Identification of novel inhibitors of p53-MDM2 interaction facilitated by pharmacophore-based virtual screening combining molecular docking strategy

Article abstract of DOI:10.1039/c2md20208e

3D pharmacophore models for inhibitors of p53-MDM2 interaction were developed. The pharmacophore-based virtual screening of an in-house compound database combined with docking studies led to the identification of compound MCL0527 as a novel lead (MDM2 binding IC₅₀ = 4.23 μM). Initial structure optimization yielded some derivatives with improved p53-MDM2 binding inhibitory activities. Furthermore, all target compounds showed potent anti-proliferative effect against human tumor cell lines with a generally selective profile on wild-type p53 cell lines. The Royal Society of Chemistry.

Full text of DOI:10.1039/c2md20208e

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Medicinal Chemistry Communications
Identification of novel inhibitors of p53-MDM2 interaction
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facilitated by pharmacophore-based virtual screening
combining molecular docking strategy
Weisi Wanga, Xiaolei Zhua, Xueqin Honga, Lin Zhengb, Hong Zhub, Yongzhou Hua*
aZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
bInstitute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
*E-mail: huyz@zju.edu.cn; Fax: +86 571 8820 8460; Tel: +086 571 8820 8460
Abstract:
3D pharmacophore models for inhibitors of p53-MDM2 interaction were developed. The
pharmacophore-based virtual screening of an in-house compound database combined with
docking studies led to the identification of compound MCL0527 as a novel lead (MDM2 binding
IC50 = 4.23μM). Initial structure optimization yielded some derivatives with improved p53-MDM2
binding inhibitory activities. Furthermore, all target compounds showed potent anti-proliferative
effect against human tumor cell lines with a generally selective profile on wild-type p53 cell lines.
Introduction
The tumor suppressor p53 plays a prominent role in the regulation of cell cycle, apoptosis,
and DNA repair via a complex network of proteins known as the p53 pathway.1, 2 It is an attractive
molecule target for cancer therapy. However, p53 is one of the most frequently altered genes in
human tumors. Approximately 50% of all human malignancies harbor mutations or deletions in
the DNA-binding domain of p53.3, 4 Although the remaining 50% of all human tumors express
wild-type p53, aberrations in p53 regulation and/or defective signaling in the p53 pathway result
in many cases of inadequate tumor suppression. One important cause involves MDM2, the
principal negative regulator of p53.5-7 Restoration of the p53 function by disruption of the
p53-MDM2 interaction through utilizing small molecule inhibitors is now recognized as a
promising strategy for tumor treatment.8-10
Pharmacophore-based virtual screening is a widely used rational drug design strategy for
identification of novel hits or leads with diverse chemical scaffolds. Although a few
pharmacophore models of p53-MDM2 binding inhibitors were established many years ago11, 12
,
their pharmacophoric features were no more suitable for discovering promising inhibitors
nowadays. Since many kinds of potent and selective inhibitors identified in recent five years were
not considered in former models. Moreover, along with the improving understanding of the
interaction mechanism of inhibitors and MDM2, the pharmacophore elements of former models
were not consistent with the generic structural features and principles for small molecule
inhibitors of p53-MDM2 interaction. So as a part of on-going effort to identify new chemotypes of
p53-MDM2 binding inhibitors for potential drug development, we performed an updated and
reliable pharmacophore modeling study that incorporated virtual screening of candidate inhibitors.
In the present study, pharmacophore-based virtual screening against an in-house database
revealed a novel series of p53-MDM2 binding inhibitors possessing a 3,4,5-trisubstituted

