Discovery of new low-molecular-weight p53-Mdmx disruptors and their anti-cancer activities

Article abstract of DOI:10.1016/j.bmc.2016.03.021

Although several p53-Mdm2-binding disruptors have been identified to date, few studies have been published on p53-Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophenol derivatives with molecular weights of 200-300 selectively inhibited the p53-Mdmx interaction. S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially inhibited the p53-Mdmx interaction over the p53-Mdm2 interaction, respectively, by using a Flag-p53 and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition, the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c. These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53-Mdmx, resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experiments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and 36.6% that of the control at 100 mg/kg and 150 mg/kg, respectively, in 40 days.

Full text of DOI:10.1016/j.bmc.2016.03.021

Bioorganic & Medicinal Chemistry 24 (2016) 1919–1926
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of new low-molecular-weight p53–Mdmx disruptors and
their anti-cancer activities
Shinichi Uesato a, , Yoshihiro Matsuura , Saki Matsue , Takaaki Sumiyoshi , Yoshiyuki Hirata ,
⇑
a
a
a
a
a
a
a
a
b
Suzuho Takemoto , Yasuyuki Kawaratani , Yusuke Yamai , Kyoji Ishida , Tsutomu Sasaki ,
c,
⇑
Masato Enari
a
Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680, Japan
Department of Neurology, Graduate School of Medicine, Osaka University, Yamadaoka 2-2, Suita, Osaka 565-0871, Japan
Division of Refractory and Advanced Cancer, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Although several p53–Mdm2-binding disruptors have been identified to date, few studies have been pub-
lished on p53–Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophe-
nol derivatives with molecular weights of 200–300 selectively inhibited the p53–Mdmx interaction.
S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression
of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with
wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected
forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially
inhibited the p53–Mdmx interaction over the p53–Mdm2 interaction, respectively, by using a Flag-p53
and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition,
the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c.
These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53–Mdmx,
resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experi-
ments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and
Received 11 February 2016
Revised 8 March 2016
Accepted 10 March 2016
Available online 11 March 2016
Keywords:
o-Aminothiophenol derivative
p53–Mdmx-interaction inhibitor
Protein–protein interaction inhibitor
Modified ELISA
GST–Mdmx
36.6% that of the control at 100 mg/kg and 150 mg/kg, respectively, in 40 days.
Ó 2016 Elsevier Ltd. All rights reserved.
1
. Introduction
The tumor suppressor p53 induces cell cycle arrest and apopto-
export of p53 through its mono-ubiquitination to inactivate
8
,10
p53-mediated transcription.
Mdm2 and Mdmx both bind to
p53 through hydrophobic interactions as follows. p53 forms a
short helix inside the p53-binding cleft of Mdm2 or Mdmx, and
the three residues: Phe19, Trp23, and Leu26, of the helix principally
sis in response to cellular stresses by activating the expression of
its downstream genes.1 However, in some cancer cells, negative
regulators such as Mdm2, Mdmx, COP1, Pirh2, Arf-BP1, and synovi-
olin impair the functions of p53.6 For example, Mdm2 exhibits E3
ubiquitin ligase activity and inactivates the functions of p53 via the
ubiquitin–proteasome system. In contrast, Mdmx, a homolog of
Mdm2, has no ubiquitin ligase activity to conjugate ubiquitin with
–5
contribute to binding to Mdm2 or Mdmx. A large number of p53–
–8
11–15
Mdm2-binding disruptors have been identified to date.
For
example, Nutlin-3a is a useful compound that inhibits the p53–
Mdm2 interaction by docking antagonistically in the p53-binding
1
6
domain of Mdm2. On the other hand, Nutlin-3a is less likely to
9
17
p53. Mdmx interacts with Mdm2 to enhance ubiquitination of
inhibit p53–Mdmx binding, and this may be interpreted in terms
p53 and ubiquitinated p53 degrades via the proteasome system
to inhibit p53 function. In addition to the degradation pathway,
the Mdm2–Mdmx complex is known as a factor in the nuclear
of differences in the sizes of the p-53 binding sites of Mdm2 and
Mdmx as follows. Leu54 in Mdm2 is replaced with the larger
Met53 in Mdmx, which makes the cavity of the p53-binding site
in Mdmx narrower than that in Mdm2, thereby poorly accommo-
1
8
dating a large molecule such as Nutlin-3a. Although double inhibi-
tors have also been developed for p53–Mdm2 and p53–Mdmx
⇑
Corresponding authors. Tel./fax: +81 753412068 (S.U.); tel.: +81 335422511;
fax: +81 335422530 (M.E.).
19–21
interactions,
for the p53–Mdmx interaction.
few studies have investigated selective inhibitors
2
2,23
We recently discovered
E-mail addresses: suesato@gaia.eonet.ne.jp, uesato@kansai-u.ac.jp (S. Uesato),
menari@ncc.go.jp (M. Enari).
o-aminothiophenol derivatives with molecular weights of
http://dx.doi.org/10.1016/j.bmc.2016.03.021
0
968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

1
920
S. Uesato et al. / Bioorg. Med. Chem. 24 (2016) 1919–1926
2
00–300 in our protein–protein interaction (PPI)-inhibitor
prepared by the reduction of the disulfides 4b and 5a with NaBH
and PPh , respectively, whereas 1b and 1d were obtained by the
alkaline hydrolysis of 1c and 1e, respectively.
4
2
4,25
library.
3
In the present study, we demonstrated that K-178 (1c)
increased the expression of p53 as well as its downstream genes
p21 and Mdm2 in IMR32 and MCF-7 cells with wild-type p53
and overexpressing Mdmx. Furthermore, K-178 (1c) dose-depen-
dently suppressed the growth of cancer cells with wild-type p53
and Mdmx, whereas it only weakly influenced the growth of cancer
cells with mutant p53. In order to analyze structure–activity rela-
tionships (SAR) in p53–Mdmx inhibitors, we further synthesized
K-178 (1c)-related compounds with different substituents on
o-aminothiophenol or o-aminophenol. The compounds obtained
were subjected to a modified enzyme-linked immunosorbent assay
3
. Determination of inhibitory activities of o-aminothiophenol
derivatives against the p53–Mdm2 or p53–Mdmx interaction
using the modified ELISA
The o-aminothiophenols were subjected to the modified
2
4,25
ELISA
in order to determine their inhibitory activities against
the complex of Flag-p53 with glutathione S-transferase (GST)–
Mdm2 or GST–Mdmx.
As shown in Table 1, S-isobutyryl-protected sulfides 1c and 3c
preferentially disrupted binding between p53 and Mdmx over that
between p53 and Mdm2. Additionally, they practically did not inhibit
binding between p53 and the other p53-binding partners, death-
2
4,25
(
ELISA)
in order to examine the selectivity of the inhibition of
p53–Mdm2 and p53–Mdmx interactions. The results obtained sug-
gested that the S-isobutyryl-protected sulfide K-178 (1c) or K-181
30
(3c), after conversion to the corresponding monomeric sulfide 1b
associated protein kinase 1 (DAPK1) and peptidylprolyl isomerase
2
6
31
or 3b , preferentially interacted with p53–Mdmx over p-53–
Mdm2 in the body. This bond may have triggered a deformation
in the three-dimensional structure of p53 or Mdmx, leading to
the dissociation of the two proteins. Oral administration
experiments on K-178 (1c) in nude mice with human colon carci-
noma (HCT116) tumor xenografts supported the results so far
obtained.
