2-(4-Chlorophenyl)-2-oxoethyl 4-benzamidobenzoate derivatives, a novel class of SENP1 inhibitors: Virtual screening, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2012.09.037

Prostate cancer is one of the most prevalent types of malignant cancers in men and has a high mortality rate among all male cancers. Previous studies have demonstrated that Sentrin/SUMO-specific protease 1 (SENP1) plays an important role in the occurrence and development of prostate cancer, and has been identified as a novel drug target for development of small molecule drugs against prostate cancer. In this paper, we used virtual screening and docking to identify compound J5 as a novel lead compound inhibiting SENP1, from SPECS library. We further investigated the SAR (structure-activity relationship) of the benzoate substituent of compound J5, and discovered compounds 8d and 8e as better small molecule inhibitors of SENP1. Both compounds are the high potent SENP1 small molecule inhibitors discovered up to date, and further lead optimization may lead to a series of novel anti-SENP1 agents. Further SAR studies are in process and will be reported in due course.

Full text of DOI:10.1016/j.bmcl.2012.09.037

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6867–6870
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-(4-Chlorophenyl)-2-oxoethyl 4-benzamidobenzoate derivatives, a novel
class of SENP1 inhibitors: Virtual screening, synthesis and biological evaluation
Yingyi Chen a, , Donghua Wen a, , Zhimin Huang a, , Min Huang a, Yu Luo a, Bin Liu a, Han Lu b, Yingli Wu a,
a,
Yuefeng Peng c, , Jian Zhang
⇑
⇑
a Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine,
Shanghai 200025, PR China
b Department of Anesthesiology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025, PR China
c Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 1068 XueYuan Blvd, Shenzhen University Town, Shenzhen, Guangdong 518055, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2012
Revised 24 August 2012
Accepted 12 September 2012
Available online 20 September 2012
Prostate cancer is one of the most prevalent types of malignant cancers in men and has a high mortality
rate among all male cancers. Previous studies have demonstrated that Sentrin/SUMO-specific protease 1
(SENP1) plays an important role in the occurrence and development of prostate cancer, and has been
identified as a novel drug target for development of small molecule drugs against prostate cancer. In this
paper, we used virtual screening and docking to identify compound J5 as a novel lead compound inhib-
iting SENP1, from SPECS library. We further investigated the SAR (structure–activity relationship) of the
benzoate substituent of compound J5, and discovered compounds 8d and 8e as better small molecule
inhibitors of SENP1. Both compounds are the high potent SENP1 small molecule inhibitors discovered
up to date, and further lead optimization may lead to a series of novel anti-SENP1 agents. Further SAR
studies are in process and will be reported in due course.
Keywords:
SENP1
Virtual screening
Structure–activity relationship
Ó 2012 Elsevier Ltd. All rights reserved.
Modification of proteins by conjugation of Small Ubiquitin-like
Modifier (SUMO) is a key mechanism for regulating many cellular
processes.1–4 SUMOylation is a dynamic process and can be re-
versed by SUMO-specific proteases (SENPs), which are critical in
maintaining the balance between the level of SUMOylated and
unmodified cellular substrates and hence play an important role
in mediating normal cellular physiology.5–7 Recently, SENP1 was
found to be over-expressed in over 50% of more than 100 prosta-
tectomy cases with high-grade prostatic intraepithelial neoplasia
(PIN) and prostate cancer.8,9 The role of SENP1 overexpression in
the development of prostate cancer has been confirmed in SENP1
transgenic mice.10–12 Thus, SENP1 could be the attractive target
of prostate cancer. Although some inhibitors designed for SENP1
were developed by us and other groups,13–16 novel SENP1 inhibi-
tors with better efficacy are still urgently needed.17,18 Thus, the
present study was carried out to seek novel leads as inhibitors of
SENP1 by (a) virtual screening and (b) structural optimization.
Here, we report the process of screen, synthesis, modification
and biological evaluation of a novel type of SENP1 inhibitors.
