A3, as shown in
Fig. 2A. In the docked poses, the A1 moiety points to either the
Phe496-His529 site or the Lys455-Thr459-Asn467-Asp468 site. The
L1-A2-L2 moiety is inserted into the binding tunnel and covered by
residues Trp465, Leu466, His529, Val532, Trp534, Gln597, and
Cys603. The A2 moiety has conserved interaction with Trp465 and/
The catalytic triad, Cys603, His533, and Asp550, of SENP1 is
critical for its catalytic activity, the corresponding mutants (C603A,
H533A, and D550A) are completely inactive in the deconjugation of
SUMO [22]. The binding tunnel of SENP1, which is composed of
amino acids Trp465, His529, Val532, Trp534, and Leu466, is
essential for interactions with the C-terminal of SUMO for the
subsequent deconjugation of SUMO from modified proteins. The
catalytic activity of SENP1 is seriously impaired or completely
abolished in the corresponding mutants including W465A, H529A,
and W534A [22]. Thus, these key residues, Trp465, Leu466, His529,
Val532, His533, Trp534 and Cys603, are all included in the docking
box (Fig.1A) for virtual screening. The centroid of T90GGH93 peptide
(red in Fig. 1A) located inside the binding tunnel is used to generate
the docking grid.
Table 1
Inhibitory effect of hit compounds from in silico screening against SENP1.
a
Compound
Structure
IC50
(
m
M)
1
O
O
15.0
Cl
Cl
N
N
H
H
S
S
F
F
2
3
HO
O
20.7
28.4
NC
H
N
N
O
OH
N
N
N
S
As shown in Fig. 1B, the compounds from Specs database were
prepared by LigPrep and screened by Glide in three consecutive
modes: high-throughput virtual screening (HTVS), standard pre-
cision (SP), and extra precision (XP). The top ranked 150 molecules
from SP screening and the top ranked 150 molecules from XP
screening were submitted to manual inspection using the following
HO
4
5
HO
H
N
14.0
17.6
N
Cl
O
Cl
H
N
H
N
O
O
O
Cl
6
Cl
47.9
O
O
H
N
N
O
H
O
N
O
Br
7
8
H
N
15.8
20.8
N
H
N
S
O
N
O
N
N
H
N
O
S
O
O
S
H2N
O
9
18.4
23.5
O
H
N
N
O2
N
N
NO2
H
O
O
10
H
N
S
O
N
N
11
21.2
NO2
OH
O
N
O
Fig. 1. A, Complex of SENP1 (gray surface) and pre-SUMO (yellow cartoon) (PDB ID:
2IY1). The key residues of SENP1 are shown in green, the T90GGH93 peptide of pre-
SUMO which is used for docking box definition is shown in red. B, Schematic repre-
sentation of our hit discovery strategy. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
N
S
H
a
IC50 measurement has an error range within 10%.

180
Y. Zhao et al. / European Journal of Medicinal Chemistry 122 (2016) 178e184
Table 2
We selected compounds 1 and 5 as the model compounds. Their
aryl groups, phenyl and benzothiophenyl, were incorporated as the
A1, A2, and A3 groups. Their amide groups with the nitrogen atom
either directly connected to or a carbon atom away from the central
phenyl were incorporated as the L1 and L2 linkage groups. This
resulted in the structure of compound 13a whose SENP1 inhibitory
Inhibitory effect of designed compounds against SENP1.
a
Compound
Structure
IC50 (mM)
13a
26.6
O
H
N
N
H
S
O
activity (IC50 ¼ 26.6
mM) was shown to be weaker than compounds
Br
Br
13b
13c
9.1
6.4
O
H
N
1 and 5. The docking structure of 13a with SENP1 showed that the
structure spans a shorter dimension than compound 1 or 5, which
might contribute to its decreased activity. After the benzothio-
phenyl was replaced by a larger group, i.e. bromobiphenyl, the
N
H
O
O
O
H
N
N
H
O
O
inhibitory activity (compound 13b, IC50 ¼ 9.1
mM) became superior
13d
13e
13f
16.4
9.2
to compounds 1 and 5. The docking structure of 13b in SENP1
showed that the phenyl group at the A3 terminus was near the
His533 and Met552 residues which potentially could form sand-
wiched hydrophobic interaction, and extension of the linkage
group may enhance this interaction (Fig. 3). Thus, compound 13c
with an extended linkage group containing a double bond was
H
N
Br
N
H
O
H
N
N
H
O
O
4.9
O
H
synthesized and showed a slightly improved IC50 value of 6.4 mM.
N
N
H
O
The docked model showed that compound 13c formed favorable
hydrophobic interaction with His533 and Met552. Both compounds
13b and 13c formed four hydrogen bonds with residues Leu466,
His529, Val532, and Gln597.