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aminothiophene scaffold. Synthesized target compounds exhibited good p53-MDM2 binding
inhibitory activities, together with potent cytotoxic activities against the tested tumor cell linVeise.w Article Online
Also, most of the compounds showed a generally selective profile on wild-type p53 cell lines. It is
the first time reported that the pharmacophore-based virtual screening hits showed good activities
both in MDM2 binding and cell growth inhibition. Further pharmacophore mapping and docking
studies indicated the interaction mode of 3,4,5-trisubstituted aminothiophenes with MDM2. The
identified leads, MCL0527 and MCL0527-1~3, provided starting points for further modification
and optimization.
Results and discussion
Pharmacophore generation and validation.
For the pharmacophore modeling studies, a set of 98 compounds was selected from
literatures and patents considering their chemical structure diversity and certain coverage of
activity range (ESI, Table. S2). Their MDM2 binding affinity data spanned over 6 orders of
magnitude (from 0.002μM to 480μM). These inhibitors were divided into a training set (ESI, Fig.
S1) of 29 compounds and a test set (ESI, Fig. S2) of 69 compounds. The pharmacophore
hypotheses were generated using the Catalyst/HypoGen program (Catalyst 4.1, Molecular
Simulations Inc., SanDiego, CA). The best pharmacophore model (Hypo_1, Fig. 1) was evaluated
in terms of cost functions and other statistical parameters such as correlation coefficient and
RMSD value (ESI, Table. S1). Hypo_1 featured five pharmacophore elements: two ring aromatic
(RA, colored by orange) and three hydrophobic group (HY, colored by cyan) (Fig. 1). With the
purpose to evaluate the reliability and accuracy of Hypo_1, prediction capability of the test set
(see ESI, Fig. S3B and Table S2), Cat-Scramble validation (see ESI, Fig. S3C) and Enrichment
Factor (EF) studies (see ESI) were performed.
Figure. 1. The topological features of the best pharmacophore model Hypo_1: five
pharmacophoric features, including two ring aromatic (RA, orange) and three hydrophobic group
(HY, cyan).
Pharmacophore-based virtual screening combining molecular docking studies.
Given the above results, Hypo_1 showed superior recognition ability for p53-MDM2 binding
inhibitors. Consequently, Hypo_l hypothesis was applied as a 3D search query to screen our
in-house database. Our in-house database is composed of the compounds synthesized in our lab
(~3000 molecules) and from Maybridge database (~60,000 molecules). All the compounds are
available with searchable 3D structures and multi-conformers. The first step was

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Medicinal Chemistry Communications
ligand-pharmacophore mapping. Over 100 compounds mapped well with the features of Hypo_1
were considered as potential hits for further exploration. The second step was to confirm the
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interaction between each compound and MDM2 protein binding site using CDOCKER (Discovery
Studio, version 2.1; Accelrys, San Diego, CA, USA, 2008). The X-ray crystal structure of MDM2
bound to the transactivation domain of p53 (PDB ID: 1YCR) was used for the docking calculation.
Finally, 10 compounds (ESI, Fig. S4) were identified as preferable hits in both
pharmacophore-based screening and molecular docking studies (ESI, Table S3).
Figure 2. The virtual evaluated results of preferable hit MCL0527 by Hypo_1 and molecular
docking studies. (a) The chemical structure of MCL0527. (b) MCL0527 was aligned to Hypo_1.
(c) Interaction mode between MDM2 and MCL0527. Three key amino acid residues of p53
(Phe19, Trp23 and Leu26) are colored in light blue.
Among them, a 3,4,5-trisubstituted aminothiophene derivative MCL0527 (synthesized by our
lab, Fig. 2a) showed the best Fit-value (11.56) and lowest docking energy (-32.26 Kcal/mol) in
virtual evaluation. As shown in Fig. 2, the two 4-chlorophenyl groups at 4-and 5-position of
thiophene ring of MCL0527 map well on RA_1, HY_2, RA_2 and HY_3 features of Hypo_1,
while the methyl ester at 3-position map well with the HY_1 feature. Based on the summarized
generic structural features for small molecule inhibitors of p53-MDM2 interaction, a rigid and
appropriate core structure projecting three lipophilic groups into the three binding pockets (Phe19,
Tpr23, and Leu2 6) in just the right directions is crucial for the construction of MDM2 antagonists
(Fig. 3). Several highly potent inhibitors fit the principle very well, such as the cis-imidazolines
(Nutlin-313), benzodiazepinediones (TDP66575914), spiro-oxindoles (MI-21915) and piperidinones
(AM-855316). The structural construction of MCL0527 is also consistent with the general features.
According to the docking simulation results, the thiophene ring serves as a core structure to
project three hydrophobic groups into the MDM2 binding cleft at suitable directions. Two