In this study, virtual screening and docking were performed
using Glide version 4.519 (Schrodinger Suite 2007) with default
docking parameter settings. The structure of SENP1 from SENP1–
SUMO2–RanGAP1 crystal complex, previously determined by Hay
group,20 was retrieved from the Protein Data Bank (PDB entry:
2IY0). Hydrogen atoms and charges were added during a brief
relaxation performed using the ‘Protein Preparation’ module in
Maestro with the ‘preparation and refinement’ option,21 and a re-
strained partial minimization was terminated when the root-
mean-square deviation (RMSD) reached a maximum value of
0.3 Å in order to relieve steric clashes of amino acid residues lo-
cated within 14 Å from the catalytic thiolate of Cys603 were de-
fined as part of the binding site for docking studies. All
crystallographic water molecules were removed from the coordi-
nate set. The compound library for screening is from SPECS com-
pany [SPECS: Chemistry Solutions for Drug Discovery, 2008;
http://www.specs.net/ (accessed October 1, 2011).]. All 180,000
compounds were desalted, neutralized, and parameterized using
the OPLS 2005 force field. Then, tautomers and ionization states
expected to occur in the pH range of 5.0–9.0 were generated using
the ‘ionize’ module. In the docking process, standard-precision (SP)
and extra-precision (XP) docking were respectively adopted to
generate the minimized pose, and the Glide scoring function (G-
Score) was used to select the final pose with the lowest energy con-
formation for each ligand.22 Thirty-eight compounds that ranked in
⇑
Corresponding authors. Tel.: +86 755 86585250 (Y.P.);tel.: +86 21 63846590
776922; fax: +86 21 64154900 (J.Z.).
E-mail addresses: yf.peng@siat.ac.cn (Y. Peng), jian.zhang@sjtu.edu.cn (J. Zhang).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.037

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Y. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6867–6870
Figure 1. Concentration gradient of our compound 8d on the inhibition of SUMO-DRanGAP cleavage by SENP1 and its IC50 curve.
Figure 2. Computational model of compound J5 (left) and 8e (right) bound to SENP1 revealed by docking simulations. For protein, surface of SENP1 are shown in grey. Side
chains of crucial residues in the binding site are shown as stick and labeled. Hydrogen bonds between compounds and SENP1 are depicted in dotted line in yellow. Figures
were generated by Pymol.
the top 100 of both SP and XP scores were eventually purchased
and dissolved in DMSO to biological test.
The purification of recombinant SENP1 was performed as de-
scribed by Hay and co-workers.20 and IC50 values against SENP1
were determined by using our bioassay (Fig. 1) described previ-
ously.16,23,24 Among these 38 compounds, five compounds show
compound J5 form 2 hydrogen bonds with the side chain nitrogen
of Gln597 and epsilon 2 nitrogen of His529, respectively, leading to
the stability of the extended binding mode of compound J5 so as to
play an inhibiting role in the function of SENP1. More importantly,
the binding mode of compound J5 implies that the pocket around
Phe496 and His529 is still unused by the binding of compound J5,
which could be a potential direction for structural optimization.
Based on our lead compound J5, we designed and synthesized a
series of derivatives to further explore the SAR (structure–activity
relationship) of the 2-(4-chlorophenyl)-2-oxoethyl 4-ben-
zamidobenzoate analogs.
SENP1 inhibition at 50
tested to have the most potent inhibition of SENP1 (IC50
2.385 M). The docking poses of compound J5 to the active site
lM (Table S1) and compound J5 was further
=
l
of SENP1 are the same in both SP and XP modes. As showed in
Figure 2, the shape of compound J5 is well stuck into the binding
site composed by Trp465, Asp468, Phe496, His529, Val532,
Gln597 and Cys603. The amide nitrogen and carbonyl oxygen of
The derivatives of compound 8 were prepared using the meth-
ods outlined in Scheme 1. a-Bromo-4-chloroacetophenone (1) was
Scheme 1. Synthesis of compounds 8a–g. Reagents and conditions: (a) 4-Aminobenzoic acid, K2CO3 (yield: 80%); (b) RCOOH, SOCl2; (c) TEA, THF (yield: 8a: 64%; 8b: 76%; 8c:
62%; 8d: 67%; 8e: 72%; 8f: 52%; 8g: 58%)

Y. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6867–6870
6869
Scheme 2. Synthesis of compound 8h. Reagents and conditions: (a) SOCl2 (b) ethyl 4-aminobenzoate, TEA, THF (yield: 75%); (c) Fe powder, NH4Cl, EtOH, H2O, cat concd HCl
(yield: 80%); (d) NaOH, EtOH; (e) 2-bromo-1-(4-chlorophenyl)ethanone, K2CO3 (two steps: 66%).
