Design, synthesis and biological evaluation of novel 3,4,5-trisubstituted aminothiophenes as inhibitors of p53-MDM2 interaction. Part 1

Article abstract of DOI:10.1016/j.bmc.2013.03.061

A series of 3,4,5-trisubstituted aminothiophenes were designed, synthesized, and evaluated for their p53-MDM2 binding inhibitory potency and anti-proliferation activities against A549 and PC3 tumor cell lines. Fourteen compounds had appreciably improved MDM2 binding affinities than lead compound MCL0527 (3) and a few compounds showed comparable activities to that of Nutlin-3. Meanwhile, most of the 3,4,5-trisubstituted aminothiophenes displayed better or equivalent anti-proliferation activities against wild-type p53 cell line A549 compared to that of Nutlin-3. Over ten compounds exhibited desirable selective profiles of p53 status. Particularly, compounds 9, 16 and 18 displayed 22-, 6- and 22-fold selectivity of p53 status, respectively, much better than that of Nutlin-3 (fourfold).

Full text of DOI:10.1016/j.bmc.2013.03.061

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel
3,4,5-trisubstituted aminothiophenes as inhibitors of p53–MDM2
interaction. Part 1
Weisi Wang a, Shihao Shangguan a, Ni Qiu b, Chunqi Hu a, Lei Zhang b, Yongzhou Hu a,
⇑
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
A series of 3,4,5-trisubstituted aminothiophenes were designed, synthesized, and evaluated for their
p53–MDM2 binding inhibitory potency and anti-proliferation activities against A549 and PC3 tumor cell
lines. Fourteen compounds had appreciably improved MDM2 binding affinities than lead compound
MCL0527 (3) and a few compounds showed comparable activities to that of Nutlin-3. Meanwhile, most
of the 3,4,5-trisubstituted aminothiophenes displayed better or equivalent anti-proliferation activities
against wild-type p53 cell line A549 compared to that of Nutlin-3. Over ten compounds exhibited desir-
able selective profiles of p53 status. Particularly, compounds 9, 16 and 18 displayed 22-, 6- and 22-fold
selectivity of p53 status, respectively, much better than that of Nutlin-3 (fourfold).
Keywords:
P53–MDM2 binding inhibitor
3,4,5-Trisubstituted aminothiophenes
Anti-cancer
SARs
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
(2) focusing on the modification at 3-carboxy group, using amides
to replace esters; (3) derivatization at the 2-amino group to adjust
The tumor suppressor p53, key node of the p53 pathway, plays
essential roles in various cellular activities, such as cell cycle, apop-
tosis and DNA repair.1–4 It is an attractive molecule target for tu-
mor treatment. 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 p53.5,6 Although the remaining 50% of all human tumors
express wild-type p53, its activity can be inhibited by over-expres-
sion of MDM2 oncoprotein (murine double minute 2, also fre-
quently referred to as HDM2 in human), the master negative
regulator of p53.7–9 Reactivation of p53 by utilizing small-molecule
p53–MDM2 binding inhibitors is now recognized as a promising
strategy for cancer therapy.10–12
the physicochemical properties. In this work, we reported the syn-
thesis and biological evaluation of this new series of p53–MDM2
binding inhibitors. Besides, the preliminary structure–activity rela-
tionships (SARs) of the target compounds were discussed.
2. Results and discussion
2.1. Chemistry
The synthetic routes for 3,4,5-trisubstituted aminothiophene
derivatives are summarized in Scheme 1. Reaction of 4-chloro-
In our previous work, a promising lead compound (MCL0527)
with a novel scaffold was identified by a pharmacophore-based
virtual screening strategy combining molecular docking studies
(Fig. 1).13 The biological characterization revealed that MCL0527
and its derivatives displayed good MDM2 binding affinities and
anti-proliferative effects against four tumor cell lines. Herein, with
the attempt to find better p53–MDM2 binding inhibitors, the
structural modification and optimization of MCL0527 was carried
out incorporating the following concepts: (1) retaining the 2-ami-
no-4,5-bis(4-chlorophenyl)thiophene-3-carboxylic acid scaffold;
⇑
Corresponding author. Tel./fax: +86 571 8820 8460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
Figure 1. Structure of the lead compound MCL0527.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.061
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.061

2
W. Wang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Scheme 1. Synthetic routes of target compounds (5–31). Reagents and conditions: (a) AlCl3, CH2Cl2, rt; (b) methyl cyanoacetate, TiCl4, pyridine, THF, rt; (c) sulfur, NHEt2, THF,
rt; (d) 2 N NaOH, EtOH, reflux; (e) EDCI, HOBt, amines, CH2Cl2, rt; (f) for 26, acetyl chloride, TEA, CH2Cl2, rt; for 27, chloropropionyl chloride, TEA, CH2Cl2, rt; morpholine, TEA,
dioxane, reflux; (g) methylsulfonyl chloride, TEA, CH2Cl2, rt; (h) ethyl carbonochloridate, K2CO3, toluene, rt; (i) 2-chloroethyl isocyanate, TEA, CH2Cl2, reflux; TEA, dioxane,
reflux, morpholine for 30, diethylamine for 31.
phenylacetylchloride with chlorobenzene gave the Friedel–Crafts
acylation product 1. According to the Gewald synthesis,14 conden-
sation of compound 1 with methyl cyanoacetate in the presence of
TiCl4 afforded the olefin intermediate 2. Treatment of 2 with ele-
mental sulfur and diethyl amine led to the lead compound 3
(MCL0527). Hydrolysis of 3 with 2 N NaOH aqueous solution pro-
duced the common intermediate 4. Then, 4 was treated with
appropriate amines in the presence of EDCI and HOBt yielded the
desired compounds (5–25).