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4-chlorophenyl groups and one methyl ester mimic the three key residues of p53 (Leu26, Trp23
and Phe19, respectively) to form the hydrophobic interactions with protein (Fig. 2c). From a
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chemical structure viewpoint, the privileged bis-chloro-aromatic structure is also contained in the
structure of MCL0527.
Figure 3. Generic structural features for small-molecule p53-MDM2 interaction inhibitor. The
core structures are colored in blue. The groups that interact with the Trp23, Leu26 and Phe19
binding pockets are shown in green, purple and pink, respectively.
Chemistry
In order to validate the reliability of a combination of Hypo_1 and docking studies,
MCL0527 was selected as a lead compound for further studies. In addition, after a careful analysis
of the docking results of MCL0527 and MDM2, a spare room not occupied by methyl ester at the
Phe19 binding site was observed. So we decided to carry out a preliminary structure optimization
at the 3-carboxyl group of thiophene ring to find out whether the MDM2 binding affinities of the
derivatives can be improved. Consequently, the pyrolidine, cyclopropylamine, cyclohexylamine
and aniline groups are introduced. MCL0527 and its derivatives were synthesized for further
biological evaluation including MDM2 binding affinities and cytotoxic activities against several
cancer cell lines.
The synthetic approach taken for the preparation of MCL series was summarized in Scheme
1. Reaction of 4-chlorophenylacetylchloride with chlorobenzene gave the Friedel–Crafts acylation
product (1). According to the Gewald synthesis 17, condensation of the acylation compound (1)
with methyl cyanoacetate or 2-cyanoacetamide derivatives in the presence of TiCl4 afforded the

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Medicinal Chemistry Communications
olefin intermediates (2). Treatment of olefin intermediates (2) with elemental sulfur and
diethylamine furnished the cyclization of thiophene ring leading to the target compounds.
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O
Cl
Cl
Cl
O
a
b
+
O
Cl
CN
1
R
Cl
Cl
O
CN
Cl
R
S
c
NH2
Cl
O
2
R
Cl
MCL0527
R= CH3O
MCL0527-1 R=
MCL0527-2 R=
N
NH
H
N
MCL0527-3
MCL0527-4
R=
R=
H
N
Scheme 1. Synthetic route of MCL0527 and its derivatives. Reagent and conditions: (a) AlCl3,
CH2Cl2, rt, 89%; (b) methyl cyanoacetate or 2-cyanoacetamide derivatives, TiCl4, pyridine, THF,
rt; (c) sulfur, NHEt2, THF, rt, two steps 54~80%.
P53-MDM2 binding inhibitory activity and cytotoxic activity against cancer cell lines
P53-MDM2 binding inhibitory activities of lead compound MCL0527 and its derivatives
(Scheme 1) were evaluated by fluorescence-polarization (FP)-based binding assay (Table 1).18
Nutlin-3 (racemate mixture) was used as a positive control, the concentration was based on
Nutlin-3a. As shown in Table 1, MCL0527 and its derivatives MCL0527-1~3 exhibited good
MDM2 binding affinities with IC50 values ranging from 0.90 to 4.23 μM. Introducing proper
bulky substituents at the carboxyl group, such as pyrrolidine (MCL0527-1), cyclopropylamine
(MCL0527-2) and cyclohexylamine (MCL0527-3) imparted positive effect on inhibitory potency
compared with that of lead (MCL0527). Among the evaluated compounds, MCL0527-3 was the
most potent inhibitor of p53-MDM2 interaction exhibiting IC50 of 0.90 μM. However, a
significant decrease in the activity was observed when the aniline group is attached at the
3-position carboxyl of the thiophene ring (MCL0527-4).
Then the 3,4,5-trisubstituted aminothiophene derivatives were tested for their cytotoxic
activities against five human cancer cell lines including two wild-type p53 cell lines (A549 and
HCT116 ), one p53 null cell line (PC3), one p53 mutated cell line (SW620) and one MDM2 gene
amplified cell line (SJSA-1, wild-type p53). The results are summarized in Table 2. Generally,
most of the derivatives (MCL0527 and MCL0527-1~3) showed better cytotoxicities in wild-type
p53 cell lines (A549 and HCT116) rather than in p53 null (PC3) and mutated cell lines (SW620).
MCL0527-1~3 were more potent in A549 and HCT116 cell lines compared with the lead
compound MCL0527. In HCT116 cell line, MCL0527 and MCL0527-1~3 exhibited
1.3~19-folds improvement on growth inhibitory activities than that of Nultin-3. Significant drop in
the activities of MCL0527-4 in all five cell lines were observed. All the tested compounds
displayed moderate cytotoxic activities in MDM2 gene amplified cell line, SJSA-1. As above