D (PPID). This was also the case for the S-isobutyryl-deprotected
forms 1b, 3b, 4b, and 5b, except for 2b. Furthermore, the replacement
of a sulfur atom (in 1b and 1c) with an oxygen atom (in 1d and 1e)
resulted in the loss of inhibitory activities for binding between p53
and Mdmx, suggesting that the SH group in 1b and 3b may have
an important role in the disruption of p53–Mdmx binding.
4
. Effects of o-aminothiophenol derivatives on cell growth in
2
. Chemistry
various cancer cells
Of the N,N -di-carbamoyl disulfide derivatives 1a–5a, 1a, 2a28
0
27
As shown in Figure 1, the cell viabilities of human mammary
adenocarcinoma (MCF-7), human lung adenocarcinoma (A427),
and HCT116 cells, all with wild-type p53 and Mdmx, were
decreased by increasing concentrations of K-178 (1c). In contrast,
the cell viabilities of cancer cells with mutant p53 such as human
epidermoid carcinoma (A431) cells and human breast carcinoma
(MDA-MB-468) cells as well as normal human embryonic lung
fibroblasts (TIG-7) cells only moderately declined. These results
suggest that the K-178 (1c)-mediated activation of the p53 path-
way plays a pivotal role in suppressing the growth of cancer cells.
Since acyl-protected sulfides are known to be metabolized into free
2
9
and 3a were known compounds. The derivatives 4a and 5a were
synthesized by the reaction of 2,2 dithioaniline with 4-chloroben-
0
zoyl chloride and 6-chloronicotinoyl chloride in the presence of
N,N-diisopropylethylamine and N,N-4-dimethylaminopyridine,
respectively (Scheme 1). The N,S-diisobutyryl- and N,O-diisobu-
tyryl-substituted monomers 1c and 1e were prepared by the reac-
tion of o-aminothiophenol and o-aminophenol with isobutyryl
chloride and pyridine (Py), respectively. The other S-isobutyryl-
protected monomer 3c was synthesized starting from the disulfide
a via the corresponding sulfide 3b. Thus, 3a was reduced with
NaBH to give 3b, which was treated with isobutyryl chloride in
the typical manner to give 3c. The sulfides 4b and 5b were
3
3
2
sulfides by intracellular esterases, the S-isobutyryl-deprotected
form 1b of K-178 (1c) appears to inhibit the growth of cancer cells.
4
O
O
O
NH
2
R
NH
R1
NH
1
R
1
NH
S
R
1
COCl
i
S
reduction
ii
SH isobutyryl chloride
iii
X
S
S
R
2
HN
R
1
NH
2
O
1
a: R
1
= isopropyl
= heptyl
2
3
4
5
a: R
1
1
1
1
2b: R
3b: R
4b: R
5b: R
1
1
1
= heptyl
a: R = phenyl
a: R
a: R
= phenyl
1 2
3c: R = phenyl; R = isobutyryl
= 4-chlorophenyl
= 6-chloropyridin-3-yl
= 4-chlorophenyl
1
= 6-chloropyridin-3-yl
O
O
NH
2
NH
X
NH
XH
isobutyryl chloride
iii
aq. KOH
iv
X
H
O
o-aminothiophenol: X = S
o-aminophenol: X = O
1c: X = S
1e: X = O
1b: X = S
1d: X = O
aReagents and conditions: (i) N,N-diisopropylethylamine, CH
ii) NaBH , THF-EtOH, rt (2b),(3b),(4b) or PPh , THF, rt (5b); (iii) Py, CH
2
Cl
2
, rt (4a) or N,N-4-dimethylaminopyridine, DMF, rt (5a);
Cl , rt; (iv) MeOH, rt.
(
4
3
2
2
Scheme 1. Synthesis of o-aminothiophenol derivatives.

S. Uesato et al. / Bioorg. Med. Chem. 24 (2016) 1919–1926
1921
Table 1
Inhibitory activities of o-aminothiophenol derivatives against the p53 and GST–protein interaction measured by the modified ELISA
Compounds
R
1
X
R
2
Competitive inhibition (IC50, l
M)a,b of
p53–Mdm2
p53–Mdmx
p53–DAPK1
p53–PPID
1
1
1
1
2
3
3
4
5
b
c
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Heptyl
Phenyl
Phenyl
4-Chlorophenyl
6-Chloropyridin-3-yl
—
S
S
O
O
S
S
S
S
S
H
>25
13.1
>25
>25
>25
>25
14.6
18.4
15.3
0.5
6.3
1.5
>25
>25
>25
7.2
4.6
3.4
8.9
8.2
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
Isobutyryl
H
Isobutyryl
H
d
e
b
b
c
H
Isobutyryl
b
b
H
H
—
Nutlin-3a
—
a
Inhibitory activities against the protein complexes were measured by the modified ELISA using Flag-p53 (10 mg) and GST–protein (1.5 mg).
The compounds were subjected to the assay at concentrations of less than or equal to 25 mM owing to their limited solubilities.
b
1
1
1
40
20
00
according to the schedules indicated in Figure 3A. K-178 (1c) sup-
pressed tumor growth in HCT116 xenografts to 49.0% and 36.6%
that of the control at 100 mg/kg and 150 mg/kg, respectively, in
TIG䠓
MCF7
4
0 days. No weight losses were observed in either experiment
(Fig. 3B).
A Western blotting analysis using proteins extracted from
A431
A427
8
6
4
2
0
0
0
0
0
tumor masses showed that p53 and its downstream proteins
Mdm2 and p21 were up-regulated, particularly in tumor masses
in nude mice administered a dose of 150 mg/kg (Fig. 4), as
observed in cells incubated with K-178 (1c) in vitro. Additionally,
cleaved caspase 3, a marker of apoptosis, was markedly increased
in tumors treated with 1c. These results strongly suggest that the
activation of p53 by K-178 (1c) occurs in vivo as detected by an
in vitro assay. Furthermore, the treatment with K-178 (1c)
increased the number of terminal transferase-mediated dUTP nick
end-labeling (TUNEL)-positive cells in sections of tumor masses
(Fig. 5A). The percentage of the apoptotic cell population was cal-
culated by counting TUNEL-positive cells in microscopic fields,
and the treatment with K-178 (1c) increased the number of
TUNEL-positive cells in a dose-dependent manner (Fig. 5B).
MDA-MB-468
HCT116
0
10
20
30
40
K-178 (1c) (µM)
Figure 1. Effects of K-178 (1c) on growth in various cancer cells. Cells harboring
wild-type or mutant p53 were incubated in the absence or presence of K-178 (1c) at
the indicated concentrations. Cell viability was assayed 48 h after the treatment
with K-178 (1c) by staining with crystal violet as described in Section 8. The
following cells were used in this assay: MCF-7, A427, and HCT116 (Human cancer
cells with wild-type p53); A431 and MDA-MB-468 (human cancer cells with
mutant p53); TIG-7 (human normal cell).
