Synthesis and characterization of radioiodinated 3-phenethyl-2-indolinone derivatives for SPECT imaging of survivin in tumors

Article abstract of DOI:10.1016/j.bmc.2018.04.034

Survivin, overexpressed in most cancers, is associated with poor prognosis and resistance to radiation therapy and chemotherapy. Herein, we report the synthesis of three 3-phenethyl-2-indolinone derivatives and their application as in vivo imaging agents

Full text of DOI:10.1016/j.bmc.2018.04.034

Accepted Manuscript
Synthesis and characterization of radioiodinated 3-phenethyl-2-indolinone de-
rivatives for SPECT imaging of survivin in tumors
Natsumi Ishikawa, Takeshi Fuchigami, Tatsuya Mizoguchi, Sakura Yoshida,
Mamoru Haratake, Morio Nakayama
PII:
S0968-0896(18)30057-9
https://doi.org/10.1016/j.bmc.2018.04.034
BMC 14317
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 January 2018
11 April 2018
16 April 2018
Please cite this article as: Ishikawa, N., Fuchigami, T., Mizoguchi, T., Yoshida, S., Haratake, M., Nakayama, M.,
Synthesis and characterization of radioiodinated 3-phenethyl-2-indolinone derivatives for SPECT imaging of
survivin in tumors, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.04.034
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Ishikawa et al. Page 1/30
Synthesis and characterization of radioiodinated 3-phenethyl-2-indolinone
derivatives for SPECT imaging of survivin in tumors
Natsumi Ishikawaa, Takeshi Fuchigamia*, Tatsuya Mizoguchia, Sakura Yoshidaa,
Mamoru Haratakeb, Morio Nakayamaa*
a
Department of Hygienic Chemistry, Graduate School of Biomedical Sciences,
Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
bFaculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto
860-0082, Japan
*corresponding author:
Takeshi Fuchigami,
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852-8521, Japan.
Tel.: +81-95-819-2443;
Fax: +81-95-819-2443;
E-mail: t-fuchi@nagasaki-u.ac.jp

Ishikawa et al. Page 2/30
Morio Nakayama,
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852-8521, Japan.
Tel.: +81-95-819-2441;
Fax: +81-95-819-2441;
E-mail: morio@nagasaki-u.ac.jp
Abstract
Survivin, overexpressed in most cancers, is associated with poor prognosis and
resistance to radiation therapy and chemotherapy. Herein, we report the synthesis of
three 3-phenethyl-2-indolinone derivatives and their application as in vivo imaging
agents for survivin. Of these, 3-(2-(benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)-3-hydroxy-5-
iodoindolin-2-one (IPI-1) showed the highest binding affinity (Kd = 68.3 nM) to
recombinant human survivin, as determined by quartz crystal microbalance (QCM). In
vitro studies demonstrated that the
[
125I]IPI-1 binding in survivin-positive
MDA-MB-231 cells was significantly higher than that in survivin-negative MCF-10A

Ishikawa et al. Page 3/30
cells. In addition, uptake of [125I]IPI-1 by MDA-MB-231 cells decreased in a
dose-dependent manner in the presence of the high-affinity survivin ligand S12; this is
indicative of specific binding of [125I]IPI-1 to cellular survivin protein in vitro.
Biodistribution studies in MDA-MB-231 tumor-bearing mice demonstrated the
moderate uptake of[125I]IPI-1 in the tumor tissue (1.37% ID/g) at 30 min that decreased
to 0.32% ID/g at 180 min. Co-injection of S12 (2.5 mg/kg) slightly reduced tumor
uptake and the tumor/muscle ratio of [125I]IPI-1. Although further structural
modifications are necessary to improve pharmacokinetic properties, our results indicate
that PI derivatives may be useful as tumor-imaging probes targeting survivin.
Key words: survivin, cancer imaging, single photon emission computed tomography
(SPECT).