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mentioned, MCL0527 and its derivatives MCL0527-1~3 can be considered as interesting and
promising candidates for further chemical optimization to obtain potent inhibitors for canVceierw Article Online
therapy.
Table 1. MDM2 binding affinities of target compounds.
MDM2 binding IC50 (μM)
Compd.
Exp.a
Pred. Error factor
MCL0527
MCL0527-1
MCL0527-2
MCL0527-3
MCL0527-4
Nutlin-3
4.23 ± 0.91
2.84 ± 0.22
2.08 ± 0.19
0.90 ± 0.03
>50
0.79
1.32
2.3
-5.4
-2.2
1.1
1.0
—
0.96
170
0.39
0.11 ± 0.009
1.8
a The mean value of at least three separate determinations.
Table 2. In vitro cytotoxicities of target compounds.
SRB IC50 (μM)
Compd.
A549a
HCT116a
1.66 ± 0.17
3.63 ± 0.34
0.24 ± 0.06
0.59 ± 0.16
14.47 ± 0.88
4.63 ± 0.78
PC3a
SW620a
SJSA-1
40.9
MCL0527
MCL0527-1
MCL0527-2
MCL0527-3
5.04 ± 0.72
0.89 ± 0.29
1.95 ± 0.14
2.41 ± 0.33
2.78 ± 0.30
6.22 ± 0.79
3.68 ± 0.31
7.3 ± 0.97
>50
3.23 ± 0.40
3.87 ± 0.29
1.74 ± 0.16
3.07 ± 0.33
39.74 ±4.91
0.38 ± 0.09
17.6
31.9
102.3
>500
1.81
MCL0527-4 25.03 ± 3.91
Nutlin-3 0.41 ± 0.09
6.37 ± 1.00
a The mean value of at least three separate determinations.
Conclusion
In this study, a pharmacophore-based virtual screening protocol was performed to identify a
series of p53-MDM2 binding inhibitors with a novel scaffold. These 3,4,5-trisubstituted
aminothiophene derivatives exhibited good p53-MDM2 binding inhibitory activities. Also, most
of the compounds showed potent cytotoxic activities against human tumor cell lines with a
generally selective profile on wild-type p53 cell lines. These results indicate that the
Hypo_1-based virtual screening is a rational and reliable strategy to discover novel p53-MDM2
binding inhibitors. Compared with other types of potent inhibitors, it is noteworthy that the
common structure of 3,4,5-trisubstituted aminothiophenes is quite different. Since the core
scaffold is the most important part of the inhibitor structural construction, and these derivatives
bear an aromatic core, thiophene ring, which is distinct from other alkyl core structures (such as
the imidazoline, diazepane, pyrrolidine and piperidinone). This indicates that aromatic core
structures can also be utilized to identify potent p53-MDM2 binding inhibitors. Chemical
synthesis of the analogous compounds of MCL0527, focusing on the modification at 2-amino and
3-carboxyl groups as well as the halogen substituents of two phenyl rings, are currently in
progress. Such studies could optimize MCL0527 into novel anti-cancer pharmaceuticals that
disrupt the p53-MDM2 interaction with higher potency in vitro/in vivo.