7
. Discussion and conclusion
We previously reported that the S-isobutyryl-protected o-
aminothiophenol K179 disturbed, albeit weakly, binding between
b-catenin and transcription factor 4 (TCF4) proteins. In the pre-
3
3
5
. Detection of p53, Mdm2, and p21 by a Western blot assay and
localization of p53 by immunofluorescence microscopy
sent study, we found that the same series of compounds, K-178
(
1c) and K-181 (3c), had the ability to inhibit binding between
Western blot analyses showed that p53, Mdm2, and p21 levels
were higher in MCF-7 cells treated with K-178 (1c) or Nutlin-3a than
in control cells, and these increases occurred in a dose-dependent
manner (Fig. 2A). These results suggest that the activation of p53 in
cells treated with K-178 (1c) up-regulates the expression of its down-
stream genes such as p21 and Mdm2, as observed in cells treated with
Nutlin-3a. The results of the immunofluorescence study confirmed
that K178 (1c)- or Nutlin-3a-activated p53 accumulated in the nuclei
of human neuroblastoma (IMR32) and MCF-7 cells (Fig. 2B).
p53 and Mdmx. We prepared the related compounds 1b, 1d, 1e,
2b, 3b, 4b, and 5b in order to analyze SAR as well as selectivity
in the inhibition of binding between p53 and Mdm2 and between
p53 and Mdmx using a modified ELISA. In the present study, not
only the S-isobutyryl-deprotected sulfides 1b, 3b, 4b, and 5b, but
also the S-isobutyryl-protected derivatives 1c and 3c, preferen-
tially inhibited the p53–Mdmx interaction over the p53–Mdm2
interaction. Furthermore, inhibitory activity against the p53–
Mdmx interaction was lost by replacing the sulfur atom in 1c
and 1b by an oxygen atom in 1e and 1d. We speculate that 1b or
3b disrupted the p53–Mdmx interaction through disulfide-bond
formation between the free thiol groups of 1b or 3b and Cys77 in
Mdmx in the p53-binding domain. However, this speculation
needs to be substantiated by spectroscopically detecting the pres-
ence of this disulfide bond. Furthermore, it is not clear why neither
6
. Oral administration study on K-178 (1c) in nude mice with
HCT116 xenografts
K-178 (1c) was orally administered to nude mice with HCT116
xenografts at a dose of 100 mg/kg or 150 mg/kg over 38 days

1
922
S. Uesato et al. / Bioorg. Med. Chem. 24 (2016) 1919–1926
(B)
(
A)
K-178 (1c) Nutlin-3a
DMSO
CHC
Mdm2
K-178 (1c)
(
10 µM)
p53
p21
Nutlin-3a
10 µM)
(
IMR32 cells
MCF7 cells
Figure 2. Detection of p53, Mdm2, and p21 by the Western blot assay and localization of p53 by immunofluorescence microscopy. (A) Cells were treated with K-178 (1c) for
4 h and expression levels of the indicated proteins in these cell lysates were determined by immunoblotting. (B) Cells were fixed with 3% paraformaldehyde 24 h after the
2
treatment with K-178 (1c) and subjected to a confocal immunofluorescent analysis using the anti-p53 antibody (green). Nuclei were stained by DAPI (blue).
Figure 3. Oral administration study on K-178 (1c) in nude mice with HCT116 cell-inoculated xenografts. The effects of K-178 (1c) on tumor volume (A) as well as relative
body weight (RBW) changes (B) at a dose of 100 mg or 150 mg/kg/day were examined as described in Section 8. K-178 (1c) was orally administered to mice every other day
until day 38. Significant differences (p <0.05) were obtained for tumor volume with K-178 (1c) vs. the control as follows: K-178 (1c) (100 mg/kg) at all time points after day
2
2; K-178 (1c) (150 mg/kg) on days 4 and 14 as well as at all time points after day 20.
1
b nor 3b inhibited the binding of p53 to Cys76 in Mdm2. The rea-
spectra were measured on HPLC–ESI–LC–MS/MS (Prominance sys-
tem, Shimadzu Co., Ltd and Q Exactive series, Thermo scientific Co.,
Ltd). H NMR spectra were recorded on JEOL EX-400 (400 MHz) in
son for this may be interpreted in terms of differences in the steric
bulkiness of the two C-terminal amino acids next to Cys between
Mdmx and Mdm2 as follows. Mdmx has less hindered Gly78-
Gly79 relative to Ser77 and Asn78 in Mdm2. Therefore, the sulfide
1
3
CDCl , unless otherwise noted, as a solvent and tetramethylsilane as
an internal standard. Analytical TLC was performed using Silica gel
60 F254 (Merck, 0.25 mm) glass plates. Column chromatography
was performed using Silica Gel 60 (70–230 mesh ASTM) or flash chro-
matographs [Smart Flash EPCLC W-Prep 2XY (Yamazen) and Isolera
1
b or 3b preferentially approaches from the less sterically hindered
side to Cys77 in Mdmx. We also assume that the heptyl group in
the sulfide 2b is too bulky to inhibit the p53–Mdm2 and p53–
Mdmx interactions.
2 4
One (Biotage)]. All solvents were dried over Na SO , and evaporated
In the present study, we demonstrated that o-aminothiophenol
series compounds with molecular weights of 200–300 had the
capabilities to inhibit the p53–Mdmx interaction. Based on the
inhibitory effects of the same series of compound K-179 on
b-catenin-TCF4 binding, the present series of compounds have
the potential to inhibit a number of PPIs by changing substituents at
the N-position or on a benzene ring. We expect o-aminothiophenol
series compounds to provide a promising basis for the design and
synthesis of new low-molecular-weight PPI inhibitors.
in vacuo. Bis(2-benzamidophenyl) disulfide (3a) was purchased from
TCI Japan, Inc. The TUNEL assay reagent, DeadEnd Fluorometric
TUNEL System, was obtained from Promega Corporation.
TM
8.2. Synthesis
8.2.1. S-2-Isobutyramidophenyl 2-methylpropanethioate K-178
(1c)
Py (0.35 mL, 4.33 mmol) and isobutyryl chloride (0.43 mL,
4
.04 mmol) were added to a solution of o-aminothiophenol
8
8
. Experimental section
(0.20 mL, 1.87 mmol) in CH Cl (5 mL). After stirring at room tem-
perature (RT) for 1 h, the reaction mixture was washed successively
with 1% HCl, satd NaHCO , and brine. The organic layer was dried
2
2
.1. General
3
and concentrated to give a residue. Chromatography (n-hexane/
EtOAc 5:1) over a silica gel and recrystallization from EtOH gave
Melting pointsweredeterminedonaYanacoMP-500Pmicromelt-
ing point apparatus and were uncorrected. High-resolution mass
K-178 (1c) (466.6 mg, 1.76 mmol, yield 94.0%) as colorless needles.

S. Uesato et al. / Bioorg. Med. Chem. 24 (2016) 1919–1926
1923
3 3
CHCl . The CHCl layer was dried and concentrated to yield a white
solid, which was rinsed with n-hexane to afford 1b (40.7 mg,
0
.21 mmol, yield 39.5%).