As shown in Table 1, most of the 3,4,5-trisubstituted aminothi-
ophene derivatives exhibited micromolar to nanomolar affinities
towards MDM2. Fourteen compounds showed improved binding
inhibitory activities than that of MCL0527 (3) and a few com-
pounds (5, 7, 8, 16, 22 and 24) displayed comparable activities to
that of Nutlin-3. Most of the compounds with secondary carbox-
amides at the 3-position of thiophene ring (5–9, 20–22, 24 and
25) showed potent MDM2 binding affinities. Among them, N-ben-
zyl carboxamide at the 3-position of thiophene ring (24,
In addition, the 2-amino group in compounds 5 and 11 was con-
verted into amides, sulfamides, carbamates and ureas, respectively.
Acylation of 11 with acetyl chloride or chloropropionyl chloride
and followed by reaction with morpholine obtained amides 26
and 27. Sulfamide 28 was prepared by reaction of 5 with methyl-
sulfonyl chloride. Treatment of 5 with ethyl chlorformate afforded
the carbamate 29. Reaction of 5 with 2-chloro ethyl isocyanate
yielded chloroethylurea, which subsequently treated with corre-
sponding secondary amines to give target compounds 30 and 31.
Ki = 0.15 lM) was the most preferred substituent for binding affin-
ity. However, in the case of N-phenyl carboxamide (23), a signifi-
cant decrease of potency was observed. On the other hand,
compounds with aliphatic tertiary carboxamides at 3-position
exhibited moderate to potent binding affinities (10–19). Compar-
ing the binding affinities of 11, 12 and 17–19 revealed that intro-
ducing heterocycles with two nitrogen-atoms at the end of
carboxamide (17–19) caused a dramatic loss in potency. All 2-ami-
no acylated derivatives (26–31) showed a remarkably lowered
potency.
2.2. p53–MDM2 binding inhibitory activities
2.3. Tumor cell growth inhibition studies
The obtained 3,4,5-trisubstituted aminothiophene compounds
were evaluated for their p53–MDM2 binding inhibitory activities
by fluorescence-polarization based binding assay (FP assay).15,16
Nutlin-3 was used as positive control. The results are summarized
in Table 1.
All the synthesized compounds were further evaluated for their
anti-proliferation activities against two human cancer cell lines,
including one wild-type p53 cell line A549 and one p53 null cell
line PC3. IC50 values were obtained using the standard SRB assay.
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.061

W. Wang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
Table 1
P53–MDM2 binding inhibitory activities and tumor cell anti-proliferation activities of 3,4,5-trisubstituted aminothiophenes (3 and 5–31)
Cl
S
R2
O
R1
Cl
Compd
R1
R2
Kia
(lM)
SRB IC50 (lM)
A549a
PC3a
3 (MCL0527)c
5c
OCH3
NH2
NH2
NH2
1.52
0.54
1.12
5.04
2.78
3.68
24.79
HN
1.95
12.48
HN
HN
HN
6
7
NH2
NH2
NH2
NH2
NH2
NH2
0.54
0.23
0.99
0.74
1.21
0.80
6.79
2.41
0.25
0.89
4.62
1.75
21.24
7.3
8c
HN
N
9
5.67
6.22
14.33
5.75
OH
10c
11
12
N
N
N
O
N
13
14
15
NH2
NH2
NH2
1.55
2.19
3.52
1.82
2.01
4.09
4.94
1.34
9.23
O
O
N
O
16
17
18
NH2
NH2
NH2
0.43
6.81
1.52
1.87
1.02
0.59
11.69
2.71
N
N
N
N
O
NH
N
13.14
NH
19
NH2
17.66
4.93
7.21
HN
HN
20
21
NH2
NH2
0.55
4.13
8.17
17.75
13.16
20.98
22
NH2
NH2
NH2
0.81
>50
8.85
15.14
>50
HN
23c
24
25.03
16.30
HN
HN
0.15
31.81
HN
25
26
NH2
0.64
3.57
18.23
6.01
29.54
10.32
OMe
O
O
N
HN
HN
N
27
NAb
>25
>25
N
O
O
S
HN
HN
28
29
>50
NAb
NTd
NTd
HN
O
O
O
39.79
27.56
HN
HN
O
O
O
30
N
N
NAb
NTd
NTd
HN
N
H
HN
31
NAb
NTd
NTd
HN
N
H
Nutlin-3
—
—
0.055
4.62
20.04
a
b
c
Values are means of two experiments.
NA, no activity.
These compounds were reported in our previous studies.13
NT, not tested.
d
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.061

4
W. Wang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Figure 2. Molecular docking analysis of 3 and 24 with MDM2. (A) 3D schematic interaction model for 3 (cyan backbone) with MDM2. (B) Surface show of 3 (cyan backbone)
bound to MDM2. (C) 3D schematic interaction model for 24 (pink backbone) with MDM2. (D) Surface show of 24 (pink backbone) bound to MDM2. Yellow circles indicated
that the 2-amino group of thiophene ring was very close to the Met62 residue of MDM2.