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Medicinal Chemistry Communications
Materials and methods
Chemistry
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Melting points were determined with a B-540 Büchi apparatus and are uncorrected. NMR
spectra were recorded on a Brüker 500 (500 MHz) spectrometer (chemical shifts are given in
ppm(δ) relative to TMS as internal standard, coupling constants (J) are in hertz (Hz), and signals
are using the following abbreviations: s, singlet; d, doublet; t, triplet; m, multiplet, etc. High
resolution mass spectra (HR-MS), ESI (positive) were recorded on a Bruker micrOTOF-QⅡ
spectrometer. Mass spectra (MS), ESI (positive) were recorded on an Esquire-LC-00075
spectrometer. The purities of compounds MCL0527 and MCL0527-1~4 were above 95% tested on
a Agilent 1100 series HPLC system. All yields are unoptimized and generally represent the result
of a single experiment.
MDM2 protein expression and purification
MDM2 (1-118) plasmid was provided by Dr. Shaomeng Wang's group, and transformed into
Escherichia coli BL-21 (DE3). Cultures were grown at 37℃ in TB medium, and induced by 0.4
mM IPTG at an OD600 of 0.6 at 18℃ for 20 h. Cells were lysed in 50 mM Tries, pH 7.5 buffer
containing 500 mM NaCl and 10% glycerol. MDM2 (1-118) was purified from the soluble
fraction using Ni-NTA resin, and desalted in PBS buffer pH 7.5, 150 mM NaCl and 10% glycerol.
The protein was purified to >95% as judged SDS–PAGE.
Fluorescence polarization competitive binding assay
Measurements were made with a Beckmancoulter DTX880 Multilabel Plate Reader using a
485 nM excitation filter and a 535 nM emission filter. Assays were performed in Microtiter
96-Well black and round bottom plates. Nutlin-3 was used as a positive control, while DMSO was
used as a negative control. Assays were performed in duplicate and repeated at least three times on
separate days. Competition experiments were carried out in a total volume of 20 μL 40 mM
Tris–HCl, pH 7.5, 150 mM NaCl, and 1 mM DTT, 4% DMSO. Probe peptide was present at a
final concentration of 1 nM, and MDM2 was present at a final concentration of 10nM. Plates were
allowed to incubate at room temperature for 1 h prior to measurement.
Cell proliferation assay
Cell proliferation was assessed by sulforhodamin B (SRB) assay. Briefly, cells were seeded
into 96-well plates and cultured overnight; exposed to serial concentrations of compounds for 72 h.
Cells were then washed with PBS and fixed with 10% (w/v) trichloroacetic acid at 4 oC for an
hour. After washing, the cells were stained for 30 min with 0.4% SRB dissolved in 1% acetic acid.
Then the cells were washed by 1% acetic acid for 5 times, and protein-bound dye was extracted
with 10 mM unbuffered Tris base. The absorbance was measured at 515 nm using a multiscan
spectrum (Thermo Electron Co., Vantaa, Finland). The inhibition rate on cell proliferation of each
well was calculated as (A515 control cells – A515 treated cells )/ A515 control cells × 100%. The
average IC50 values were determined by Logit method from at least three independent tests.
Synthetic procedures of target compounds
Synthesis of 1,2-bis(4-chlorophenyl)ethanone19 and 2-cyanoacetamide derivatives20 are exactly
following the procedures of reference 19 and 20.