Mp 83.3–87.0 °C. H NMR d : 1.31 (6H, d, J = 6.8 Hz, (CH
1
3
2
) CHA),
2
3 2
.62 (1H, septet, J = 6.8 Hz, (CH ) CHA), 3.09 (1H, s, SH), 7.02 (1H, t,
J = 8.0 Hz, Ar-H), 7.31 (1H, t, J = 8.0 Hz, Ar-H), 7.51 (1H, d, J = 8.0 Hz,
Ar-H), 8.11 (1H, br s, NH), 8.30 (1H, d, J = 8.0 Hz, Ar-H). HR-LC–ESI-
ꢀ
MS m/z: (MꢀH) calcd for C10
H12NOS, 194.0640; found 194.0637.
Mdmx
Mdm2
8
.2.3. N-(2-Mercaptophenyl)octanamide (2b)
NaBH
4
(20.6 mg, 0.54 mmol) was added in one portion at 0 °C to a
0
solution of N,N -(disulfanediylbis(2,1-phenylene)dioctanamide (2a)
(151 mg, 0.30 mmol) in THF (2.4 mL) and EtOH (0.6 mL). After stir-
ring at rt for 3 h, 1 M aq NaOH (5 mL) and then 1 M aq citric acid
(2 mL) was added to the reaction mixture, which was extracted with
EtOAc. The EtOAc layer was dried and concentrated. The crude pro-
duct was purified by flash column chromatography on a silica gel
p53
p21
caspase 3
(EtOAc/n-hexane) to yield 2b as a yellowish oil (3.9 mg, 0.016 mmol,
1
yield 2.6%). H NMR d: 0.86–0.88 (3H, m, CH
3
(CH
A), 1.55–1.64 (1H, m, ACH
CO), 2.28 (1H, t, J = 7.6 Hz, ACH
CH CO), 4.03 (1H, s, SH), 6.82 (1H, dd, J = 8.0,
.1 Hz, Ar-H), 7.01 (1H, td, J = 7.6, 1.2 Hz, Ar-H), 7.17 (1H, td, J = 7.6,
2
)
4
CH
CH CO), 1.86–1.95
CH CO), 3.39 (1H,
2
A), 1.27–1.31
(
8H, m, CH
(CH ) CH
3 2 4 2
2
2
cleaved caspase3
(1H, m, ACH
2
CH
2
2
2
dd, J = 8.7, 6.0 Hz, ACH
1
1
2
2
β-actin
.4 Hz, Ar-H), 7.31–7.33 (1H, m, Ar-H), 8.07 (1H, s, NH). HR-LC–ESI-
ꢀ
MS m/z: (MꢀH) calcd for C14
H20NOS, 250.1266; found 250.1272.
Figure 4. Western blot analysis of proteins derived from tumor masses. Tumor
masses were removed from mice 40 days after initiating the administration of K-
1
78 (1c), and expression levels of the indicated proteins in the lysates were
8
.2.4. N-(2-mercaptophenyl)benzamide (3b)
To a solution of bis(2-benzamidophenyl)disulfide (3a) (140 mg,
.31 mmol) in THF (2.4 mL) and EtOH (0.8 mL) was added NaBH
12 mg, 0.32 mmol) in one portion. After stirring at rt for 1.5 h, 2 M
determined by immunoblotting. The numbers (1) and (2) in parentheses indicate
the numbering of arbitrary masses.
0
(
4
1
Mp 92–94 °C. H NMR d: 1.25 (6H, br d, J = 7.2 Hz, (CH
3
)
2
CHA),
CHA), 2.51 (1H, septet, J = 7.2 Hz,
CHA), 2.94 (1H, septet, J = 7.2 Hz, (CH CHA), 7.13 (1H, br
aq NaOH (10 mL) was added to the reaction mixture, which was then
washed with n-hexane/EtOAc 2:1. The water layer was successively
adjusted to pH 4 with 1 M aq citric acid (10 mL) and extracted with
EtOAc. The EtOAc layer were dried and concentrated to yield a white
1
.30 (6H, br d, J = 7.2 Hz, (CH
3 2
)
(
CH
3
)
2
3 2
)
t, J = 7.6 Hz, Ar-H), 7.38 (1H, br d, J = 7.6 Hz, Ar-H), 7.46 (1H, br t,
J = 7.2 Hz, Ar-H), 7.77 (1H, br s, NH), 8.34 (1H, d, J = 7.6 Hz, Ar-H).
2
solid. The resulting solid was washed with Et O to afford 3b (15 mg,
ꢀ
HR-LC–ESI-MS m/z: (MꢀH) calcd for C14
H20NO
2
S, 264.1058;
1
0.065 mmol, yield 11%). Mp 98.0–101.3 °C. H NMR d: 3.17 (s, 1H,
found, 264.1065.
SH), 7.08 (1H, td, J = 7.4, 1.2 Hz, Ar-H), 7.39 (1H, t, J = 7.8 Hz, Ar-H),
7
.61-7.51 (4H, m, Ar-Hꢁ4), 7.98–7.96 (2H, m, Ar-H
2
), 8.48 (1H, d,
8
.2.2. N-(2-Mercaptophenyl)isobutyramide (1b)
KOH (151 mg, 2.69 mmol) was added to a solution of 1c
140 mg, 0.53 mmol) in THF (0.8 mL) and MeOH (0.8 mL). After
stirring at rt for 4 h, the reaction mixture was diluted with water
10 mL) and concentrated to remove THF. The resulting aqueous
ꢀ
J = 8.4 Hz, Ar-H), 8.94 (1H, br s, NH). HR-LC–ESI-MS m/z: (MꢀH)
calcd for C13 10NOS, 228.0483; found 228.0485.
H
(
8
.2.5. S-2-Benzamidophenyl 2-methylpropanethioate K-181 (3c)
NaBH (113.5 mg, 3.00 mmol) was added under ice-cooling to a
(
4
solution was successively washed with n-hexane/EtOAc 1:1,
adjusted to pH 4 with 1 M aq citric acid (2 mL), and extracted with
solution of bis(2-benzoylaminophenyl)disulfide (3a) (456.6 mg,
(
A)
(B)
1
1
1
40
20
00
*
*
control
K-178 (1c) 100mg
K-178 (1c) 150mg
80
60
40
20
0
control
*
*
K-178 (1c) 100mg/kg
**p<0.01 vs control
K-178 (1c) 150mg/kg
Figure 5. Detection of TUNEL-positive cells in sections prepared from tumor masses. Tumor masses were treated as indicated in Section 8. (A) TUNEL-positive cells (brown in
color) were visualized with fluorescent microscopy according to the procedure indicated in Section 8. (B) TUNEL-positive cells as a percentage of all cells in the microscopic
field were determined by examining eight fields per view at ꢁ20 magnification and then expressed in the form of bar graphs.

1
924
S. Uesato et al. / Bioorg. Med. Chem. 24 (2016) 1919–1926
1
2
.00 mmol) in THF (4 mL) and EtOH (20 mL). After stirring at rt for
h, the reaction mixture was quenched by a few drops of acetic
were dried and concentrated to yield a white solid, which was
washed with n-hexane to afford 4b (178 mg, 0.67 mmol, yield
68%) as a white solid.