Ishikawa et al. Page 4/30
1. Introduction
Survivin, the smallest protein (16.5 kDa) of the inhibitor of apoptosis protein (IAP)
family, is highly expressed in several cancers but shows undetectable expression in
non-dividing tissues.1,2 The expression of survivin contributes to the resistance against
chemotherapy and radiotherapy and is associated with the malignancy and prognosis in
cancer patients.3 This protein forms a homodimer or heterometric complexes with
various proteins with multifunctional roles, including cancer survival, proliferation,
progression, and angiogenesis.4-6 As survivin has been proposed to be an ideal target for
the specific diagnosis and treatment of most cancer types, various anticancer agents and
intervention targeting survivin have been developed, including small-molecules,
antisense oligonucleotides, and vaccines.7-10 Radioligands such as 99mTc-labeled
antisense oligonucleotide targeting survivin mRNA have been developed for the
diagnosis of cancer; however, these agents have limitations such as the lack of in vivo
metabolic stability and specificity.11 We recently reported a radioiodinated
4,6-diaryl-3-cyano-2-pyridinone derivative ([125I]IDCP) that directly binds to survivin
and demonstrated its application in single-photon emission computed tomography
(SPECT) imaging of survivin (Fig. 1).12 125I]IDCP exhibited significantly higher
[
accumulation in MDA-MB-231 cells that display high expression of survivin as

Ishikawa et al. Page 5/30
compared with MCF-10A cells with low expression of survivin. However, the
biodistribution study of [125I]IDCP in tumor-bearing mice revealed its inadequate uptake
by the cancer tissue.
In our continuing effort to explore more useful chemical backbones, we selected
3-phenethyl-2-indolinone (PI) as a new core structure for survivin imaging. PI
derivative S12 (Fig. 1) has been reported to exhibit high binding affinity for survivin
dimer interface (Ka = 447 × 106 M−1) and inhibit tumor growth in vitro and in vivo based
on survivin degradation.13 Nuclear magnetic resonance (NMR) analysis of S12 and
survivin-binding interaction showed that the bromine atom of S12 with other
hydrophobic functional groups maintained its binding affinity for survivin. We designed
an iodine analog of PI (IPI-1), wherein the bromine atom was replaced with an iodine
atom at position 5 of S12 for its application as a SPECT imaging probe (Fig. 2). In
addition, benzo[1,3]dioxolyl group at the 3-position of PI backbone was replaced with
dimethoxyphenyl or benzoxazolyl group. Thus, we designed IPI-2 and IPI-3 as new
candidate agents for survivin imaging (Fig. 2).
In this study, we performed the synthesis of IPI derivatives and radioiodination and
evaluated their efficiencies as in vivo imaging agents for survivin.

Ishikawa et al. Page 6/30
Fig. 1. Chemical structures of survivin-targeting small molecules.
Fig. 2. Design of PI derivatives for their application in SPECT imaging.

Ishikawa et al. Page 7/30
2. Results and discussion
2.1. Chemistry
The synthesis of PI derivatives was accomplished according to the pathway
illustrated in Schemes 1 and 2. The nucleophilic addition of 3’,4’-(methilenedioxyl)
acetophenone to 5-bromoisatin produced the lead compound S12. Bromo-to-tributyltin
exchange reaction of 5-bromoisatin catalyzed by Pd(0) yielded tributyltin derivative 1.
Compound 2 was synthesized from compound 1 using the procedure for S12 synthesis.
Finally, iodine (I2)/chloroform (CHCl3) solution was added to compound 2 to yield the
target IPI-1 by tributyltin-to-iodine exchange reaction (Scheme 1). IPI-2 and IPI-3 could
be obtained by a one-step nucleophilic addition of 3’,4’-dimethoxyacetophenone or
5-acetylbenzoxazole to 5-iodoisatin (Scheme 2).

Ishikawa et al. Page 8/30
Scheme 1. Reagents and conditions; (a) 3’,4’-(Methylenedioxy)acetophenone, MeOH,
Et2NH2, r.t. 6-30 h, 21% for S12 and 22% for 2, (b) (Bu3Sn)2, (Ph3P)4Pd, Dioxane, TEA,
r.t.. 6 h, 11%. (c) I2, CHCl3, r.t. 2 hr,quant.
Scheme 2. Reagents and conditions: (a) 3',4'-dimethoxy acetophenone, Et2NH, MeOH,
r.t., 31 hr, 58%. (b) 5-acetylbenzoxazole Et2NH, MeOH, r.t., 31 hr, 19%.
2.2. Evaluation of the binding of PI derivatives to survivin
The binding affinities of PI derivatives to survivin were evaluated by quartz crystal