13g
13h
13i
13j
6.2
7.6
O
H
BnO
N
N
H
O
O
Next, compounds 13de13j with various substituents at the A1
terminus were synthesized in order to further improve the SENP1
inhibitory activity from compound 13c. The meta-bromo derivative
H
N
O
N
H
O
12.4
7.1
O
H
13d showed a 2.5-fold decrease of activity (IC50 ¼ 16.4
Removal of the bromo-atom gave comparable activity (13e,
IC50 ¼ 9.2 M). The para-methoxy compound 13f showed the best
potency (IC50 ¼ 4.9 M) among the derivatives of 13c. When the
mM).
N
N
H
m
O
H
N
m
N
H
S
O
biphenyl group was replaced with various aryl rings (compounds
13ge13j), the SENP1 inhibitory activity was not improved.
Br
13k
15.3
O
We next tested the effect of the flexibility of linkage group L2 on
the inhibitory activity. Compounds 13ke13n which are the single
bond derivatives of the double bond-containing compounds 13c,
13e, 13f, and 13g were synthesized. For compounds 13k, 13l, and
13n, more flexibility led to either reduced or comparable activity.
However, compound 13m gave the most potent inhibitory activity
H
N
N
H
O
13l
8.5
3.5
O
H
N
N
H
O
O
13m
O
H
N
N
H
(IC50 ¼ 3.5
mM) among all compounds in this work.
O
The synthesis of these compounds are described below. As
shown in Scheme 1, aminomethylaniline was used as the common
starting material. After the benzyl amino group was protected by
Boc, the phenylamino group was coupled with various carboxylic
acids in the presence of EDCI/DMAP followed by treatment with
TFA to give compounds 12ae12c. Compounds 13ae13n were sub-
sequently prepared by coupling compounds 12ae12c with the
corresponding carboxylic acids.
13n
8.0
O
H
BnO
N
N
H
O
a
IC50 measurement has an error range within 10%.
or Trp534, while the L1 and L2 linkages form hydrogen bond in-
teractions. The A3 moiety points to the His533-Met552 site.
The predicted binding poses of compounds 1 and 5 are shown in
Fig. 2. Both compounds assume binding poses that closely resemble
the binding conformation of peptide E88QTGGHS94 derived from
pre-SUMO. Compound 1 showed hydrogen bonds with Lys455,
His529, Val532, Leu466, and Gln597, pep interaction with Trp465,
His533, and Trp534, and hydrophobic interaction with Met552.
Compound 5 showed hydrogen bonds with Leu466, His529,
Val532, and Gln597, pep interaction with Trp465, His529, His533,
3. Conclusions
The SENPs which catalyze the deconjugation of SUMO from
their target proteins play essential roles in a variety of critical
cellular events. Dysfunction of the equilibrium of SUMOylation and
deSUMOylation is associated with numerous pathological condi-
tions including cancer. Specifically, SENP1 has been found to be
closely related to the prostate and colon cancer. As a potential
therapeutic target, the discovery of SENP1 inhibitors remains in its
infancy. In this study, we identified 11 SENP1 inhibitors with
various scaffolds through in silico screening. Based on the structural
features and the docked poses of the initial hits, compounds
13ae13n were designed and synthesized, which gave improved
and Trp534, and hydrophobic interaction with Phe496 and Met552.
2.3. Structural optimization and structure-activity relationship
(SAR) study
In order to improve their SENP1 inhibitory activity, we designed
and synthesized compounds 13ae13n (Table 2) based on the core
structural features of A1-L1-A2-L2-A3 which is derived from the
initial hits.
SENP1 inhibitory activity with compound 13m (IC50 ¼ 3.5
mM) as
the most potent compound in this series. We believe these new
SENP1 inhibitors will contribute to the further development of
SENP1 inhibitors as potential therapeutics and functional probes.

Y. Zhao et al. / European Journal of Medicinal Chemistry 122 (2016) 178e184
181
Fig. 2. A, Representative structure of SENP1 inhibitors. B, Overlap of compounds 1 (green stick), 5 (salmon stick) and E88QTGGHS94 of pre-SUMO (purple stick) in SENP1 (gray
cartoon) pocket. C, Detailed interactions between compound 1 and SENP1 (gray surface). D, Detailed interactions between compound 5 and SENP1 (gray surface). The residues
interacting with compound 1 or 5 are shown in gray stick. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Material and methods
4.1. Preparation of protein and small molecule database for
computational study
The 3D structure of the SENP1-pre-SUMO complex (PDB ID:
2IY1) was used to derive the SENP1-T90GGH93 peptide complex that
was used in the study and is described as follows. Using the
workflows of Maestro [23], the A603 was mutated back to C603, the
hydrogen atoms were added, the bond orders were assigned, the
added hydrogen atoms were optimized with exhaustive sampling
method, and all atoms were energy minimized to reach the
convergent RMSD of 0.30 Å with the OPLS_2005 force field [24].