Mp 110.1–113.6 °C. H NMR d : 3.17 (1H, s) 7.09 (1H, t, J = 8.0 Hz,
Ar-H) 7.39 (1H, t, J = 8.0 Hz, Ar-H) 7.50 (2H, td, J = 2.4, 8.8 Hz,
acid and concentrated. The residue was dissolved in EtOAc, and
the solution was washed with water. The organic layer was dried
and concentrated to give a yellowish solid. This substance, without
purification, was, in turn, subjected to isobutyrylation with isobu-
tyryl chloride (0.422 mL, 4.00 mmol) and Py (0.322 mL, 3.98 mmol)
1
Ar-H
Ar-H
2
), 7.58 (1H, d, J = 8.0 Hz, Ar-H), 7.90 (2H, td, J = 2.4, 8.8 Hz,
2
), 8.44 (1H, d, J = 8.0 Hz, Ar-H), 8.90 (1H, br s, NH).
ꢀ
in CH
2
Cl
2
(5 mL) in the same manner as that described for the
HR-LC–ESI-MS m/z: (MꢀH) calcd for C13
8 2
H ClN OS, 263.0046;
preparation of 2c, giving 3c (205.0 mg, 0.68 mmol, yield 34.2%).
found 263.0047.
1
Mp 104.6–105.7 °C.
J = 6.8 Hz, (CH CHA), 2.65 (1H, septet, J = 6.8 Hz, (CH
.33 (1H, t, J = 8.0 Hz, Ar-H), 7.47–7.54 (4H, m, Ar-H ), 7.58 (1H,
d, J = 7.2 Hz, Ar-H), 7.62 (1H, d, J = 7.6 Hz, Ar-H), 7.92 (2H, d, A
of A , J = 7.6 Hz, Ar-H ), 9.93 (1H, br s, NH). HR-FAB-MS m/z:
, 300.1059; found, 300.1054.
H
NMR (DMSO-d
6
)
d: 1.09 (6H, d,
0
3
)
2
3
)
2
CHA),
8.2.10. N,N -(Disulfanediylbis(2,1-phenylene))bis(6-
chloronicotinamide) (5a)
7
4
2
N,N-4-Dimethylaminopyridine (0.590 mL, 4.82 mmol) and 6-
chloronicotinoyl chloride (850 mg, 4.82 mmol) were added to a
2
B
2
2
+
0
(
M+H) calcd for C22
H
18
N
3
O
9
solution of 2,2 -dithiodianiline (500 mg, 2.01 mmol) in N,N-
dimethylformamide (DMF) (10 mL). After stirring at rt for 9 h,
DMF was removed and washed successively with water and EtOH.
8
.2.6. 2-Isobutyramidophenyl isobutyrate (1e)
Py (2.21 mL, 27.3 mmol) and isobutyryl chloride (2.90 mL,
3
Recrystallization from CHCl gave 5a (600 mg, 1.14 mmol, yield
2
9
7.5 mmol) were added to a solution of o-aminophenol (1.00 g,
56.6%) as colorless needles.
.16 mmol) in CH
2
Cl
2
(5 mL). After stirring at rt for 30 min, the
Mp 216.1–218.0 °C. 1H NMR d: 7.01 (2H, td, J = 7.7, 1.6 Hz, Ar-
Hꢁ2), 7.27 (2H, td, J = 8.0, 1.4 Hz, Ar-Hꢁ2), 7.44 (2H, dd, J = 8.4,
0.8 Hz, Ar-Hꢁ2), 7.53 (2H, dd, J = 7.8, 1.4 Hz, Ar-Hꢁ2), 7.90 (2H,
dd, J = 8.4, 2.4 Hz, Ar-Hꢁ2), 8.34 (2H, dd, J = 8.4, 1.2 Hz, Ar-Hꢁ2),
reaction mixture was washed successively with 1% HCl, satd
NaHCO , and brine. The organic layer was dried and concentrated
to give a solid. Recrystallization from CHCl and n-hexane gave
e (1.65 g, 6.62 mmol, yield 72.2%) as colorless needles.
3
3
1
8.63 (2H, br d, J = 2.4 Hz, Ar-Hꢁ2), 8.77 (2H, br s, NHꢁ2). HR-ESI-
1
+
Mp 88.5–90.6 °C. H NMR d: 1.25 (6H, d, J = 6.8 Hz, (CH
.37 (6H, J = 6.8 Hz, (CH CHA), 2.51 (1H, septet, J = 6.8 Hz,
CHA), 2.94 (1H, septet, J = 6.8 Hz, (CH CHA), 7.11–7.30
). HR-LC–ESI-MS m/z: (MꢀH) calcd for C14
48.1287; found 248.1292.
3
)
2
CHA),
MS m/z: (M+H) calcd for C24
H17Cl
2
N
O
4 2
2
S , 527.0171; found,
1
3
)
2
527.0169.
(
CH
3
)
2
3 2
)
ꢀ
(
3H, m, Ar-H
3
H
3
18NO ,
8.2.11. 6-Chloro-N-(2-mercaptophenyl)nicotinamide (5b)
PPh (102 mg, 0.39 mmol) was added in one portion at 0 °C to a
2
3
solution of 5a (102 mg, 0.19 mmol) in THF (1.9 mL). After stirring
at rt for 3 h, the reaction mixture was concentrated in vacuo to give
a crude product, which was purified by flash column chromatogra-
phy on a silica gel (EtOAc/n-hexane) to yield 5b as a white solid
(12.9 mg, 0.049 mmol, yield 12.9%). Mp 135.1–136.4 °C. H NMR
d: 3.75 (1H, s, SH), 7.26–7.28 (1H, m, Ar-H), 7.43–7.56 (3H, m,
8
.2.7. N-(2-Hydroxyphenyl)isobutyramide (1d)
A total of 5 N aq KOH (1.21 mL, 6.03 mmol) was added to a solu-
tion of 1e (500.0 mg, 1.70 mmol) in EtOH (10 mL). The progress of
the reaction was monitored by TLC. When the reaction ceased, the
reaction mixture was adjusted to pH 6.0 with 1 N HCl and concen-
trated. The residue was dissolved in EtOAc and washed succes-
1
3
Ar-H ), 7.95 (1H, d, J = 7.8 Hz, Ar-H), 8.11 (1H, d, J = 8.0 Hz, Ar-H),
sively with satd NaHCO
and concentrated to give 1d (169.1 mg, 0.94 mmol, yield 55.5%)
as colorless needles. Mp 107.4–109.2 °C. H NMR d: 1.31 (6H, d,
3
and brine. The organic layer was dried
8.36 (1H, dd, J = 8.3, 2.7 Hz, Ar-H), 9.07 (1H, s, NH). HR–ESI-MS
ꢀ
m/z: (MꢀH) calcd for C13
9
H ClNOS, 262.0093; found 262.0100.
1
J = 7.2 Hz, (CH
3
)
2
CHA), 2.65 (1H, septet, J = 6.8 Hz, (CH
3
)
2
CHA),
8.3. Cell lines, cultures, and antibodies
6
.86 (1H, t, J = 7.2 Hz, Ar-H), 6.97 (1H, d, J = 8.0 Hz, Ar-H), 7.02
(
1H, d, J = 8.0 Hz, Ar-H), 7.13 (1H, t, J = 7.2 Hz, Ar-H), 7.40 (1H, br
HCT116 (wild-type p53), TIG7 (wild-type p53), MCF-7 (wild-
type p53), A427 (wild-type p53), A431 (mutant p53), and MDA-
MB-468 (mutant p53) cell lines were purchased from the American
Type Culture Collection (ATCC). IMR32 (wild-type p53) was a gift
from Dr. Nakagawara (Saga-ken Medical Centre Koseikan).