Ishikawa et al. Page 9/30
microbalance (QCM)—a highly sensitive and accurate mass measuring technique based
on the linear decrease in the resonance frequency in response to the mass increase on a
QCM plate at a nanogram level.14 This method is used to evaluate the binding affinity of
small molecules to biomolecules.15,16 Fig. 3 shows the representative QCM sensorgram
of the binding interaction between IPI-1 and QCM electrodes coated with survivin. The
binding of IPI-1 to survivin protein showed saturation and well-fitted one binding site
model (R2 = 0.955). By using the same procedure, QCM assays of S12, IPI-2, and IPI-3
were performed and similar sensorgrams were obtained (data not shown). The value of
Kd for each compound was calculated from the frequency changes of QCM plates, and
the binding affinities of all compounds to survivin were compared. As shown in Table 1,
IPI-1 showed slightly higher binding affinity (Kd = 68.3 nM) as compared with lead
compound S12 (Kd = 77.0 nM), indicating that the substitution from bromine to iodine
retains the binding affinity to survivin. On the other hand, IPI-2 and IPI-3 showed a Kd
value of 169 and 140 nM, respectively, indicating that the substitution of
benzo[1,3]dioxolyl with dimethoxyphenyl or benzoxazolyl group at 3-position of IPI-1
resulted in a decreased binding affinity for survivin.

Ishikawa et al. Page 10/30
Fig. 3. Typical response curves of IPI-1 bound to QCM electrodes coated with survivin.
The delta F value represents the frequency change of electrodes.
Table 1. Dissociation constant (Kd) of PI derivatives for survivin determined by QCM
assay.
a
Kd (nM)
Compounds
S12
77.0
68.3
23.2
2.60
IPI-1
169
46.2
22.6
IPI-2
140
IPI-3

Ishikawa et al. Page 11/30
*Values are mean SEM, n = 3.
2.3. In vitro binding assay of [125I]IPI-1 in cell lines
Based on the results of the binding assay to recombinant survivin, we selected IPI-1
as the radioiodinated imaging agent for survivin in further experiments. The
radiosynthesis of [125I]IPI-1 was performed by an iododestannylation reaction of
tributyltin derivative 2 with an oxidant chloramine T to obtain a radiochemical yield of
95% and purity of > 95% (Scheme 3). The yield of purified [125I]IPI-1 was confirmed
by analytical HPLC using the cold reference IPI-1 (Fig. 4). Next, we evaluated the
binding properties of [125I]IPI-1 to intracellular survivin using two cell lines exhibiting
different expression level of survivin. MDA-MB-231 is a human breast carcinoma cell
line with high survivin expression level, while MCF-10A is a human breast
non-tumorigenic epithelial cell line with low expression of survivin.17 We evaluated the
in vitro binding of [125I]IPI-1 to cellular survivin using fixed and permeabilized cells to
abolish cell membrane permeability factors of the radioligand. As shown in Fig. 5A,
[
125I]IPI-1 demonstrated a significantly higher accumulation in MDA-MB-231 cells as
compared with MCF-10A cells (88.4% versus 15.3% ID/mg protein, P < 0.05). The
binding to MDA-MB-231 cells was significantly decreased by co-incubation with 5.0

Ishikawa et al. Page 12/30
µM S12 (88.4% versus 37.2% ID/mg protein, P < 0.05). On the other hand, no
significant blocking effect of S12 was observed in MCF-10A cells. These results
indicate that the binding level of [125I]IPI-1 corresponded to the cellular survivin
expression. These results are consistent with our previous reports on [125I]IDCP.12
However, [125I]IPI-1 showed higher specific binding to survivin-positive cells as
compared to [125I]IDCP. Next, we evaluated the uptake of [125I]IPI-1 in the living cells,
wherein the binding of the radioligand to the intracellular survivin relied on its cell
permeability (Fig. 5B). Consistent with the results of permeabilized cells, the amount of
[
125I]IPI-1 binding was significantly higher in MDA-MB-231 cells as compared with
MCF-10A cells (35.9% versus 17.7% ID/mg protein, P < 0.001). In addition,
accumulation of [125I]IPI-1 was blocked by S12 only in MDA-MB-231 cells in a
dose-dependent manner. This suggests that uptake of [125I]IPI-1 is dependent on the
expression level of survivin in living cells. The decrease in the uptake of [125I]IPI-1 in
the living cells as compared to permeabilized cells may be associated with the cell
permeability, efflux,. and/or metabolism factors. Taken together, these in vitro results
suggest that [125I]IPI-1 displays the potential to bind specifically to survivin in living
cells.