According to the results (Table 1), several compounds (such as 7–
13, 16, 18 and 22) showed a good selectivity on wild-type p53 cell
line A549 over p53 null cell line PC3. In particular, compounds 9,
16 and 18 showed 22-, 6- and 22-fold selectivity of p53 status,
respectively, much better than that of Nutlin-3 (fourfold). Besides,
more than 10 compounds exhibited improved anti-proliferation
activities against A549 cell line than that of Nutlin-3. Among them,
Phe19 binding site was observed, and it could be perfectly satisfied
by the N-benzyl group in compound 24 (Fig. 2D). In addition, the 2-
amino group of thiophene ring was found very close to the Met62
residue in both molecule simulation results (highlighted by yellow
circles, Fig. 2B and D). Derivatization at this position would possi-
bly lead to direct contact between compounds and protein surface,
which consequently influences the binding affinity. Thus, the re-
sults of molecular docking studies gave an explanation to the dra-
matic drop of potency observed in compounds with substituents at
2-amino group.
compound 9 showed the best activity (IC50 = 0.25 lM). Unfortu-
nately, although compound 24 was potent for MDM2 binding affin-
ity, it did not exhibit satisfactory performance in tumor cell growth
inhibition studies, possibly due to its poor solubility. Based on the
above experimental results, it is noteworthy that compound 9 and
16 displayed potent activities not only in p53–MDM2 binding inhi-
bition but also in tumor cell anti-proliferation, and had excellent
selective profile on p53 status. Therefore, they can be used as
promising lead compounds for further investigation.
3. Conclusion
A series of novel 3,4,5-trisubstituted aminothiophene deriva-
tives were designed and synthesized as p53–MDM2 binding inhib-
itors. In the MDM2 binding assay, most derivatives showed largely
improved potency compared to lead compound MCL0527. Eleven
compounds displayed potent binding affinities at nanomolar level,
equivalent to that of Nutlin-3. In addition, more than ten com-
pounds exhibited better anti-proliferation activities against A549
cell line than that of Nutlin-3. Several compounds showed at least
3-fold inhibitory selectivity on wild-type p53 cell line A549 over
p53 null cell line PC3. This study validated the potential of devel-
oping 3,4,5-trisubstituted aminothiophene series as a tool for can-
cer therapy by targeting p53–MDM2 interaction. Further studies
are still under investigation.
2.4. Molecular docking studies
To examine possible binding modes of lead compound
MCL0527 (3) and the most potent compound 24 with MDM2, a
docking analysis utilizing the C-DOCKER program within Discovery
Studio 2.1 software package was performed. The published X-ray
crystal structure of MDM2 (PDB ID:1YCR) was used for the docking
calculation. Similar binding modes of 3 and 24 with MDM2 were
indicated in the docking results (Fig. 2). Herein, the thiophene ring,
serving as an important core structure, played the role in project-
ing three hydrophobic substituents into the MDM2 binding cleft
in proper directions. Two 4-chlorophenyl groups and one methyl
ester/N-benzyl group mimiced the three key residues of p53
(Leu26, Trp23 and Phe19, respectively) to form the hydrophobic
interactions with protein (Fig. 2A and C). For the lead compound
3 (Fig. 2B), a spare room not occupied by methyl ester at the
4. Experimental
4.1. Chemistry
Melting points were determined with a B-540 Büchi apparatus
and are uncorrected. NMR spectra were recorded on a Brüker 500
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.061

W. Wang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
(500 MHz) spectrometer (chemical shifts are given in ppm (d) rel-
ative to TMS as internal standard, coupling constants (J) are in
hertz (Hz), and signals are using the following abbreviations: s, sin-
glet; d, doublet; t, triplet; m, multiplet, etc. Mass spectra (MS), ESI
(positive) were recorded on an Esquire-LC-00075 spectrometer.
Thin layer chromatography was carried out using plate silica gel
F254 Merck. Reagents and solvents were purchased from common
commercial suppliers and were used without further purification.
All yields are unoptimized and generally represent the result of a
single experiment.
1H NMR (500 MHz, CDCl3) d = 7.25 (d, J = 8.5 Hz, 2H, Ar-H), 7.18 (d,
J = 8.5 Hz, 2H, Ar-H), 7.12 (d, J = 8.5 Hz, 2H, Ar-H), 7.01 (d,
J = 8.5 Hz, 2H, Ar-H), 3.31 (m, 4H, 2CH2), 1.42 (m, 6H, 3CH2). ESI-
MS: m/z = 431 [M+1]+.
4.1.2.5.
2-Amino-4,5-bis(4-chlorophenyl)thiophen-3-yl)(4-
Yellow solid (68%),
methylpiperidin-1-yl)methanone (12).
mp: 188–190 °C. 1H NMR (500 MHz, CDCl3) d = 7.24 (d, J = 7.2 Hz,
1H, Ar-H), 7.21 (d, J = 8.1 Hz, 1H, Ar-H), 7.17 (d, J = 6.9 Hz, 2H, Ar-
H), 7.13 (d, J = 8.2 Hz, 1H, Ar-H), 7.07 (d, J = 8.1 Hz, 1H, Ar-H),
7.01 (d, J = 7.9 Hz, 2H, Ar-H), 2.64 (m, 4H, 2CH2), 1.49 (m, 1H,
CH), 1.36 (m, 2H, CH2), 1.26 (m, 2H, CH2), 0.68 (d, J = 6.2 Hz, 3H,
CH3). ESI-MS: m/z = 445 [M+1]+.
4.1.1. Synthesis of 2-amino-4,5-bis(4-chlorophenyl)thiophene-
3-carboxylic acid (4)
2 N aqueous NaOH (75 mL) was added to a solution of methyl 2-
amino-4,5-bis (4-chlorophenyl)thiophene-3-carboxylate (3, 5 g,
13 mmol) in 125 mL ethanol. The resulting mixture was refluxed
for 2 h. Upon cooling, the reaction mixture was evaporated under
reduced pressure. The residue was suspended in H2O, and the sus-
pension was acidified to pH 6.0 with 1 N HCl aqueous solution. The
mixture was extracted with ethyl acetate (2 Â 200 mL) and organic
layer was washed with water (2 Â 200 mL) and brine (2 Â 200 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure to give the crude product as a yellow solid. Since com-
pound 4 was not very stable, the crude product was used in the
next step without purification.