HCT116 cells were cultured in McCoy’s medium (GIBCO), supple-
mented with 10%fetal bovine serum (FBS) (Biowest or GIBCO),
ꢀ
s, OH), 8.86 (1H, br s, NH). HR-LC–ESI-MS m/z: (MꢀH) calcd for
C
10
H12NO
2
, 178.0868; found 178.0861.
0
8
.2.8. N,N -(Disulfanediylbis(2,1-phenylene))bis(4-
chlorobenzamide) (4a)
4
-Chlorobenzoyl chloride (1.08 mL, 8.46 mmol) was added to a
0
solution of 2,2 -dithioaniline (1000 mg, 4.03 mmol) in N,N-diiso-
propylethylamine (1.75 mL, 10.1 mmol) and CH
After stirring at rt for 2 h, the resulting white precipitate was col-
lected and washed with water (20 mL). The resulting solid was
dried at rt to afford 4a as a white solid (1780 mg, 3.40 mmol, yield
50
in a 5% CO
tured in Dulbecco’s Modified Eagle Medium (Sigma–Aldrich) sup-
plemented with 10% FBS, 50 g/mL penicillin G, and 50 g/mL
streptomycin sulfate (GIBCO) in a 5% CO and 95% air atmosphere
at 37 °C. Other cells were cultured in RPMI-1640 (Sigma–Aldrich)
supplemented with 10% FBS, 50 g/mL penicillin G, and 50 g/mL
streptomycin sulfate (GIBCO) in a 5% CO and 95% air atmosphere
l
g/mL penicillin G, and 50
lg/mL streptomycin sulfate (GIBCO)
2
Cl
2
(13.4 mL).
2
and 95% air atmosphere at 37 °C. TIG-7 cells were cul-
l
l
2
1
8
H
4%). Mp 178.0–178.8 °C. H NMR d: 6.97 (4H, td, J = 7.6, 1.2 Hz, Ar-
2
ꢁ2), 7.30 (2H, td, J = 8.6, 1.0 Hz, Ar-H
2
ꢁ2), 7.41, 7.57 (8H, A
2
B
2
,
l
l
J = 8.4 Hz, Ar-H
4
ꢁ2), 7.46 (4H, dd, J = 7.8, 1.8 Hz, Ar-H
2
ꢁ2), 8.43
2
ꢀ
(
4H, dd, J = 8.0, 1.0 Hz, ANHꢁ2). HR-LC-ESI-MS m/z: (MꢀH) calcd
at 37 °C. Trypsin was purchased from GIBCO, and CELLBANKER 2
was from Nippon Zenyaku Kogyo Co., Ltd. The anti-actin antibody
was purchased from Sigma–Aldrich. The anti-p53 antibody was
from Santa Cruz Biotechnology, antibodies specific to Mdm2 were
from Abcam and Merck Millipore (Calbiochem), and the anti-
Mdmx antibody was from Abcam. The anti-p21 antibodies were
from BioLegend and Abcam, and the anti-caspase 3 antibody was
from BioLegend. The antibody to cleaved caspase 3 (Asp175) was
for C26
H18Cl
2
N
O
2 2
2
S , 523.0109; found 523.0121.
8
.2.9. 4-Chloro-N-(2-mercaptophenyl)benzamide (4b)
NaBH (34 mg, 0.90 mmol) was added in one portion to a solu-
4
tion of 4a (262 mg, 0.50 mmol) in THF (4.0 mL) and EtOH (1.0 mL).
After stirring at rt for 8 h, 1 M aq NaOH (10 mL) was added to the
reaction mixture, which was then washed with n-hexane/EtOAc
TM
2
:1. The water layer was successively adjusted to pH 4 with 1 M
from Cell Signaling Technology. ECL Anti-Rabbit IgG, Horseradish
TM
aq citric acid (4 mL) and extracted with CHCl . The CHCl layer
3
3
Peroxidase-linked whole antibody, ECL
Anti-mouse IgG,

S. Uesato et al. / Bioorg. Med. Chem. 24 (2016) 1919–1926
1925
TM
Horseradish Peroxidase-linked whole antibody, and ECL Prime
Western Blotting Detection Reagent were from GE Healthcare.
SuperSignal West Femto Chemiluminescent Substrate was from
Thermo SCIENTIFIC.
the following proteins were used: b-actin, p53, Mdm2, Mdmx,
p21, and cleaved caspase 3 at a 1:1000 dilution; full-length caspase
3 at a 3:1000 dilution). Secondary antibodies conjugated to horse-
radish peroxidase (at a 1:1000–2000 dilution) (GE Healthcare, UK)
TM
were used, and blots were further incubated with ECL Prime Wes-
Ò
8
.4. Animals
tern Blotting Detection Reagent (GE Healthcare) or SuperSignal
West Femto Maximum Sensitivity Substrate (Thermo Fisher Scien-
tific). Immunoreactive proteins were detected using ECL chemilu-
minescence (Image Quant LAS 3000 detection system) (GE
Healthcare).
Female BALB/c-nu.nu nude mice (5 weeks old) were purchased
from Japan SLC Inc. Animals were housed and experiments were
performed according to the guidelines stipulated by the Osaka
University of Pharmaceutical Sciences Animal Care and Use
Committee. Mice were used at 6 weeks of age.
8.8. Detection of apoptotic cells by the TUNEL method
8
.5. Cell viability assays using crystal violet
The TUNEL assay to detect apoptosis in the tumor masses was
outsourced to Applied Medical Research Co., Ltd, Japan. Tumor
masses were collected and fixed in 4% paraformaldehyde over-
night, dehydrated, and embedded in paraffin the next day. Tumor
sections were deparaffinized, then rehydrated and treated with
an Apop Tag Peroxidase In Situ Apoptosis Detection Kit (Millipore)
according to the manufacturer’s protocol. TUNEL-positive cells as a
percentage of all cells in a microscopic field were determined by
examining eight fields of a view at ꢁ20 magnification with a
Keyence BZ-9000 microscope.
4
Cells were plated at a density of 2 ꢁ 10 cells/well on 24-well
microtiter plates. After incubating in an atmosphere of 5% CO
2
and 95% air at 37 °C for 24 h, cells were grown in the presence of
serially diluted samples for 48 h and then stained for 15 min with
a crystal violet solution (0.75% crystal violet, 0.25% NaCl, 1.75%
formaldehyde, 50% EtOH, and 47.25% MilliQ). The absorbance of
the solutions was measured spectrophotometrically at 600 nm
using a SH-1000 Microplate Reader (Corona Electric Co., Ltd), and
cell viability was assessed. Cell viability (%) relative to the control
at each concentration point (0, 10, 15, 20, 25, or 30
lM) was calcu-
8.9. Immunofluorescence
lated by the number of tested cells/number of control cells ꢁ 100.