Ishikawa et al. Page 13/30
O
O
O
O
O
O
125I
SnBu3
a
HO
O
HO
O
N
H
N
H
[
125I]IPI-1
2
Scheme 3. Reagents and conditions: (a) Na125I, Chloramine T, 1 M HCl, EtOH, r.t.,
95%.
Fig. 4. Analytical HPLC chromatogram of UV (A) and radioactivity (B) after
co-injection of purified [125I]IPI-1 with the cold reference IPI-1.

Ishikawa et al. Page 14/30
Fig. 5. In vitro binding of [125I]IPI-1 to permeabilized (A) and living cells (B).
Nonspecific binding was determined by co-incubation of S12. * P < 0.05, ** P < 0.01,
*** P < 0.001 (ANOVA, Bonferroni t test). Data are shown as means SEM, n = 6 (A),
n= 6-10 (B).
2.4. Biodistribution of [125I]IPI-1 in tumor-bearing mice
Next, we assessed the biodistribution of [125I]IPI-1 in BALB/c mice bearing
MDA-MB-231 xenografts at 30, 60, and 180 min after tail vein injection. As shown in
Table 2, the highest uptake of [125I]IPI-1 in most organs was observed at 30 min after
injection and decreased with time. Furthermore, [125I]IPI-1 exhibited hepatobiliary
excretion and its highest uptake was observed in the intestine (12.2% and 19.7% ID/g at
30 and 60 min). Uptake of [125I]IPI-1 derivatives by the thyroid gland increased with
time, which indicated gradual deiodination. The accumulation of [125I]IPI-1 in the tumor

Ishikawa et al. Page 15/30
tissue was 1.37% ID/g at 30 min and decreased to 0.32% ID/g at 180 min. The
tumor/muscle ratio of [125I]IPI-1 increased over time and a maximum value of 2.41 was
obtained at 180 min after injection. The co-injection of S12 (2.5 mg/kg) and [125I]IPI-1
decreased the tumor uptake (1.37% versus 0.99% ID/g) and tumor/muscle ratio from
1.15 to 0.78 at 30 min. Tumor/blood ratio was less than 1.0 at all times; however, a
small alteration in tumor/blood ratio was observed following co-injection with S12
(0.90 versus 0.87). Although pretreatment with S12 slightly reduced the tumor/muscle
ratio, the effect of S12 on inhibition of tumor accumulation was minimal. It is possible
that the S12-mediated reduction in tumor/muscle ratio was mainly caused by a
reduction in circulating [125I]IPI-1. In addition, the low tumor/blood contrast and the
small inhibitory effect on [125I]IPI-1 may be attributed to insufficient binding between
IPI-1 and survivin, nonspecific cellular binding, and/or insufficient metabolic stability.
The significantly reduced [125I]IPI-1 level in the kidneys, intestines, and brain during
S12 pretreatment may also be due to reduced [125I]IPI-1 concentration in the blood.
Interestingly, the tissue/blood contrast of the kidneys, intestines, and brain was not
significantly different between the control and the blocking groups. On the other hand,
our previous report demonstrated that [125I]IDCP showed 0.51% ID/g of tumor uptake
with 0.37 of tumor/blood at 30 min after injection.12 In comparison with [125I]IDCP,