The docking grid was then generated using Glide 5 [25]. The
centroid of the T90GGH93 peptide was defined as the active site
Fig. 3. Overlap of compounds 13b (cyan stick) and 13c (yellow stick) in SENP1 (gray
center and the bounding box which contains the acceptable posi-
tions for the ligand center was set to 14 Å ꢀ 14 Å ꢀ 14 Å. The Specs
database which includes approximately 197,000 commercially
surface) pocket. The residues interacting with compounds 13b and 13c are shown in
gray stick. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
H
N
NH2
R1
NH2
H2N
H2N
BocHN
ii, iii
i
12a: R1 = phenyl
O
12b: R1 = cinnamenyl
12c: R1 = phenethyl
12a-12c
O
H
N
R1
iv
R2
N
H
O
13a-13n
Scheme 1. Reagents and conditions: (i) Boc2O, TEA, DCM, rt, 20 h; (ii) R1COOH, DCM, EDCI, DMAP, rt, overnight; (iii) TFA, DCM, rt, 2 h; (iv) R2COOH, DCM, EDCI, DMAP, rt, overnight.

182
Y. Zhao et al. / European Journal of Medicinal Chemistry 122 (2016) 178e184
available compounds was used for in silico screening. LigPrep [26]
was used to prepare these compounds in OPLS_2005 force field.
The possible ionization state at pH ¼ 7.0 2.0 was generated for
each compound. About 350,000 small molecule conformations
were generated.
used. Purity was based on the integrated UV chromatogram
(254 nm). The purity of all biological assay compounds was >95%.
4.4.1. N-[3-(Aminomethyl)phenyl]benzamide (12a)
The solution of EDCI (3.14 g, 16.38 mmol) and DMAP (100 mg,
0.82 mmol) in 10 mL DCM was added dropwise to a mixture of tert-
butyl-3-aminobenzyl-carbamate (2.0 g, 9.01 mmol) and benzoic
acid (1.0 g, 8.19 mmol) in 20 mL DCM at 0 ꢁC under nitrogen at-
mosphere, and the mixture was warmed to r.t. and stirred over-
night. After quenched with water and extracted with DCM, the
organic layer was washed with 1 M NaOH (20 mL), 1 M HCl (20 mL),
and saturated NaHCO3 (10 mL), dried over Na2SO4, and concen-
trated under reduced pressure. The Boc-protected 12a was dis-
solved in DCM (5 mL), trifluoroacetic acid (5 mL) was added. The
reaction mixture was allowed to stir for 2 h at r.t. and concentrated
under reduced pressure. The residue was purified by column
chromatography over silica gel and further purified by recrystalli-
zation (ethyl ether) to obtain the title compound as a white solid
4.2. In silico screening
The Glide module [25] was employed to screen the Specs
database in order to identify SENP1 inhibitors. The screening was
done by three consecutive flows: HTVS (350,000 compounds
screened), SP (top ranked 40,000 compounds from HTVS screened),
and XP (top ranked 5000 compounds from SP screened). The li-
gands were set to be flexible, the ring conformations were sampled,
and the non-planar conformations for amide bonds were penalized.
The docked conformations were ranked by Glide scoring function.
Both top ranked 150 molecules after SP sieve and top ranked 150
molecules after XP sieve were manually analyzed. Finally, 30
compounds were purchased and tested.
(1.2 g, yield 64.8%). 1H NMR (600 MHz, DMSO-d6)
d: 10.42 (1H, s),
8.34 (3H, s), 8.01e7.96 (3H, m), 7.70 (1H, d, J ¼ 8.4 Hz), 7.61 (1H, t,
4.3. Bioactivity assay
J ¼ 8.4 Hz), 7.54 (2H, t, J ¼ 7.8 Hz), 7.41 (1H, t, J ¼ 7.8 Hz), 7.22 (1H, d,
J ¼ 7.8 Hz), 4.04 (2H, s). 13C NMR (150 MHz, DMSO-d6)
d: 166.1,
The pET28a-SENP1 plasmid (catalytic core, residues 419e644)
was transformed into Escherichia coli BL21 (DE3). Expression of
SENP1 was induced with 0.5 mM IPTG at 16 ꢁC for 16 h. Cell pellets
were resuspended in lysis buffer (50 mM Na2HPO4/NaH2PO4 pH 7.4,
139.9, 135.2, 134.9, 132.1, 129.4, 128.8, 128.1, 124.4, 121.3, 121.0, 42.9.
4.4.2. N-[3-(Aminomethyl)pheny]cinnamamide (12b)
300 mM NaCl, 10% glycerol, 10 mM
b
-mercaptoethanol, and 10 mM
Compound 12b was prepared following a similar procedure to
imidazole) and sonicated. SENP1 was purified using Ni-NTA resin
(Qiagen) and eluted with elution buffer containing 150 mM imid-
azole. The pET28a-RanGAP (residues 403e585) and pSAE1/SAE2-
UBC9-SUMO plasmids were cotransformed into E. coli BL21 (DE3),
and similar methods were used in the expression and purification
of fusion protein RanGAP-SUMO.
compound 12a. Yield: 53.5%. 1H NMR (600 MHz, DMSO-d6)
d: 10.24
(1H, s), 7.67 (1H, s), 7.65e7.54 (4H, m), 7.49e7.38 (3H, m), 7.28 (1H,
t, J ¼ 8.0 Hz), 7.08 (1H, d, J ¼ 8.0 Hz), 6.88 (1H, d, J ¼ 16 Hz), 3.76 (2H,
s). 13C NMR (150 MHz, DMSO-d6)
d: 163.9, 143.6, 140.4, 139.6, 135.2,
130.2, 129.4, 129.1, 128.2, 122.9, 122.8, 118.6, 118.1, 45.6.