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Synthesis of 3,4,5-trisubstituted aminothiophene derivatives (MCL0527, and MCL0527-1~V4)ie:w Article Online
general procedures
A mixture of 1,2-bis(4-chlorophenyl)ethanone (1, 1.1mmol) and methyl cyanoacetate or
2-cyanoacetamide derivatives (2.2mmol) in THF (10mL) was slowly added to a solution of TiCl4
(5.5mmol) in dry THF (20mL) at 0℃. After removing the ice bath, pyridine (0.2mL) was added.
And the reaction mixture was stirred at room temperature overnight. The progress of the reaction
was monitored by TLC. After completion of the reaction, THF was removed under reduced
pressure and the reaction mixture was dissolved in ethyl acetate (30 mL), washed with water (2 x
30 mL) and brine (2 x 30 mL). The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The crude olefin intermediate (2) was used for the next step
without purification. To a stirred solution of the crude product (2) in THF (20mL), elemental
sulfur (2.2mmol) and diethylamine (0.2mL) were added. The reaction mixture was stirred at room
temperature for 2~4h. After completion of the reaction, THF was removed under reduced pressure
and the reaction mixture was dissolved in ethyl acetate (20 mL), washed with water (2 x 20 mL)
and brine (2 x 20 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The crude products were purified by column chromatography to give the
target compounds (PE: EtOAc: CH2Cl2=2:1:1~1:3:1, v/v/v).
methyl 2-amino-4,5-bis(4-chlorophenyl)thiophene-3-carboxylate, MCL0527
White solid (two steps 65%). mp: 181 ~ 183 °C. 1H NMR (500 MHz, CDCl3): δ = 7.26 (d, J = 8.4
Hz, 2H, Ar-H), 7.11 (d, J = 6.4 Hz, 2H, Ar-H), 7.07 (d, J = 6.2 Hz, 2H, Ar-H), 6.91 (d, J = 8.5 Hz,
2H, Ar-H), 6.17 (br, 2H, NH2), 3.51 (s, 3H, CH3). 13C NMR (500 MHz, DMSO): δ = 164.57,
163.54, 135.99, 135.12, 132.37, 131.59, 131.44, 131.08, 129.94, 128.31, 127.52, 117.73, 104.92,
50.17. HR-ESIMS: m/z = 399.9933 [M+Na]+. ESI-MS: m/z = 378 [M+1]+.
(2-amino-4,5-bis(4-chlorophenyl)thiophen-3-yl)(pyrrolidin-1-yl)methanone, MCL0527-1
White solid (two steps 54%). mp: 203 ~ 205 °C. 1H NMR (500 MHz, CDCl3): δ = 7.23 (d, J = 8.3
Hz, 2H, Ar-H), 7.16 (d, J = 8.3 Hz, 2H, Ar-H), 7.10 (d, J = 8.3 Hz, 2H, Ar-H), 6.99 (d, J = 8.2 Hz,
2H, Ar-H), 6.20 (br, 2H, NH2), 3.43 (m, 4H, pyrolidine-H), 1.40 (m, 4H, pyrolidine-H). ESI-MS:
m/z = 417 [M+1]+.
2-amino-4,5-bis(4-chlorophenyl)-N-cyclopropylthiophene-3-carboxamide, MCL0527-2
Yellow solid (two steps 80%). mp: 167 ~ 169 °C. 1H NMR (500 MHz, CDCl3):δ = 7.39 (d, J = 8.4
Hz, 2H, Ar-H), 7.18 (d, J = 8.4 Hz, 2H, Ar-H), 7.13 (d, J = 8.6 Hz, 2H, Ar-H), 6.91 (d, J = 8.6 Hz,
2H, Ar-H), 6.26 (br, 2H, NH2), 4.87 (m, 1H, NH), 2.60 (dt, J = 10.4, 1H, CH), 0.64 (m, 2H, CH2),
0.03 (m, 2H, CH2)；ESI-MS: m/z = 403 [M+1]+.
2-amino-4,5-bis(4-chlorophenyl)-N-cyclohexylthiophene-3-carboxamide, MCL0527-3
Yellow solid (two steps 70%). mp: 179 ~ 181 °C. 1H NMR (500 MHz, CDCl3): δ = 7.40 (d, J = 8.4
Hz, 2H, Ar-H), 7.24 (d, J = 8.4 Hz, 2H, Ar-H), 7.13 (d, J = 8.5 Hz, 2H, Ar-H), 6.91 (d, J = 8.5 Hz,
2H, Ar-H), 6.05 (br, 2H, NH2), 4.81 (d, J = 7.5 Hz, 1H, NH), 3.72 (m, 1H, cyclohexane-H), 1.63
(m, 2H, cyclohexane-H), 1.45 (m, 3H, cyclohexane-H), 1.27 (m, 2H, cyclohexane-H), 1.09 (m, 1H,
cyclohexane-H), 0.75 (d, J = 9.9 Hz, 2H, cyclohexane-H). ESI-MS: m/z = 445 [M+1]+.
2-amino-4,5-bis(4-chlorophenyl)-N-phenylthiophene-3-carboxamide, MCL0527-4
1
Yellow solid (two steps 54%). mp: 168 ~ 170 °C. H NMR (500 MHz, CDCl3) δ 7.46 (d, J = 8.3
Hz, 2H, Ar-H), 7.32 (d, J = 8.3 Hz, 2H, Ar-H), 7.21 (t, J = 7.8 Hz, 2H, Ar-H), 7.14 (d, J = 8.5 Hz,