4.1.2.6.
bonyl)piperidin-2-one (13).
1-(2-Amino-4,5-bis(4-chlorophenyl)thiophene-3-car-
Yellow solid (48%), mp: 252–
254 °C. 1H NMR (500 MHz, CDCl3) d = 7.24 (d, J = 8.4 Hz, 2H, Ar-
H), 7.18 (d, J = 8.5 Hz, 2H, Ar-H), 7.10 (d, J = 8.4 Hz, 2H, Ar-H),
6.99 (d, J = 8.5 Hz, 2H, Ar-H), 4.01 (m, 2H, CH2), 3.74 (m, 2H,
CH2), 3.23 (m, 2H, CH2), 2.83 (m, 2H, CH2). ESI-MS: m/z = 445
[M+1]+.
4.1.2.7.
1-(2-Amino-4,5-bis(4-chlorophenyl)thiophene-3-car-
bonyl)piperidin-4-one (14).
Yellow solid (40%), mp: 290–
293 °C. 1H NMR (500 MHz, CDCl3) d 7.24 (d, J = 8.3 Hz, 2H, Ar-H),
7.18 (d, J = 8.2 Hz, 2H, Ar-H), 7.13 (d, J = 8.1 Hz, 2H, Ar-H), 6.99
(d, J = 8.4 Hz, 2H, Ar-H), 3.62 (m, 2H, CH2), 3.43 (m, 2H, CH2),
2.24 (m, 2H, CH2), 1.87 (m, 2H, CH2). ESI-MS: m/z = 445 [M+1]+.
4.1.2. General procedure for the synthesis of 3,4,5-trisubstituted
aminothiophene derivatives (5–25)
2-Amino-4,5-bis(4-chlorophenyl)thiophene-3-carboxylic acid
(4, 0.4 mmol), EDCI (1.2 mmol) and HOBt (0.8 mmol) were dis-
solved in anhydrous CH2Cl2 (20 mL) and stirred for 30 min. Then
the corresponding amine (0.8 mM) was added dropwise. After stir-
red overnight at room temperature, the reaction mixture was
washed with water (2 Â 20 mL) and brine (2 Â 20 mL), and dried
over Na2SO4. The solvent was evaporated under reduced pressure
and the residue obtained was purified by silica gel column chroma-
tography (petroleum ether/ethyl acetate/CH2Cl2 = 10:1:1–1:1:1 v/
v/v) to get target compounds 5–25.
4.1.2.8.
1-(2-Amino-4,5-bis(4-chlorophenyl)thiophene-3-car-
Yellow solid (43%), mp:
bonyl)piperidine-2,4-dione (15).
261–263 °C. 1H NMR (500 MHz, CDCl3) d 7.37 (d, J = 8.3 Hz, 2H,
Ar-H), 7.24 (d, J = 8.3 Hz, 2H, Ar-H), 7.12 (d, J = 8.5 Hz, 2H, Ar-H),
6.89 (d, J = 8.5 Hz, 2H, Ar-H), 6.46 (br s, 2H, NH2), 4.90 (m, 2H,
CH2), 3.43 (m, 2H, CH2), 1.65 (m, 2H, CH2). ESI-MS: m/z = 459
[M+1]+.
4.1.2.9. (2-Amino-4,5-bis(4-chlorophenyl)thiophen-3-yl)(mor-
pholino)methanone (16).
White solid (80%), mp: 186–
4.1.2.1. 2-Amino-4,5-bis(4-chlorophenyl)-N-cyclobutylthioph-
187 °C. 1H NMR (500 MHz, CDCl3) d 7.26 (d, J = 8.4 Hz, 2H, Ar-H),
7.17 (d, J = 8.4 Hz, 2H, Ar-H), 7.09 (d, J = 8.4 Hz, 2H, Ar-H), 6.98
(d, J = 8.4 Hz, 2H, Ar-H), 3.44 (m, 8H, 4CH2). ESI-MS: m/z = 433
[M+1]+.
ene-3-carboxamide (6).
Yellow solid (73%), mp: 175–
177 °C. 1H NMR (500 MHz, CDCl3) d = 7.41 (d, J = 8.1 Hz, 2H, Ar-
H), 7.23 (d, J = 8.1 Hz, 2H, Ar-H), 7.12 (d, J = 8.3 Hz, 2H, Ar-H),
6.91 (d, J = 8.3 Hz, 2H, Ar-H), 6.35 (br s, 2H, NH2), 4.92 (d,
J = 7.0 Hz, 1H, NH), 4.25 (dd, J = 15.9, 8.0 Hz, 1H, CH), 2.17 (m, 2H,
CH2), 1.61 (m, 2H, CH2), 1.32 (m, 2H, CH2). ESI-MS: m/z = 417
[M+1]+.
4.1.2.10. (2-Amino-4,5-bis(4-chlorophenyl)thiophen-3-yl)(pip-
erazin-1-yl)methanone (17).
Yellow solid (50%), mp: 182–
183 °C. 1H NMR (500 MHz, CDCl3) d = 7.24 (d, J = 8.4 Hz, 2H, Ar-
H), 7.17 (dd, J = 8.8, 2.2 Hz, 2H, Ar-H), 7.09 (d, J = 8.4 Hz, 2H, Ar-
H), 6.99 (dd, J = 8.4, 6.6 Hz, 2H, Ar-H), 4.57 (s, 1H, NH), 3.25 (m,
4H, 2CH2), 2.2 (m, 4H, 2CH2). ESI-MS: m/z = 432 [M+1]+.