Ishikawa et al. Page 16/30
[
125I]IPI-1 exhibited higher tumor uptake and contrast to non-target region, indicating
that PI derivatives may exhibit superior pharmacokinetic properties as compared with
DCP derivatives for in vivo imaging of survivin-expressing tumor tissues. Further
structure-activity relationship studies are needed for the optimization of in vivo
pharmacokinetics and to discover survivin-imaging agents with superior
survivin-positive tumor targeting efficiencies and higher contrast against non-targeting
tissues.
Table 2. Biodistribution of [125I]IPI-1 in MDA-MB-231 tumor-bearing mice.a
Time after injection (min)
Organ
30
60
180
30 (blocking)b
+ S12
[
125I]IPI-1
Blood
1.53 (0.13)
8.82 (0.93)
5.40 (0.77)
12.17 (2.04)
1.16 (0.18)
0.82 (0.18)
3.40 (1.27)
1.94 (0.77)
19.68 (5.07)
0.37 (0.13)
0.54 (0.06)
1.84 (0.83)
0.59 (0.12)
8.35 (0.78)
0.25 (0.09)
1.01 (0.16)*
6.15 (2.60)
3.51 (0.40)*
5.38 (1.57)*
0.81 (0.24)
Liver
Kidney
Intestine
Spleen

Ishikawa et al. Page 17/30
Lung
1.97 (0.27)
5.75 (3.90)
4.87 (5.09)
1.68 (0.27)
0.62 (0.05)
2.08 (0.16)
1.21 (0.21)
1.37 (0.07)
0.76 (0.16)
3.73 (1.87)
0.69 (0.36)
0.48 (0.21)
0.27 (0.07)
2.85 (0.23)
0.33 (0.10)
0.61 (0.15)
0.45 (0.03)
2.26 (0.48)
0.19 (0.03)
0.14 (0.01)
0.04 (0.01)
11.8 (8.23)
0.13 (0.04)
0.32 (0.08)
1.51 (0.63)
4.71 (2.52)
1.71 (0.61)
1.23 (0.31)
0.45 (0.02)**
1.84 (0.22)
1.13 (0.11)
0.99 (0.41)
Stomach
Pancreas
Heart
Brain
Thyroid
Muscle
Tumor
Tumor-to-nontumor ratios
Tumor/Blood
Tumor/Muscle
0.90 (0.10)
1.15 (0.24)
0.74 (0.05)
1.87 (0.31)
0.57 (0.08)
2.41 (0.69)
0.87 (0.20)
0.78 (0.21)
aExpressed as % of injected dose per gram (% ID/g). Each value represents mean (SD)
for three mice at each interval.
bBlocking group was treated with the co-injection of S12 (2.5 mg/kg) and [125I]IPI-1.
* P < 0.05, ** P < 0.01 compared with corresponding control group (unpaired t test).
3. Conclusion
Here, we demonstrated the successful synthesis of PI derivatives as candidate

Ishikawa et al. Page 18/30
survivin-imaging probes. Of these, [125I]IPI-1 displayed high binding affinity for
recombinant survivin and its in vitro accumulation corresponded with the expression
level of survivin. Biodistribution studies in tumor-bearing mice revealed moderate
uptake of [125I]IPI-1 by the tumor tissue. In addition, small inhibitory effects of S12 on
tumor uptake and tumor-to-muscle ratio were observed. Therefore, although further
structural modifications are necessary to improve pharmacokinetic properties of PI
derivatives, this study demonstrates the feasibility with the use of PI backbone as a
scaffold for the development of tumor-imaging agents targeting survivin.
4. Experimental
4.1. General information
All reagents were commercial products and used without further purification unless
otherwise indicated. Na[125]I was obtained from MuromachiYakuhin (Tokyo, Japan) or
1
Perkin Elmer Life Sciences (Boston, MA, USA). The H NMR spectra was obtained
using a Jeol JNMAL-400 spectrometer with TMS as the internal standard. The mass
spectra were obtained using JMS－700N or JMS-T100TD instruments (JEOL, Japan).
The HPLC analysis was performed by a Shimadzu HPLC system (LC-10AT pump with
SPD-10A UV detector, λ= 254 nm). NaI(Tl) scintillation survey meter (TCS-172B)