The SENP1 inhibitory effect of the compounds was tested using
deSUMOylation assay. SENP1 (4 nM) was incubated with com-
pounds at 37 ꢁC for 10 min in reaction buffer (50 mM Tris-HCl pH
4.4.3. N-[3-(Aminomethyl)phenyl]-3-phenylpropanamide (12c)
Compound 12c was prepared following a similar procedure to
compound 12a. Yield: 66.8%. 1H NMR (600 MHz, DMSO-d6)
d: 10.08
8.0, 20 mM NaCl, 2 mM DTT, 2 mM CaCl2). Then 5
mL RanGAP-SUMO
(1H, s), 8.25 (3H, s), 7.78 (1H, s), 7.48 (1H, d, J ¼ 8.4 Hz), 7.34 (1H, t,
J ¼ 8.4 Hz), 7.32e7.24 (4H, m), 7.18 (1H, t, J ¼ 7.2 Hz), 7.13 (1H, d,
J ¼ 7.8 Hz), 3.99 (2H, q, J ¼ 6.0 Hz), 2.91 (2H, t, J ¼ 7.8 Hz), 2.65 (2H, t,
(2 g/
m
m
L) was added and incubated for another 25 min at 37 ꢁC. The
reaction was terminated by adding loading buffer and boiling for
5 min. The protein samples were subjected to SDS-PAGE and coo-
massie staining. The RanGAP-SUMO bands were quantified on the
Odyssey infrared scanner (lex ¼ 680 nm and lem ¼ 720 nm).
Percentage inhibition was calculated using the following equa-
tion: %inhibition ¼ [(RFUsample-RFUblank)/(RFUsubstrate-RFUblank)] x
100%, sample - RanGAP-SUMO, SENP1, and compounds in assay
buffer, blank - RanGAP-SUMO, SENP1 in assay buffer, substrate -
RanGAP-SUMO in assay buffer without SENP1. For IC50 measure-
ment, a test compound at interval concentrations (50, 25, 12.5, 6.25,
J ¼ 7.8 Hz). 13C NMR (150 MHz, DMSO-d6)
d: 171.0, 141.5, 139.9,
134.9, 129.5, 128.7, 128.6, 126.4, 123.8, 120.0, 119.6, 42.9, 38.2, 31.2.
4.4.4. Benzo[b]thiophene-2-carboxylic acid 3-benzoylamino-
benzylamide (13a)
The solution of EDCI (107 mg, 0.56 mmol) and DMAP (3 mg,
0.03 mmol) in 5 mL DCM was added dropwise to a mixture of
compound 12a (69 mg, 0.31 mmol) and benzo[b]thiophene-2-
carboxylic acid (50 mg, 0.28 mmol) in 5 mL DCM at 0 ꢁC under
nitrogen atmosphere, and the mixture was warmed to r.t. and
stirred overnight. The mixture was quenched with water and
extracted with DCM. The organic layer was washed with 1 M NaOH
(20 mL),1 M HCl (20 mL), and saturated NaHCO3 (10 mL), dried over
Na2SO4, and concentrated under reduced pressure. The residue was
purified by column chromatography over silica gel and further
purified by recrystallization (ethyl ether) to obtain the title com-
pound as a white solid (45 mg, yield 41.5%) Mp: 258e260 ꢁC. 1H
3.12, 1.56, 0.78, 0.39 mM) was used and the data were fitted with
GraphPad to give the IC50 value.
4.4. Chemistry
All solvents and reagents were purchased from commercial
sources and used without further purification unless otherwise
noted. NMR spectra were recorded on Bruker Avance III 400 MHz or
600 MHz. Chemical shifts are expressed in parts per million (ppm)
relative to residual solvent as an internal reference (DMSO-d6:
2.50). High resolution mass spectra were obtained on Agilent 6530
Accurate Mass Q-TOF LC-MS. Column chromatography was per-
NMR (400 MHz, DMSO-d6)
d
: 10.31 (1H, s), 9.43 (1H, t, J ¼ 6.0 Hz),
8.20 (1H, s), 8.02 (1H, d, J ¼ 7.2 Hz), 7.99e7.91 (3H, m), 7.79 (1H, s),
7.73 (1H, d, J ¼ 8.0 Hz), 7.56 (1H, m), 7.55e7.41 (4H, m), 7.33 (1H, t,
J ¼ 8.0 Hz), 7.10 (1H, d, J ¼ 7.2 Hz), 4.51 (2H, d, J ¼ 6.0 Hz). 13C NMR
formed using Huanghai silica gel (45e75 mm). HPLC analysis was
performed on an Agilent 200 with a flow rate of 1 mL/min and a
gradient of 10% v/v MeOH in H2O (t ¼ 0 min) to 100% MeOH
(t ¼ 15.0 min) stopping at 20 or 25 min using a DAD detector. An
(100 MHz, DMSO-d6) d: 166.1, 161.9, 140.6, 140.4, 140.2, 139.7, 139.6,
135.3, 131.9, 129.0, 128.8, 128.1, 126.6, 125.6, 125.3, 123.3, 123.2,
119.7, 119.5, 43.2. HRMS calcd for C23H19N2O2S (MþH)þ 387.1167,
found 387.1164. HPLC purity: 97.4% (14.0 min).