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Medicinal Chemistry Communications
2H, Ar-H), 7.01 (t, J = 7.2 Hz, 3H, Ar-H), 6.94 (d, J = 8.5 Hz, 2H, Ar-H), 6.66 (s, 1H, NH).
ESI-MS: m/z = 439 [M+1]+.
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Acknowledgments
This study was financially supported by the National Natural Science Foundation of China
(30873163). The authors also thank Dr. Shaomeng Wang for providing the plasmid of MDM2
(1-118) and the tracer, and Dr. Jia Li for helping with the protein purification experiment.
References:
1. S. L. Harris and A. J. Levine, Oncogene., 2005, 24, 2899-2908.
2. B. Vogelstein, D. Lane and A. J. Levine, Nature., 2000, 408, 307-310.
3. M. Hollstein, K. Rice, M. S. Greenblatt, T. Soussi, R. Fuchs, T. Sorlie, E. Hovig, B.
Smith-Sorensen, R. Montesano and C. C. Harris, Nucleic Acids Res., 1994, 22, 3551-3555.
4. P. Hainaut, T. Soussi, B. Shomer, M. Hollstein, M. Greenblatt, E. Hovig, C. C. Harris and R.
Montesano, Nucleic Acids Res., 1997, 25, 151-157.
5. J. D. Oliner, J. A. Pietenpol, S. Thiagalingam, J. Gyuris, K. W. Kinzler and B. Vogelstein, Nature.,
1993, 362, 857-860.
6. J. D. Oliner, K. W. Kinzler, P. S. Meltzer, D. L. George and B. Vogelstein, Nature., 1992, 358,
80-83.
7. S. S. Fakharzadeh, S. P. Trusko and D. L. George, Embo J., 1991, 10, 1565-1569.
8. M. P. Dickens, R. Fitzgerald and P. M. Fischer, Semin Cancer Biol., 2010, 20, 10-18.
9. S. Shangary and S. Wang, Annu Rev Pharmacol Toxicol., 2009, 49, 223-241.
10. W. Wang and Y. Hu, Med Res Rev., 2011, DOI: 10.1002/med.20236.
11. R. Sheng, C. Q. Hu, W. H. Huang and Y. Z. Hu, Acta Phy Chim Sin., 2007, 23, 1815-1820.
12. P. S. Galatin and D. J. Abraham, J Med Chem., 2004, 47, 4163-4165.
13. L. T. Vassilev, B. T. Vu, B. Graves, D. Carvajal, F. Podlaski, Z. Filipovic, N. Kong, U. Kammlott,
C. Lukacs, C. Klein, N. Fotouhi and E. A. Liu, Science., 2004, 303, 844-848.
14. H. K. Koblish, S. Zhao, C. F. Franks, R. R. Donatelli, R. M. Tominovich, L. V. LaFrance, K. A.
Leonard, J. M. Gushue, D. J. Parks, R. R. Calvo, K. L. Milkiewicz, J. J. Marugan, P. Raboisson, M. D.
Cummings, B. L. Grasberger, D. L. Johnson, T. Lu, C. J. Molloy and A. C. Maroney, Mol Cancer
Ther., 2006, 5, 160-169.
15. S. Shangary, D. Qin, D. McEachern, M. Liu, R. S. Miller, S. Qiu, Z. Nikolovska-Coleska, K.
Ding, G. Wang, J. Chen, D. Bernard, J. Zhang, Y. Lu, Q. Gu, R. B. Shah, K. J. Pienta, X. Ling, S.
Kang, M. Guo, Y. Sun, D. Yang and S. Wang, Proc Natl Acad Sci U S A., 2008, 105, 3933-3938.
16. Y. Rew, D. Sun, D. T. F. Gonzalez-Lopez, M. D. Bartberger, H. P. Beck, J. Canon, A. Chen, D.
Chow, J. Deignan, B. M. Fox, D. Gustin, X. Huang, M. Jiang, X. Jiao, L. Jin, F. Kayser, D. J. Kopecky,
Y. Li, M. C. Lo, A. M. Long, K. Michelsen, J. D. Oliner, T. Osgood, M. Ragains, A. Y. Saiki, S.
Schneider, M. Toteva, P. Yakowec, X. Yan, Q. Ye, D. Yu, X. Zhao, J. Zhou, J. C. Medina and S. H.
Olson, J Med Chem., 2012, 55, 4936-4954.
17. K. Gewald, E. Schinke and H. Böttcher, Chemische Berichte., 1966, 99, 94-100.
18. K. Ding, Y. Lu, Z. Nikolovska-Coleska, G. Wang, S. Qiu, S. Shangary, W. Gao, D. Qin, J.
Stuckey, K. Krajewski, P. P. Roller and S. Wang, J Med Chem., 2006, 49, 3432-3435.
19. H. Lutjens, A. Zickgraf, H. Figler, J. Linden, R. A. Olsson and P. J. Scammells, J Med Chem.,
2003, 46, 1870-1877.

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20. K. Wang, K. Nguyen, Y. Huang and A. Domling, J Comb Chem., 2009, 11, 920-927.
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