4.1.2.2. 2-Amino-4,5-bis(4-chlorophenyl)-N-cyclopentylthioph-
ene-3-carboxamide (7).
Yellow solid (69%), mp: 182–183 °C.
1H NMR (500 MHz, CDCl3) d = 7.38 (d, J = 8.3 Hz, 2H, Ar-H), 7.22 (m,
4H, Ar-H), 6.94 (d, J = 8.5 Hz, 2H, Ar-H), 6.43 (br s, 2H, NH2), 6.32 (d,
J = 6.8 Hz, 1H, NH), 4.63 (m, 1H, CH), 1.90 (m, 2H, CH2), 1.54 (m, 2H,
CH2), 1.35 (m, 2H, CH2), 1.00 (m, 2H, CH2). ESI-MS: m/z = 431 [M+1]+.
4.1.2.11.
(2-Amino-4,5-bis(4-chlorophenyl)thiophen-3-yl)(4-
methylpiperazin-1-yl)methanone (18).
Yellow solid (52%),
mp: 184–186 °C. 1H NMR (500 MHz, CDCl3) d 7.24 (d, J= 8.4 Hz,
2H, Ar-H), 7.18 (d, J = 8.5 Hz, 2H, Ar-H), 7.09 (d, J = 8.3 Hz, 2H, Ar-
H),6.98 (d, J = 8.4 Hz, 2H, Ar-H), 3.23 (m, 4H, 2CH2), 2.01 (s, 3H,
CH3), 1.67 (m, 4H, 2CH2). ESI-MS: m/z = 446 [M+1]+.
4.1.2.3.
2-Amino-4,5-bis(4-chlorophenyl)-N-(4-hydrox-
Yellow solid
ycyclohexyl)thiophene-3-carboxamide (9).
(55%), mp: 221–223 °C. 1H NMR (500 MHz, CDCl3) d 7.38 (d,
J = 8.4 Hz, 2H, Ar-H), 7.21 (d, J = 8.4 Hz, 2H, Ar-H), 7.11 (d,
J = 8.5 Hz, 2H, Ar-H), 6.89 (d, J = 8.6 Hz, 2H, Ar-H), 6.32 (br s, 2H,
NH2), 4.65 (d, J = 7.3 Hz, 1H, NH), 4.57 (s, 1H, OH), 3.65 (m, 1H,
CH), 3.49 (m, 1H, CH), 1.76 (m, 4H, 2CH2), 1.31 (m, 2H, CH2), 0.70
(m, 2H, CH2). ESI-MS: m/z = 461 [M+1]+.
4.1.2.12.
(2-Amino-4,5-bis(4-chlorophenyl)thiophen-3-yl)(3-
Yellow solid (47%),
methylpiperazin-1-yl)methanone (19).
mp: 184–187 °C. 1H NMR (500 MHz, CDCl3) d 7.25 (d, J = 7.9 Hz,
2H, Ar-H), 7.16 (d, J = 8.5 Hz, 2H, Ar-H), 7.08 (d, J = 8.5 Hz, 2H, Ar-
H), 6.98 (d, J = 7.7 Hz, 2H, Ar-H), 4.62 (m, 2H, CH2), 3.34 (m, 1H,
CH), 2.80 (m, 3H, CH2), 2.44 (m, 1H, CH2), 1.13 (m, 3H, CH3). ESI-
MS: m/z = 446 [M+1]+.
4.1.2.4. 2-Amino-4,5-bis(4-chlorophenyl)thiophen-3-yl)(piperi-
din-1-yl)methanone (11).
White solid (83%), mp: 188–189 °C.
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.061

6
W. Wang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
4.1.2.13. 2-Amino-N-butyl-4,5-bis(4-chlorophenyl)thiophene-3-
carboxamide (20).
Yellow solid (63%), mp: 157–159 °C. 1H
NMR (500 MHz, CDCl3) d 7.38 (d, J = 8.5 Hz, 2H, Ar-H), 7.22 (d,
J = 8.3 Hz, 2H, Ar-H), 7.11 (d, J = 8.9 Hz, 2H, Ar-H), 6.90 (d,
J = 8.5 Hz, 2H, Ar-H), 4.83 (s, 1H, NH), 3.10 (dd, J = 12.2, 6.7 Hz,
2H, CH2), 1.13 (dt, J = 14.8, 6.9 Hz, 2H, CH2), 1.05 0.93 (m, 2H,
CH2), 0.74 (t, J = 7.0 Hz, 3H, CH3). ESI-MS: m/z = 419 [M+1]+.
pressure. Then, the obtained residue was dissolved in 10 mL diox-
ane, to which was added triethylamine (1.6 mmol) and morpholine
(139 mg, 3.2 mmol), refluxed for 1 h. Upon cooling, the reaction
mixture was evaporated under reduced pressure and the residue
obtained was dissolved in ethyl acetate. The organic phase was
washed with water (2 Â 15 mL) and brine (2 Â 15 mL), dried over
anhydrous Na2SO4, and concentrated under reduced pressure.
The obtained residue was purified by silica gel column chromatog-
raphy (petroleum ether/ethyl acetate = 1:3, v/v) to give a yellow
solid 27 (69%). mp: 178–180 °C. 1H NMR (500 MHz, CDCl3) d
11.40 (s, 1H, NH), 7.25 (m, 4H, Ar-H), 7.19 (m, 4H, Ar-H), 4.08
(m, 4H, 2CH2), 3.63 (m, 4H, 2CH2), 2.76 (m, 4H, 2CH2), 2.63 (m,
2H, CH2), 2.59 (m, 2H, CH2) 1.54 (m, 6H, 3CH2). ESI-MS: m/
z = 572 [M+1]+.