Agilent Eclipse XDB-C18 column (4.6 mm ꢀ 150 mm, 5
mm) was

Y. Zhao et al. / European Journal of Medicinal Chemistry 122 (2016) 178e184
183
4.4.5. 40-Bromo-biphenyl-3-carboxylic acid 3-benzoylamino-
benzylamide (13b)
4.4.9. 40-Methoxy-biphenyl-3-carboxylic acid 3-(3-phenyl-
acryloylamino)-benzylamide (13f)
Compound 13b was prepared following a similar procedure to
Compound 13f was prepared following a similar procedure to
compound 13a. Yield: 37.5%. Mp: 253e256 ꢁC. 1H NMR (600 MHz,
compound 13c. Yield: 30.8%. Mp: 195e197 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 10.27 (1H, s), 9.22 (1H, t, J ¼ 6.0 Hz), 8.22 (1H, s), 7.94
DMSO-d6)
d
: 10.22 (1H, s), 9.21 (1H, t, J ¼ 6.0 Hz), 8.19 (1H, s), 7.86
(3H, m), 7.85 (1H, d, J ¼ 7.8 Hz), 7.78 (1H, s), 7.75e7.65 (5H, m),
7.62e7.55 (2H, m), 7.51 (1H, t, J ¼ 7.8 Hz), 7.32 (1H, t, J ¼ 7.8 Hz), 7.10
(1H, d, J ¼ 7.8 Hz), 4.54 (2H, d, J ¼ 6.0 Hz). 13C NMR (150 MHz,
(1H, d, J ¼ 7.6 Hz), 7.80 (1H, d, J ¼ 8.0 Hz), 7.74e7.64 (4H, m),
7.64e7.52 (4H, m), 7.48e7.38 (3H, m), 7.31 (1H, t, J ¼ 7.6 Hz),
7.09e7.04 (3H, m), 6.84 (1H, d, J ¼ 16 Hz), 4.53 (2H, d, J ¼ 6.0 Hz),
DMSO-d6)
d
: 166.4, 166.0, 140.6, 139.7, 139.4, 139.4, 135.6, 135.4,
3.81 (3H, s). 13C NMR (100 MHz, DMSO-d6)
d: 166.6, 163.9, 159.6,
132.3, 132.0, 129.8, 129.6, 129.4, 129.0, 128.8, 128.1, 127.4, 125.7,
123.1, 121.8, 119.5, 119.3, 43.2. HRMS calcd for C27H22N2O2Br
(MþH)þ 485.0865, found 385.0861. HPLC purity: 95.7% (15.7 min).
140.8, 140.5, 140.3, 139.8, 135.3, 135.1, 132.3, 130.2, 129.4, 129.3,
129.2, 128.4, 128.1, 126.2, 125.3, 122.7, 118.2, 114.8, 55.6, 43.1. HRMS
calcd for C30H27N2O3 (MþH)þ 463.2022, found 463.2021. HPLC
purity: 99.1% (15.4 min).
4.4.6. 40-Bromo-biphenyl-3-carboxylic acid 3-(3-phenyl-
acryloylamino)-benzylamide (13c)
4.4.10. 3-Benzyloxy-N-[3-(3-phenyl-acryloylamino)-benzyl]-
benzamide (13g)
The solution of EDCI (107 mg, 0.56 mmol) and DMAP (3 mg,
0.03 mmol) in 5 mL dichloromethane was added dropwise to a
mixture of compound 12b (78 mg, 0.31 mmol) and 40-bromo-
biphenyl-3-carboxylic acid (50 mg, 0.18 mmol) in 5 mL DCM at 0 ꢁC
under nitrogen atmosphere, and the mixture was warmed to r.t.