HCT116. or MCF-7 cells were seeded at a concentration of
4
8
.6. In vivo antitumor tests
3.0 ꢁ 10 /mL in a 4-well chamber slide with 0.4 mL medium per
well and incubated for 24 h. After the addition of each test sample
6
TM
HCT116 cells (1.5 ꢁ 10 /100
l
L of Matrigel (BD Biosciences)
(10 lM), the incubation was continued for a further 24 h. Cells
were fixed with 3% paraformaldehyde in PBS at 25 °C for 30 min
were injected subcutaneously into the backs of nude mice (6 weeks
old). K-178 (1c) (2.0 or 3.0 mg/200
lL) (n = 6 or 9) as well as vehi-
and then permeabilized with 0.05% Triton X-100 in PBS at 25 °C
cle (0.1% Tween 80, 200 L) (n = 7) were administered orally to
nude mice with tumors (tumor size: 70–143 mm ) every other
l
for 5 min. The chamber slide was rinsed with PBS (200
l
L ꢁ 4),
3
and cells were blocked with 3% BSA in PBS for 10 min. Cells were
then exposed to a 1:50 dilution of the p53 Antibody (FL-393)
(Santa Cruz) overnight at 4 °C followed by a treatment with a
1:200 dilution of the goat anti-mouse IgG (H+L) secondary anti-
body conjugated to Alexa FluorÒ 488 (Molecular Probes) contain-
day until day 38. Tumor length and width as well as body weight
3
were monitored. Tumor volume (mm ) was calculated by measur-
ing tumor length and width (in mm) as described.34 Antitumor
activity was expressed as the percentage of mean tumor volumes
for treated mice to mean tumor volumes for control mice. At day
ing propidium iodide (Dojindo, Japan) (35 lg/mL) at RT for
4
0, the cervical spine was dislocated and the chest was opened.
30 min. PBS containing 3% BSA was used to dilute antibodies. After
Tumor masses were removed and weighed as they were.
removal of the PBS solution containing the unbound secondary
antibody, a few drops of DAPI (Dojindo, Japan) and PBS (500 lL)
8
.7. Western blot analysis
Cells were plated on 60-mm diameter dishes (1.0 ꢁ 10 /dish) in
were added successively to each well. Cells in each well
were viewed under fluorescence microscopy (magnification ꢁ40)
(Biorevo, Keyence, Japan).
6
medium supplemented with FBS, penicillin (20 U/mL), and strepto-
mycin (20 mg/mL). After being incubated for 24 h, the cells were
washed twice with medium (1 mL each). After the addition of med-
ium (5 mL), the culture was incubated for another 24 h and then
treated with the tested compound, Nutrin-3a or control (0.1%
DMSO) for 24 or 48 h. Medium was discarded, and floating and
adhered cells were washed with PBS (2 ꢁ 1 mL), scraped, and
8.10. Preparation of GST–Mdm2, GST–Mdmx, GST–DAPK1,
GST–PPID, and Flag-p53
GST–Mdm2, GST–Mdmx, GST–DAPK1, GST–PPID, and Flag-p53
were prepared in an analogous manner to the reported proce-
2
4,25
dures.
Thus, GST–Mdm2, GST–Mdmx, GST–DAPK1, GST–PPID,
homogenized for 1 min in 400
buffer saline, 15 mM NaCl, 0.2 mM MgCl
EGTA, 0.01% NP-40, 1 mM Na VO , 10 mM NaF, 1 mM DTT, and
mM PMSF) containing proteinase inhibitor cocktail (Nacalai Tes-
l
L of lysis buffer (5 mM Tris–HCl
and GST–Flag-p53 constructs in pGEX6P-1 vectors (GE Healthcare)
were transformed into Escherichia coli BL21 (DE3) (Novagen) by the
heat shock method. Cells were grown at 37 °C overnight on LB/agar
2
, 0.1 mM EDTA, 0.1 mM
3
4
1
(Merck) containing 100
lg/ml ampicillin. Cells were then trans-
que) at 0 °C. Tumor masses were also frozen with liquid nitrogen
and immediately ground into pieces. The resulting powder was
suspended in the above-described lysis buffer. The lysates from
cells or tumor masses were centrifuged at 15,000ꢁg at 4 °C for
ferred into LB liquid medium containing 100
l
g/ml ampicillin, sus-
pended at 37 °C until OD600 reached to 0.6, and induced with
0.1 mM IPTG (Nacalai Tesque) at 30 °C for 3 h. The bacteria pellet
was collected, resuspended and sonicated in PBST (1ꢁ PBS contain-
ing 0.1% Tween 20). The lysates were centrifuged at 7000 rpm for
10 min. The supernatants were collected and dialyzed against
20 mM PBS, and the GST fusion proteins were purified by an
ÄKTA Explorer system with the pre-loaded GSTrap column (GE
Healthcare) using an elution buffer [10 mM glutathione–20 mM
PBS (pH 8.2)]. GST–Flag-p53 was treated with Turbo3C protease
1
5 min. Protein concentrations were determined with a BCA pro-
tein assay kit (Thermo Fisher Scientific). An equal amount (10 lg)
of protein was then resolved by SDS–PAGE and transferred to a
PVDF membrane (Millipore). Blots (1ꢁ Tris–HCl buffer saline, 5%
nonfat milk, and 0.05% Tween 20) were probed with an antibody
specific to each protein. Primary monoclonal antibodies against

1
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S. Uesato et al. / Bioorg. Med. Chem. 24 (2016) 1919–1926
(
Nacalai Tesque), and the resultant Flag-p53 was purified by the
References and notes
ÄKTA Explorer system in the typical manner.
1
2
3
.
.
.
Ryan, K. M.; Phillips, A. C.; Vousden, K. H. Curr. Opin. Cell Biol. 2001, 13, 332.
Evan, G. I.; Vousden, K. H. Nature 2001, 411, 342.
Ingrid Herr, I.; Debatin, K. M. Blood 2001, 98, 2603.
8
.11. Measurement of the inhibition of o-aminothiophenols
using the modified ELISA
4. Haupt, S.; Berger, M.; Goldberg, Z.; Haupt, Y. J. Cell Sci. 2003, 116, 4077.
5.
Beckerman, R.; Prives, C. Cold Spring Harb. Perspect. Biol. 2010, 2, a000935.
http://dx.doi.org/10.1101/cshperspect.a000935.
Brooks, C. L.; Gu, W. Mol. Cell 2006, 21, 307.
In the preparation of the ELISA system, glutathione-coated
plates [8-well Strip (Thermo Scientific)] were saturated with 1ꢁ
PBS containing 1% skim milk (Nacalai Tesque) at rt for 1 h. After
removal of the buffer, a solution of the GST-fused protein in 1ꢁ
PBS containing 1% skim milk was immobilized on the glu-
tathione-coated plates at RT for 1 h. The plates were then washed
with PBST and incubated with FLAG-p53. The o-aminothiophenols
6
.
7. Yamasaki, S.; Yagishita, N.; Sasaki, T.; Nakazawa, M.; Kato, Y.; Yamadera, T.;
Bae, E.; Toriyama, S.; Ikeda, R.; Zhang, L.; Fujitani, K.; Yoo, E.; Tsuchimochi, K.;
Ohta, T.; Araya, N.; Fujita, H.; Aratani, S.; Eguchi, K.; Komiya, S.; Maruyama, I.;
Higashi, N.; Sato, M.; Senoo, H.; Ochi, T.; Yokoyama, S.; Amano, T.; Kim, J.; Gay,
S.; Fukamizu, A.; Nishioka, K.; Tanaka, K.; Nakajima1, T. The EMBO Journal 2007,
26, 113.