4.1.2.14.
ene-3-carboxamide (21).
2-Amino-4,5-bis(4-chlorophenyl)-N-isobutylthioph-
Yellow solid (70%), mp: 151–
153 °C. 1H NMR (500 MHz, CDCl3) d = 7.41 (d,J = 8.5 Hz, 2H, Ar-H),
7.23 (d,J = 6.5 hz, 2H, Ar-H), 7.13 (d, J = 6.4 hz, 2H, Ar-H), 6.91 (d,
J = 8.4 Hz, 2H, Ar-H), 6.32 (br s, 2H, NH2), 4.93 (s, 1H, NH), 3.00
(m, 2H, CH2), 1.49 (m, 1H, CH), 0.68 (m, 6H, 2CH3). ESI-MS: m/
z = 419 [M+1]+.
4.1.5. Synthesis of 4,5-bis(4-chlorophenyl)-N-cyclopropyl-2-
(methylsulfonamido)thiophene-3-carboxamide (28)
4.1.2.15. 2-Amino-N-(tert-butyl)-4,5-bis(4-chlorophenyl)thio-
phene-3-carboxamide (22).
Yellow solid (66%), mp: 187–
A solution of methanesulfonyl chloride (64 mg, 0.56 mmol) in
anhydrous CH2Cl2 (5 mL) was slowly added to a cooled (0 °C) mix-
ture of 5 (150 mg, 0.37 mmol) and triethylamine (1.11 mmol) in
anhydrous CH2Cl2 (10 mL). After stirred at room temperature for
additional 1 h, the reaction was quenched with water (10 mL).
The mixture was extracted with CH2Cl2 (2 Â 15 mL). The organic
phase was washed with water (2 Â 15 mL) and brine (2 Â 15 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The obtained residue was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 5:1, v/v) to give
a yellow solid 28 (100%). mp: 191–193 °C. 1H NMR (500 MHz,
CDCl3) d 7.29 (d, J = 8.4 Hz, 2H, Ar-H), 7.23 (d, J = 8.5 Hz, 2H, Ar-
H), 7.14 (d, J = 8.4 Hz, 2H, Ar-H), 7.08 (d, J = 8.5 Hz, 2H, Ar-H),
6.03 (d, J = 2.0 Hz, 1H, NH), 3.54 (s, 3H, CH3), 2.65 (tq, J = 7.2,
3.7 Hz, 1H, CH), 0.77 (m, 2H, CH2), 0.36 (m, 2H, CH2). ESI-MS: m/
z = 481 [M+1]+.
189 °C. 1H NMR (500 MHz, CDCl3) d = 7.41(d,J = 8.4 Hz, 2H, Ar-H),
7.23 (d,J = 6.4 Hz, 2H, Ar-H), 7.14 (d, J = 6.5 Hz, 2H, Ar-H), 6.93(d,
J = 8.4 Hz, 2H, Ar-H), 6.77 (br s 2H, NH2), 4.72 (s, 1H, NH), 1.06 (s,
9H, 3CH3). ESI-MS: m/z = 419 [M+1]+.
4.1.2.16. 2-Amino-N-benzyl-4,5-bis(4-chlorophenyl)thiophene-
3-carboxamide (24).
White solid (81%), mp: 213–215 °C. 1H
NMR (500 MHz, CDCl3) d 7.26 (m, 3H, Ar-H),7.19 (d, J = 8.3 Hz,
2H, Ar-H), 7.12 (d, J = 7.5 Hz, 2H, Ar-H), 7.10 (d, J = 8.7 Hz, 2H, Ar-
H), 6.92 (d, J = 7.2 Hz, 2H, Ar-H), 6.88 (d, J = 8.6 Hz, 2H, Ar-H),
5.06 (d, J = 3.7 Hz, 1H, NH), 4.26 (d, J = 4.0 Hz, 2H, CH2). ESI-MS:
m/z = 453 [M+1]+.
4.1.2.17. 2-Amino-4,5-bis(4-chlorophenyl)-N-(4-methoxyben-
zyl)thiophene-3-carboxamide (25).
White solid (77%), mp:
204–206 °C. 1H NMR (500 MHz, CDCl3) d 7.30 (d, J = 8.3 Hz, 2H,
Ar-H), 7.23 (d, J = 8.5 Hz, 2H, Ar-H), 7.16 (d, J = 8.3 Hz, 2H, Ar-H),
7.02 (d, J = 7.8 Hz, 2H, Ar-H), 6.93 (d, J = 8.5 Hz, 2H, Ar-H), 6.77
(d, J = 7.8 Hz, 2H, Ar-H), 6.18 (t, J = 5.3 Hz, 1H, NH), 4.11 (d,
J = 5.5 Hz, 2H, CH2), 2.26 (s, 3H, CH3). ESI-MS: m/z = 483 [M+1]+.
4.1.6. Synthesis of ethyl (4,5-bis(4-chlorophenyl)-3-
(cyclopropylcarbamoyl)thiophen-2-yl)carbamate (29)
A solution of ethyl carbonochloridate (78 mg, 0.74 mmol) in tol-
uene (5 mL) was added to a mixture of 5 (150 mg, 0.37 mmol) and
anhydrous potassium carbonate (255 mg, 1.85 mmol) in dry tolu-
ene (10 mL). After stirred at room temperature for additional 3 h,
toluene was removed under reduced pressure and the obtained
residue was dissolved in ethyl acetate (20 mL), washed with water
(2 Â 20 mL) and brine (2 Â 20 mL). The organic phase was dried
over anhydrous Na2SO4, and concentrated under reduced pressure.