and stirred overnight. After quenched with water and extracted
with DCM, the organic layer was washed with 1 M NaOH (20 mL),
1 M HCl (20 mL), and saturated NaHCO3 (10 mL), dried over Na2SO4,
and concentrated under reduced pressure. The residue was purified
by column chromatography over silica gel and further purified by
recrystallization (ethyl ether) to obtain the title compound as a
white solid (43 mg, yield 30.3%). Mp: 236e238 ꢁC. 1H NMR
Compound 13g was prepared following a similar procedure to
compound 13c. Yield: 22.1%. Mp: 206e208 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 10.22 (1H, s), 9.07 (1H, t, J ¼ 6.0 Hz), 7.66 (1H, d,
J ¼ 8.0 Hz), 7.64e7.54 (5H, m), 7.52 (1H, d, J ¼ 8.0 Hz), 7.50e7.38
(8H, m), 7.37e7.27 (2H, m), 7.19 (1H, dd, J ¼ 8.0, 2.0 Hz), 7.03 (1H, d,
J ¼ 8.0 Hz), 6.84 (1H, d, J ¼ 15.6 Hz), 5.17 (2H, s), 4.48 (2H, d,
J ¼ 6.0 Hz). 13C NMR (100 MHz, DMSO-d6)
d: 166.4, 164.0, 159.8,
140.8, 140.5, 139.8, 137.3, 136.3, 135.2, 130.2, 129.9, 129.5, 129.1,
128.9, 128.3, 128.1, 122.8, 122.7, 120.3, 118.4, 118.3, 118.2, 114.1, 69.9,
43.1. HRMS calcd for
463.2021. HPLC purity: 99.5% (15.4 min).
C
30H27N2O3 (MþH)þ 463.2022, found
(400 MHz, DMSO-d6)
d
: 10.21 (1H, s), 9.23 (1H, t, J ¼ 6.0 Hz), 8.23
4.4.11. N-(3-Cinnamamidobenzyl)-2-naphthamide (13h)
(1H, s), 7.99 (1H, s), 7.95 (1H, d, J ¼ 8.0 Hz), 7.89 (1H, d, J ¼ 8.0 Hz),
7.78 (1H, d, J ¼ 7.6 Hz), 7.71e7.53 (7H, m), 7.51e7.38 (4H, m), 7.31
(1H, t, J ¼ 7.6 Hz), 7.07 (1H, d, J ¼ 7.6 Hz), 6.83 (1H, d, J ¼ 15.6 Hz),
Compound 13h was prepared following a similar procedure to
compound 13c. Yield: 25.9%. Mp: 215e216 ꢁC. 1H NMR (400 MHz,
4.54 (2H, d, J ¼ 6.0 Hz). 13C NMR (100 MHz, DMSO-d6)
d
: 166.4,
DMSO-d6)
d
: 10.21 (1H, s), 9.25 (1H, t, J ¼ 6.0 Hz), 8.54 (1H, s),
8.07e7.98 (4H, m), 7.68 (1H, d, J ¼ 8.0 Hz), 7.65 (1H, s), 7.64e7.54
(5H, m), 7.48e7.38 (3H, m), 7.31 (1H, t, J ¼ 7.6 Hz), 7.09 (1H, d,
J ¼ 7.6 Hz), 6.83 (1H, d, J ¼ 15.6 Hz), 4.55 (2H, d, J ¼ 6.0 Hz). 13C NMR
163.9, 140.8, 140.6, 139.8, 139.4, 139.4, 135.5, 135.2, 132.3, 132.0,
130.3, 129.8, 129.7, 129.5, 129.4, 129.2, 128.2, 127.4, 125.7, 122.8,
121.8, 118.2, 118.2, 43.1. HRMS calcd for C29H24N2O2Br (MþH)þ
511.1021, found 511.1021. HPLC purity: 98.7% (13.8 min).
(100 MHz, DMSO-d6) d: 166.7, 163.9, 140.8, 140.5, 139.8, 135.2,134.6,
132.6, 130.2, 129.5, 129.3, 129.2, 128.4, 128.2, 128.1, 128.0, 127.2,
124.6, 122.8, 122.7, 118.3, 118.2, 43.2. HRMS calcd for C27H23N2O2
(MþH)þ 407.1760, found 407.1764. HPLC purity: 98.5% (14.8 min).
4.4.7. 30-Bromo-biphenyl-3-carboxylic acid 3-(3-phenyl-
acryloylamino)-benzylamide (13d)
Compound 13d was prepared following a similar procedure to
4.4.12. (E)-N-(3-Cinnamamidobenzyl)-1-naphthamide (13i)
Compound 13i was prepared following a similar procedure to
compound 13c. Yield: 26.7%. Mp: 210e212 ꢁC. 1H NMR (400 MHz,
compound 13c. Yield: 47.4%. Mp: 231e233 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 10.21 (1H, s), 9.24 (1H, t, J ¼ 6.0 Hz), 8.24 (1H, s), 7.94
(1H, d, J ¼ 8.0 Hz), 7.86 (1H, d, J ¼ 8.0 Hz), 7.77e7.65 (6H, m),
7.64e7.57 (4H, m), 7.48e7.38 (3H, m), 7.31 (1H, t, J ¼ 7.6 Hz), 7.07
(1H, d, J ¼ 7.6 Hz), 6.84 (1H, d, J ¼ 15.6 Hz), 4.54 (2H, d, J ¼ 6.0 Hz).