Scheme 3. Synthesis of compound 8i. Reagents and conditions: (a) 10% Pd/C, H2, MeOH/THF (yield: 54%).
reacted with 4-aminobenzoic acid (2) in the presence of K2CO3 to
afford compound 3, which was then reacted with acid derivatives
in the presence of TEA to afford compounds 8a–g. The compounds
were purified by silica gel chromatography.
The synthesis of compound 8h was started from 3-nitrobenzoic
acid (4) which was converted to the corresponding acid chloride,
and then reacted with ethyl-4-aminobenzoate to afford intermedi-
ate 5 in a high yield. Reduction of compound 5 afforded compound
6, which was further hydrolyzed by treatment with aqueous NaOH
solution, and the resulting compound 7 was converted to com-
pound 8h in the presence of K2CO3 (Scheme 2). Scheme 3 described
the preparation of benzoic acid derivative 8i, which was prepared
by deprotection with 10% Pd/C under H2 atmosphere.
Considering that the methoxy is an electron-donating group, we
designed compounds 8a, 8b and 8d,26 in which the meta-methoxy
is replaced with a nitro, a fluorine and a bromine respectively. After
replacing with an electron-withdrawing group, all these three
compounds showed an improved potency in inhibiting SENP1
protein.
Our modeling studies predicted that a substitute at the para site
of the ring A may clash with the phenyl ring of Phe496, leading to a
decrease in SENP1 inhibiting potency. To further testify our
hypothesis, we designed compounds 8g and 8f, in which para
and meta sites of the ring A are replaced with a pair of methoxy
Table 1
In the work presented here, we focus on the SAR studies of the
ring A, as shown in the Table 1. A number of analogues (com-
pounds 8a–i) were prepared and representative synthetic routes
used to yield compounds are shown in Schemes 1 and 2. In our lead
compound J5, the meta position of the left benzene ring is substi-
tuted by a methoxy. We first designed compound 8c, in which the
methoxy was removed and replaced a hydrogen atom. As tested by
using our inhibition assay, compound 8c showed a slightly de-
creased potency in inhibiting SENP1 protein. By replacing the
methoxy at the meta position with more polar amino and hydroxyl
groups, we obtained compounds 8h and 8i. However, both com-
pounds lost their potency in inhibiting SENP1 protein completely
Binding data for compounds J5 and 8a–i on SENP1
R2
H
N
A
R1
O
B
O
O
C
O
Cl
Compound
R1
R2
SENP1 IC50 (lM)
J5
–OMe
NO2
F
H
Br
–OCH2Ph
–OMe
–OEt
NH2
OH
H
H
H
H
H
H
–OMe
–OEt
H
2.385 0.059
1.856 0.205
1.735 0.020
3.542 0.007
1.175 0.033
1.080 0.010
>50
>50
>50
>50
8a
8b
8c
8d
8e
8f
8g
8h
8i
(IC50 > 50 lM). This result suggests that polar substitutes are not
suitable at the meta site of the ring A.
As predicted by our modeling studies, a meta benzene-ring sub-
stitute may introduce a perpendicular
p–p interaction with the
phenyl ring of Phe496, we designed compound 8e,25 in which the
meta methoxy was replaced by a benzoxy. As expected, compound
H
8e (IC50 = 1.080
lM) was more potent than compound J5 in inhib-
iting SENP1 protein.
IC50 values reflect the average from at least three separate experiments.

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Y. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6867–6870
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and ethoxy groups respectively. As expected, both compounds lost
their SENP1 inhibiting potency completely (IC50 > 50 M).
In conclusion, we explored the preliminary SAR of the ring A. In
this aromatic ring, its meta site disfavors hydrogen donor substi-
tutes, but a benzoxyl group also bears a promising SENP1 inhibit-
l
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ing activity due to the increase of a perpendicular p–p interaction
between its phenyl ring with the phenyl ring of Phe496. An intro-
duction of substitutes at para site based on J5 can result the loss of
SENP1 inhibiting potency, due to the spatial clash with Phe496.
Further SAR investigations are in progress, and will be reported
in due course.