The obtained residue was purified by silica gel column chromatog-
raphy (petroleum ether/ethyl acetate = 2:1, v/v) to give a yellow
solid 29 (82%). mp: 151–153 °C. 1H NMR (500 MHz, CDCl3) d
11.40 (s, 1H, NH), 7.39 (d, J = 8.3 Hz, 2H, Ar-H), 7.16 (m, 4H, Ar-
H), 6.98 (d, J = 8.5 Hz, 2H, Ar-H), 5.13 (s, 1H, NH), 4.29 (q,
J = 7.1 Hz, 2H, CH2), 2.61 (m, 1H, CH), 1.33 (t, J = 7.2 Hz, 3H, CH3),
0.66 (m, 2H, CH2), 0.05 (m, 2H, CH2). ESI-MS: m/z = 475 [M+1]+.
4.1.3. Synthesis of N-(4,5-bis(4-chlorophenyl)-3-(piperidine-1-
carbonyl)thiophen-2-yl)acetamide (26)
A solution of acetyl chloride (37 mg, 0.48 mmol) in anhydrous
CH2Cl2 (5 mL) was slowly added to a cooled (0 °C) mixture of 11
(150 mg, 0.32 mmol) and triethylamine (0.96 mmol) in anhydrous
CH2Cl2 (10 mL). After stirred at room temperature for additional
1 h, the reaction was quenched with water (10 mL). The mixture
was extracted with CH2Cl2 (2 Â 15 mL). The organic phase was
washed with water (2 Â 15 mL) and brine (2 Â 15 mL), dried over
anhydrous Na2SO4, and concentrated under reduced pressure.
The obtained residue was purified by silica gel column chromatog-
raphy (petroleum ether/ethyl acetate = 5:1, v/v) to give a white so-
lid 26 (98%). mp: 261–263 °C. 1H NMR (500 MHz, CDCl3) d 9.40 (s,
1H, NH), 7.25 (m, 4H, Ar-H), 7.11 (m, 4H, Ar-H), 3.47 (m, 2H, CH2),
2.65 (m, 2H, CH2), 2.23 (s, 3H,CH3), 2.08 (m, 2H, CH2), 1.67 (m, 2H,
CH2), 1.49 (m, 2H, CH2). ESI-MS: m/z = 473 [M+1]+.
4.1.7. Synthesis of 4,5-bis(4-chlorophenyl)-N-cyclopropyl-2-(3-
(2-morpholinoethyl)ureido)thiophene-3-carboxamide (30)
A solution of 5 (150 mg, 0.32 mmol) in anhydrous CH2Cl2
(10 mL) was added triethylamine (0.64 mmol) and 2-chloroethyl
isocyanate (506 mg, 4.8 mmol). The mixture was refluxed over-
night. The reaction was quenched with water (10 mL). The mixture
was extracted with CH2Cl2 (2 Â 15 mL). The organic phase was
washed with water (2 Â 15 mL) and brine (2 Â 15 mL), dried over
anhydrous Na2SO4, and concentrated under reduced pressure.
Then, the obtained residue was dissolved in 10 mL dioxane, to
which was added triethylamine (1.6 mmol) and morpholine
(139 mg, 3.2 mmol), refluxed for 1 h. After cooling to room temper-
ature, the reaction mixture was evaporated under reduced pres-
sure. The residue was extracted with ethyl acetate (2 Â 15 mL).
4.1.4. Synthesis of N-(4,5-bis(4-chlorophenyl)-3-(piperidine-1-
carbonyl)thiophen-2-yl)-3-morpholinopropanamide (27)
A solution of chloropropionyl chloride (60 mg, 0.48 mmol) in
anhydrous CH2Cl2 (5 mL) was slowly added to a cooled (0 °C) mix-
ture of 11 (150 mg, 0.32 mmol) and triethylamine (0.96 mmol) in
anhydrous CH2Cl2 (10 mL). After stirred at room temperature for
additional 1 h, the reaction was quenched with water (10 mL).
The mixture was extracted with CH2Cl2 (2 Â 15 mL). The organic
phase was washed with water (2 Â 15 mL) and brine (2 Â 15 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.061

W. Wang et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
The organic phase was washed with water (2 Â 15 mL) and brine
(2 Â 15 mL), dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The obtained residue was purified by silica gel
column chromatography (petroleum ether/ethyl acetate = 1:5, v/
v) to give a yellow solid 30 (56%). mp: 129–130 °C. 1H NMR
(500 MHz, CDCl3) d 11.60 (s, 1H, NH), 7.39 (d, J = 8.4 Hz, 2H, Ar-
H), 7.16 (d, J = 8.4 Hz, 2H, Ar-H), 7.15 (d, J = 8.4 Hz, 2H, Ar-H),
6.99 (d, J = 8.4 Hz, 2H, Ar-H), 5.73 (br s 1H, NH), 5.16 (s, 1H, NH),
3.82 (m, 4H, 2CH2), 3.51 (m, 2H, CH2), 2.66 (m, 2H, CH2), 2.64 (m,
4H, 2CH2), 2.47 (m, 1H, CH), 0.74 (m, 2H, CH2), 0.05 (m, 2H, CH2).
ESI-MS: m/z = 559 [M+1]+.
times, and protein-bound dye was extracted with 10 mmol unbuf-
fered 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 cellsÀ A515 treated cells)/A515 control cells  100%.
The average IC50 values were determined by Logit method from at
least two independent tests.