DMSO-d6)
d
: 10.26 (1H, s), 9.12 (1H, t, J ¼ 6.0 Hz), 8.26 (1H, m), 8.04
(1H, d, J ¼ 8.4 Hz), 7.98 (1H, m),7.77 (1H, s), 7.73e7.54 (8H, m),
7.48e7.38 (3H, m), 7.34 (1H, t, J ¼ 7.6 Hz), 7.12 (1H, d, J ¼ 7.6 Hz),
6.87 (1H, d, J ¼ 15.6 Hz), 4.55 (2H, d, J ¼ 6.0 Hz). 13C NMR (100 MHz,
13C NMR (100 MHz, DMSO-d6)
d: 166.4, 163.9, 142.4, 140.7, 140.6,
139.8,139.1,135.5, 135.2,131.6, 131.0,130.2,130.1,129.9,129.7, 129.5,
129.0, 129.2, 128.2, 127.8, 126.5, 125.9, 122.9, 122.8, 118.2, 118.2, 43.1.
HRMS calcd for C29H24N2O2Br (MþH)þ 511.1021, found 511.1026.
HPLC purity: 97.4% (15.1 min).
DMSO-d6) d: 169.1, 164.0, 140.8, 140.6, 139.9, 135.2, 135.1, 133.6,
130.3, 130.3, 130.3, 129.5, 129.3, 128.7, 128.2, 127.2, 126.7, 125.9,
125.7, 125.5, 122.8, 118.4, 118.2, 43.0. HRMS calcd for C27H23N2O2
(MþH)þ 407.1760, found 407.1763. HPLC purity: 95.9% (14.8 min).
4.4.8. Biphenyl-3-carboxylic acid 3-(3-phenyl-acryloylamino)-
benzylamide (13e)
4.4.13. Benzo[b]thiophene-2-carboxylic acid 3-(3-phenyl-
acryloylamino)-benzylamide (13j)
Compound 13e was prepared following a similar procedure to
Compound 13j was prepared following a similar procedure to
compound 13c. Yield: 49.4%. Mp: 191e193 ꢁC. 1H NMR (400 MHz,
compound 13c. Yield: 34.6%. Mp: 163e165 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 10.21 (1H, s), 9.23 (1H, t, J ¼ 6.0 Hz), 8.23 (1H, s), 7.92
DMSO-d6)
d
: 10.24 (1H, s), 9.37 (1H, t, J ¼ 6.0 Hz), 8.17 (1H, s), 8.04
(1H, d, J ¼ 8.0 Hz), 7.86 (1H, d, J ¼ 7.6 Hz), 7.77 (2H, d, J ¼ 7.2 Hz),
7.68 (1H, d, J ¼ 8.0 Hz), 7.66e7.54 (5H, m), 7.51 (2H, t, J ¼ 7.6 Hz),
7.48e7.38 (4H, m), 7.31 (1H, t, J ¼ 7.6 Hz), 7.06 (1H, d, J ¼ 7.6 Hz),
6.83 (1H, d, J ¼ 15.6 Hz), 4.53 (2H, d, J ¼ 6.0 Hz). 13C NMR (100 MHz,
(1H, d, J ¼ 7.2 Hz), 7.96 (1H, dd, J ¼ 6.4, 2.0 Hz), 7.68 (1H, d,
J ¼ 8.0 Hz), 7.65 (1H, s), 7.63e7.54 (3H, m), 7.51e7.38 (5H, m), 7.32
(1H, t, J ¼ 7.6 Hz), 7.06 (1H, d, J ¼ 7.6 Hz), 6.84 (1H, d, J ¼ 15.6 Hz),
4.50 (2H, d, J ¼ 6.0 Hz). 13C NMR (100 MHz, DMSO-d6)
d: 164.0,
DMSO-d6)
d
: 166.5, 164.0, 140.9, 140.7, 140.6, 140.0, 139.9, 135.4,
162.0, 140.7, 140.6, 140.4, 140.3, 139.9, 139.6, 135.2, 130.2, 129.4,
129.3, 128.2, 126.7, 125.6, 125.4, 125.3, 123.3, 122.9, 122.8, 118.4,
118.3, 43.2. HRMS calcd for C25H21N2O2S (MþH)þ 413.1324, found
413.1324. HPLC purity: 99.5% (14.5 min).
135.2, 130.2, 129.9, 129.6, 129.5, 129.2, 128.3, 128.2, 127.4, 127.0,
125.9, 122.8, 118.3, 118.2, 43.1. HRMS calcd for C29H25N2O2 (MþH)þ
433.1916, found 433.1914. HPLC purity: 98.1% (15.0 min).

184
Y. Zhao et al. / European Journal of Medicinal Chemistry 122 (2016) 178e184
4.4.14. 40-Bromo-biphenyl-3-carboxylic acid 3-(3-phenyl-
propionylamino)-benzylamide (13k)
and the E-Institutes of Shanghai Universities (EISU) Chemical Biology
Division (E-Institutes of Shanghai Municipal Education Commission
Project E09013) for financial support of this work. We thank Prof. Yingli
Wu (SJTU) for his generous provision of plasmids of pET28a-SENP1,
pET28a-RanGAP and pSAE1/SAE2-UBC9-SUMO1.