17. Borodovsky, A.; Ovaa, H.; Meester, W. J. N.; Venanzi, E. S.; Bogyo, M. S.;
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Acknowledgments
18. Dobrota˘, C.; Fasci, D.; Ha˘dade, N. D.; Roiban, G. D.; Pop, C.; Meier, V. M.;
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19. Glide, version 5.5, Schrödinger, LLC, New York, NY, 2009.
20. Shen, L.; Tatham, M. H.; Dong, C.; Zagórska, A.; Naismith, J. H.; Hay, R. T. Nat.
Struct. Mol. Biol. 2006, 1069, 13.
21. Maestro, version 9.0, Schrödinger, LLC, New York, NY, 2009.
22. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47(7), 1739.
23. The DNA sequence of the catalytic domain of SENP1 (a.a419–a.a643) (SENP1C)
amplified by PCR from PC3 cDNA library was cloned into PET28a(+) vector. The
This work was supported in part by grants from National Basic
Research Program of China (973 Program) (2011CB504001,
2010CB912104), National Natural Science Foundation of China
(21002062, 21102090, 91013008, 81102513), Innovative Research
Team of Shanghai Municipal Education Commission, the Program
for Professor of Special Appointment (Eastern Scholar) at Shanghai
Institutions of Higher Learning and Shanghai PuJiang Program
(10PJ406800). The NMR data was completed in East China Univer-
sity of Science And Technology (ECUST) and mass data was tested
in Shanghai Institute of Organic Chemistry (SIOC).
SUMO2-
China). Both plasmids were transfected into E. coli BL21, and the expression of
SENP1C was induced with 0.5 mM isopropyl- -thiogalactoside (IPTG) at 16 °C
for 12 h. SUMO2- RanGAP was induced with 1 mM (IPTG) at 25 °C for 12 h.
DRanGAP plasmid was a gift from Dr. Jinke Cheng (SJTU, Shanghai
D
D
Supplementary data
Cell pellets were resuspended in lysis buffer (300 mM NaCl, 50 mM PBS pH 8.0,
10 mM imidazole, 10 mM b-mercaptoethanol and 10% glycerol) and sonicated.
His-taged proteins SENP1C and SUMO2-DRanGAP were purified using Ni-NTA-
agarose and eluted with 50–250 mM gradient of imidazole in 300 mM NaCl,
50 mM PBS pH8.0, 10 mM b-mercaptoacetic ethanol and 10% glycerol.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.
037.
24. 5 nM SENP1C was incubated with compounds for 10 min at 37 °C. Then 6
lg
SUMO2- RanGAP was added and incubated for another 45 min at 37 °C. The
D
reaction was terminated by adding loading buffer and boiling on heat block for
5 min. The proteins were separated by SDS-PAGE and visualized by coomassie
brilliant blue G25.
References and notes
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25. Compound 8e: white solid, yield 58%. 1H NMR (400 MHz, DMSO-d6): d 5.21 (s,
2H); 5.73 (s, 2H); 7.27–7.51 (m, 7H); 7.59–7.68 (m, 4H); 8.00–8.06 (m, 6H);
10.59 (s, 1H). 13C NMR (100 MHz, DMSO-d6) d 67.3, 69.9, 114.6, 118.7, 120.2,
120.7, 124.2, 128.2, 128.4, 128.9, 129.5, 130.1, 130.2, 130.8, 133.0, 136.3, 137.2,
139.3, 144.4, 158.8, 165.3, 166.1, 192.5 ppm. HR-ESI-MS: 522.10704
(C29H22ClNO5, [M+Na+])
26. Compound 8d: white solid, yield 54%. 1H NMR (400 MHz, DMSO-d6): d 5.73 (s,
2H); 7.51–7.55 (m, 1H); 7.66–7.68 (d, J = 8.4 Hz, 2H); 7.82–7.84 (d, J = 8 Hz,
1H); 7.98–8.07 (m, 7H); 8.18 (s, 1H); 10.71 (s, 1H). 13C NMR (100 MHz, DMSO-
d6) d 67.4, 120.2, 122.2, 124.4, 127.5, 129.5, 130.2, 130.8, 130.9, 131.2, 133.0,
135.1, 137.1, 139.3, 144.1, 164.9, 165.2, 192.5 ppm. HR-ESI-MS: 493.97708
(C22H15BrClNO4, [M+Na+])
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