4.3. Molecular docking
Docking simulations were carried out by using C-DOCKER mod-
ule (Discovery Studio, version 2.1; Accelrys, San Diego, CA, USA,
2008). The X-ray crystal structure of MDM2 bound to the transac-
tivation domain of p53 (PDB ID:1YCR) was used for the docking
calculation. After removing the ligand and solvent molecules, the
CHARMm-force field was applied to the protein. And the area
around p53 peptide was chosen as the active site with a radius
set as 10 Å. Each compound was generated random conformations
using CHARMm-based molecular dynamics (1000 steps), and then
docked into the defined MDM2 binding site. The other parameters
were set as default. The final binding conformation of 3 and 24 was
determined based on the calculated CDOCKING ENERAGE. The
most stable binding mode among the top 10 docking poses of each
compound was presented in Fig. 2, respectively.
4.1.8. Synthesis of 4,5-bis(4-chlorophenyl)-N-cyclopropyl-2-(3-
(2-(diethylamino)ethyl)ureido)thiophene-3-carboxamIde (31)
Same as 30 above, except with diethylamine. Target compound
31 was obtained as a yellow solid (50%). mp: 120–121 °C. 1H NMR
(500 MHz, CDCl3) d 11.53 (s, 1H, NH), 7.37 (d, J = 8.1 Hz, 2H, Ar-H),
7.15 (d, J = 8.3 Hz, 2H, Ar-H), 7.13 (d, J = 8.5 Hz, 2H, Ar-H), 6.96 (d,
J = 8.3 Hz, 2H, Ar-H), 6.74 (br s, 1H, NH), 5.09 (s, 1H, NH), 3.61 (m,
2H, CH2), 2.94 (m, 6H, 3CH2), 2.60 (d, J = 3.3 Hz, 1H, CH), 1.25 (s, 6H,
2CH3), 0.63 (m, 2H, CH2), 0.02 (m, 2H, CH2). ESI-MS: m/z = 545
[M+1]+.
4.2. Biological evaluation
4.2.1. MDM2 protein expression and purification
Acknowledgments
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 °C in TB medium, and induced by 0.4 mM IPTG at
an OD600 of 0.6 at 18 °C 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.
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 on the protein purification
experiment.
References and notes
1. Harris, S. L.; Levine, A. J. Oncogene 2005, 24, 2899.
2. Levine, A. J. Cell 1997, 88, 323.
4.2.2. 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 posi-
tive 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
3. Vogelstein, B.; Lane, D.; Levine, A. J. Nature 2000, 408, 307.
4. Vousden, K. H.; Lu, X. Nat. Rev. Cancer 2002, 2, 594.
5. Hainaut, P.; Soussi, T.; Shomer, B.; Hollstein, M.; Greenblatt, M.; Hovig, E.;
Harris, C. C.; Montesano, R. Nucleic Acids Res. 1997, 25, 151.
6. Hollstein, M.; Rice, K.; Greenblatt, M. S.; Soussi, T.; Fuchs, R.; Sorlie, T.; Hovig,
E.; Smith-Sorensen, B.; Montesano, R.; Harris, C. C. Nucleic Acids Res. 1994, 22,
3551.
7. Fakharzadeh, S. S.; Trusko, S. P.; George, D. L. EMBO J. 1991, 10, 1565.
8. Oliner, J. D.; Kinzler, K. W.; Meltzer, P. S.; George, D. L.; Vogelstein, B. Nature
1992, 358, 80.
9. Oliner, J. D.; Pietenpol, J. A.; Thiagalingam, S.; Gyuris, J.; Kinzler, K. W.;
Vogelstein, B. Nature 1993, 362, 857.
10. Dickens, M. P.; Fitzgerald, R.; Fischer, P. M. Semin. Cancer Biol. 2010, 20, 10.
11. Shangary, S.; Wang, S. Annu. Rev. Pharmacol. Toxicol. 2009, 49, 223.
12. Wang, W.; Hu, Y. Med. Res. Rev. 2012, 32, 1159.
volume of 20 lL 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 10 nM.
Plates were allowed to incubate at room temperature for 1 h prior
to measurement. The Ki values for inhibitors were calculated by a
web-based computer program.17
13. Wang, W.; Zhu, X.; Hong, X.; Zheng, L.; Zhu, H.; Hu, Y. Med. Chem. Commun.
2013, 4, 411.
14. Gewald, K.; Schinke, E.; Böttcher, H. Chemische Berichte 1966, 99, 94.
15. Ding, K.; Lu, Y.; Nikolovska-Coleska, Z.; Qiu, S.; Ding, Y.; Gao, W.; Stuckey, J.;
Krajewski, K.; Roller, P. P.; Tomita, Y.; Parrish, D. A.; Deschamps, J. R.; Wang, S. J.
Am. Chem. Soc. 2005, 127, 10130.
16. Ding, K.; Lu, Y.; Nikolovska-Coleska, Z.; Wang, G.; Qiu, S.; Shangary, S.; Gao, W.;
Qin, D.; Stuckey, J.; Krajewski, K.; Roller, P. P.; Wang, S. J. Med. Chem. 2006, 49,
3432.
17. Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita, Y.; Li, P.; Roller, P.
P.; Krajewski, K.; Saito, N. G.; Stuckey, J. A.; Wang, S. Anal. Biochem. 2004, 332,
261.
4.2.3. Cell proliferation assay
Cell proliferation was assessed by sulforhodamin B (SRB) assay.
Briefly, A549 and PC3 cells were seeded into 96-well plates and
cultured overnight and then 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 °C for an hour. After wash-
ing, 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
Please cite this article in press as: Wang, W.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.03.061