Compound 13k was prepared following a similar procedure to
compound 13c. Yield: 37.2%. Mp: 191e193 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 9.90 (1H, s), 9.19 (1H, t, J ¼ 6.0 Hz), 8.21 (1H, s), 7.92
(1H, d, J ¼ 7.6 Hz), 7.86 (1H, d, J ¼ 8.0 Hz), 7.73 (2H, d, J ¼ 8.4 Hz),
7.69 (2H, d, J ¼ 8.4 Hz), 7.59 (1H, t, J ¼ 8.0 Hz), 7.52 (2H, d, J ¼ 7.2 Hz),
7.29e7.22 (5H, m), 7.16 (1H, t, J ¼ 7.2 Hz), 7.01 (1H, d, J ¼ 7.2 Hz),
4.49 (2H, d, J ¼ 6.0 Hz), 2.89 (2H, t, J ¼ 7.2 Hz), 2.60 (2H, t, J ¼ 7.2 Hz).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.06.018.
13C NMR (100 MHz, DMSO-d6)
d: 170.8, 166.4, 141.7, 140.6, 139.8,
139.2, 135.5, 132.4, 129.8, 129.7, 129.4, 129.1, 128.8, 128.7, 127.4,
126.4,125.7,122.4,121.8,118.2,118.0, 43.1, 38.4, 31.3. HRMS calcd for
C
29H26BrN2O2 (MþH)þ 513.1178, found 513.1174. HPLC purity: 99.2%
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Compound 13l was prepared following a similar procedure to
compound 13k. Yield: 30.1%. Mp: 159e161 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 9.9 (1H, s), 9.18 (1H, t, J ¼ 6.0 Hz), 8.21 (1H, s), 7.9 (1H,
d, J ¼ 8.0 Hz), 7.85 (1H, d, J ¼ 8.0 Hz), 7.76 (2H, d, J ¼ 7.2 Hz), 7.58
(1H, t, J ¼ 8.0 Hz), 7.55e7.48 (4H, m), 7.41 (1H, t, J ¼ 7.6 Hz), 7.3e7.21
(5H,m), 7.16 (1H, td, J ¼ 7.6, 1.6 Hz), 7.01 (1H, d, J ¼ 7.6 Hz), 4.49 (2H,
d, J ¼ 6.0 Hz). 2.89 (2H, t, J ¼ 7.6 Hz), 2.60 (2H, t, J ¼ 7.6 Hz). 13C NMR
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132.6, 130.2, 129.5, 129.3, 129.2, 128.4, 128.2, 128.1, 128.0, 127.2,
124.6, 122.8, 122.7, 118.3, 118.2, 43.2. HRMS calcd for C29H27N2O2
(MþH)þ 435.2073, found 435.2069. HPLC purity: 97.3% (15.3 min).
4.4.16. 40-Methoxy-biphenyl-3-carboxylic acid 3-(3-phenyl-
propionylamino)-benzylamide (13m)
Compound 13m was prepared following a similar procedure to
compound 13c. Yield: 37.2%. Mp: 195e197 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 9.90 (1H, s), 9.16 (1H, t, J ¼ 5.6 Hz), 8.16 (1H, s), 7.84
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DMSO-d6) d: 170.8, 166.6, 159.6, 141.7, 140.7, 139.8, 135.4, 132.4,
129.4, 129.4, 129.1, 128.8, 128.7, 128.5, 126.4, 126.3, 125.3, 122.4,
118.2, 118.0, 114.9, 55.7, 43.1, 38.4, 31.3. HRMS calcd for C30H29N2O3
(MþH)þ 465.2178, found 465.2177. HPLC purity: 99.2% (15.6 min).
4.4.17. 3-Benzyloxy-N-[3-(3-phenyl-propionylamino)-benzyl]-
benzamide (13n)
Compound 13n was prepared following a similar procedure to
compound 13c. Yield: 24.7%. Mp: 175e177 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
: 9.88 (1H, s), 9.02 (1H, t, J ¼ 6.0 Hz), 7.55e7.54 (1H, m),
7.51e7.46 (4H, m), 7.45 (1H, s), 7.41e7.36 (3H, m), 7.34e7.31 (1H, m),
7.27e7.20 (5H, m), 7.18e7.13 (2H, m), 6.96 (1H, d, J ¼ 8.0 Hz), 6.15
(1H, s), 4.42 (2H, d, J ¼ 6.0 Hz), 2.88 (2H, t, J ¼ 7.6 Hz), 2.58 (2H, t,
J ¼ 7.6 Hz). 13C NMR (100 MHz, DMSO-d6)
d: 170.8, 166.3, 158.8,
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141.7,140.7,139.8,137.4,136.3,130.0,129.1,128.9,128.8,128.7,128.4,
128.2, 126.4, 122.4, 120.2, 118.3, 118.2, 118.0, 114.0, 69.9, 43.1, 38.4,
31.3. HRMS calcd for
456.2181. HPLC purity: 96.3% (15.1 min).
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30H29N2O3 (MþH)þ 465.2178, found
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€
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Acknowledgements
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We thank National Science Foundation of China (81222042
and 81573264), the Ministry of Science and Technology of China
(2012CB518001), the Young Teacher Start Plan of SJTU (14X100040051),