8
9
.
.
Hock, A. K.; Vousden, K. H. Biochim. Biophys. Acta 2014, 137, 1843.
Wade, M.; Li, Y. C.; Wahl, G. M. Nat. Rev. Cancer 2013, 13, 83.
1
b–1e, 2b, 3b–3c, 4b, and 5b, as well as the positive control Nutlin-
10. Wang, X.; Jiang FEBS Lett. 2012, 586, 1390.
3
a, were added to these plates. After being incubated for 1 h, the
11. Zhao, Y.; Bernard, D.; Wang, S. BioDiscovery 2013, 8, 4.
1
1
2. Mohammad, R. M.; Wu, J.; Azmi, A. S.; Aboukamee, A.; Sosin, A.; Wu, S.; Yang,
D.; Wang, S.; Al-Katib, A. M. Mol. Cancer 2009, 8, 115.
plates were washed with PBST and incubated with the anti-Flag
antibody (at a 1:10,000 dilution with 1ꢁ PBS containing 1% skim
milk) at rt for 1 h. After washing with PBST to remove the buffer,
absorbance at 492 nm was measured by a microplate reader
3. Ding, Q.; Zhang, Z.; Liu, J.-J.; Jiang, N.; Zhang, J.; Ross, T. M.; Chu, X.-J.;
Bartkovitz, D.; Podlaski, F.; Janson, C.; Tovar, C.; Filipovic, Z. M.; Higgins, B.;
Glenn, K.; Packman, K.; Vassilev, L. T.; Graves, B. J. Med. Chem. 2013, 56, 5979.
4. Wang, W.; Qin, J.-J.; Voruganti, S.; Srivenugopal, K. S.; Nag, S.; Patil, S.; Sharma,
H.; Wang, M.-H.; Wang, H.; Buolamwini, J. K.; Zhang, R. Nat. Commun. 2014, 5,
5086. http://dx.doi.org/10.1038/ncomms6086.
1
(
Model 680 XR, Bio-Rad) using a HRP-peroxidase kit (Sumitomo
24,25
Bakelite) as reported previously.
concentrations (IC50) of the interaction between Flag-p53 and
GST–protein were calculated using data obtained at a concentra-
The half maximal inhibitory
15. Kumar, S. K.; Hager, E.; Pettit, C.; Gurulingappa, H.; Davidson, N. E.; Khan, S. R. J.
Med. Chem. 2003, 46, 2813.
16. Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong,
N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A. Science 2004, 303,
tion of 0.25
.12. Statistical analysis
At each time point, the heterogeneity of results across the stud-
lM, 2.5 lM, or 25.0 M.
8
44.
7. Hu, B.; Gilkes, D. M.; Farooqi, B.; Sebti, S. M.; Chen, J. J. Biol. Chem. 2006, 281,
3030.
8. Joseph, T. L.; Madhumalar, A.; Brown, C. J.; Lane, D. P.; Verma, C. Cell Cycle 2010,
9, 1167.
1
1
8
3
1
2
9. Dömling, A. Curr. Opin. Chem. Biol. 2008, 12, 281.
0. Phan, J.; Li, Z.; Kasprzak, A.; Li, B.; Sebti, S.; Guida, W.; Schönbrunn, E.; Chen, J. J.
Biol. Chem. 2010, 285, 2174.
ies was tested using the Cochran Q test. The significance of differ-
ences in the effects of drugs vs. the control was analyzed with the
Student’s t-test.
21. Popowicz, G. M.; Dömling, A.; Holak Angew. Chem., Int. Ed. 2011, 50, 2680.
2
2. Reed, D.; Shen, Y.; Shelat, A. A.; Arnold, L. A.; Ferreira, A. M.; Zhu, F.; Mills, N.;
Smithson, D. C.; Regni, C. A.; Bashford, D.; Cicero, S. A.; Schulman, B. A.;
Jochemsen, A. G.; Guy, R. K.; Dyer, M. A. J. Biol. Chem. 2010, 285, 10786.
3. Zhang, Q.; Zeng, S. X.; Lu, H. Sub-Cell Biochem. 2014, 85, 281.
4. Enari, M.; Kawaratani, Y.; Uesato, S.; Yokota, 69th Annual Meeting of the
Japanese Cancer Association, 2010, Osaka.
Acknowledgments
2
2
We are very grateful to Professor Masahiko Taniguchi and Asso-
ciate Professor Makio Shibano (Osaka University of Pharmaceutical
Sciences) for their kind support with the in vivo experiments. We
also thank Dr. Nakagawara for providing IMR32 cells. Part of this
work was supported by the following grants: Project for the Devel-
opment of Innovative Research on Cancer Therapeutics (P-DIRECT)
from Ministry of Education, Culture, Sports, Science, and Technol-
ogy – Japan, Applied Research for Innovative Treatment of Cancer
from Ministry of Health, Labour and Welfare – Japan (M.E.),
Grant-in-Aid for Scientific Research (B) (24390222) from Ministry
of Education, Culture, Sports, Science, and Technology – Japan (T.S.)
and MEXT – Supported Program for the Strategic Research Founda-
tion at Private Universities (2013–2017) – Japan (S.U.).
25. Enari, M.; Uesato, S. JP Patent Application No. 2011-178704.
26. Ito, A. Yakugaku Zasshi 1962, 82, 875.
2
2
7. Davis, A. R. Ind. Eng. Chem. 1947, 39, 94.
8. Shinkai, H.; Maeda, K.; Yamasaki, T.; Okamoto, H.; Uchida, I. J. Med. Chem. 2010,
4
3, 3566.
29. Bogert, M. T.; Snell, F. D. J. Amer. Chem. Soc. 1924, 46, 1308.
30. Pei, L.; Shang, Y.; Jin, H.; Wang, S.; Wei, N.; Yan, H.; Wu, Y.; Yao, C.; Wang, X.;
Zhu, L. Q.; Lu, Y. J. Neurosci. 2014, 34, 6546.
3
3
1. Zhao, L. P.; Ji, C.; Lu, P. H.; Li, C.; Xu, B.; Gao, H. Neurochem. Res. 2013, 38, 705.
2. Baud, M. G. J.; Leiser, T.; Petrucci, V.; Gunaratnam, M.; Neidle, S.; Meyer-Almes,
F.-J.; Fuchter, M. J. Beilstein J. Org. Chem. 2013, 9, 81.
33. Yamakawa, S.; Demizu, A.; Kawaratani, Y.; Nagaoka, Y.; Terada, Y.; Maruyama,
S.; Uesato, S. Biol. Pharm. Bull. 2008, 31, 916.
34. Komatsu, Y.; Tsukamoto, M.; Kato, T.; Nishino, N.; Sato, S.; Yamori, T.; Tsuruo,
T.; Furumai, R.; Yoshida, M.; Horinouchi, S.; Hayashi, H. Cancer Res. 2001, 61,
4459.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.03.021.