Ishikawa et al. Page 19/30
manufactured by Hitachi–Aloka Medical,was used for detection of radioactivity.
An automated gamma counter with a NaI (Tl) detector (2470 WIZARD2,
Perkin-Elmer) was used to measure the radioactivity. Female BALB/c nu/nu mice were
purchased from Japan SLC (Hamamatsu, Japan). The experiments with animals were
conducted in accordance with our institutional guidelines and were approved by the
Nagasaki UniversityAnimal Care Committee.
4.2. Chemistry
4.2.1. 3-(2Benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)-5-bromo-3-hydroxyindolin-2-one
(S12)
Diethylamine (0.3 mL) and 3’,4’-(methilenedioxyl acetophenone) (1.64 g, 10 mmol)
were added to a solution of 5-bromoisatin (1.13 g, 5.0 mmol) in methanol (MeOH; 30
mL). The mixture was stirred at room temperature for 11 h. The reaction mixture was
evaporated to dryness and the residue was treated with water and extracted thrice with
ethyl acetate (EtOAc). The combined organic layer was washed with brine, dried over
sodium sulfate (Na2SO4), and the solvent removed. The residue was recrystallized from
a mixture of dichloroethane and hexane to obtain a yellow solid of S12 (405 mg, 21%

Ishikawa et al. Page 20/30
yield). 1H NMR(DMSO-d6): δ 3.53(d, J=17.6 Hz, 1 H), 4.07(d, J=17.6 Hz, 1 H), 6.12(s,
2 H), 6.77(d, J=8.4 Hz, 1 H), 7.02(d, J=8.4 Hz, 1 H), 7.32(d, J=1.6 Hz, 1 H), 7.34(dd,
J=2.0, 8.4 Hz, 1 H), 7.47(d, J=2.0 Hz, 1 H), 7.56(dd, J=1.8, 8.4 Hz, 1 H). MS (DART)
m/z: 390 [M+H] +.
4.2.2. 5-(Tributylstannyl)indoline-2,3-dione (1)
Triethylamine (0.5 mL), bis(tributyltin) (1.2 mL, 2.40 mmol), and
tetrakis(triphenylphosphine)palladium (PPh3)4Pd (10 mg, 8.65 µmol) were added to a
solution of 5-bromoisatin (180 mg, 0.80 mmol) in 1,4-dioxane (5.0 mL). The mixture
was heated at 110 °C for 14 h. After the reaction mixture was cooled to room
temperature, the solution was passed through celite and silica gel (1:3) and washed with
EtOAc:hexane. The solvent was removed, and the crude product was chromatographed
on silica gel with a 1:5 mixture of EtOAc:hexane to yield a colorless oil of 1 (38 mg,
11% yield). 1H NMR (CDCl3): δ 0.89 (t, J= 14.4 Hz, 9H), 1.05-1.09 (m, 6 H), 1.27-1.37
(m, 6 H) 1.48-1.54 (m, 6 H), 6.88 (d, J=7.6 Hz, 1 H), 7.62 (d, J= 7.6 Hz, 1 H), 7.69 (s, 1
H). MS (DART) m/z: 436 [M+H] +.
4.2.3.

Ishikawa et al. Page 21/30
3-(2-(Benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)-3-hydroxy-5-(tributylstannyl)indolin-2-
one (2)
Diethylamine (0.2 mL) and 3’,4’-(methilenedioxyl acetophenone) (65 mg, 0.411
mmol) were added to the solution of 1 (60 mg, 0.137 mmol) in MeOH (3.0 mL). The
mixture was stirred at room temperature for 30 h. The solvent was removed and the
crude product chromatographed on silica gel with a 49:1 mixture of CHCl3 and MeOH
to yield a white solid of 2 (18 mg, 22% yield).1H NMR (CDCl3): δ 0.85 (t, J= 14.4 Hz,
9 H), 0.97-1.01 (m, 6 H), 1.24-1.33 (m, 6 H), 1.44-1.52 (m, 6 H), 3.42(d, J= 17.2 Hz, 1
H), 3.68 (d, J=17.2 Hz, 1 H), 6.03 (s, 2 H), 6.81 (d, J= 8.4 Hz, 1 H), 6.87 (d, J=7.6 Hz,
1 H), 7.31 (dd, J=1.0, 8.4 Hz, 1 H), 7.39 (d, J=1.6 Hz, 1 H),7.43(s,1 H), 7.49 (dd, J=8.0,
1.6 Hz, 1 H). MS (DART) m/z: 602 [M+H]+.
4.2.4.
3-(2-(Benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)-3-hydroxy-5-iodoindolin-2-one
(IPI-1)
A solution of iodine in CHCl3 (100 mg/mL, 1.0 mL) was added to a solution of 2
(14.9 mg) in CHCl3 (1.0 mL) at room temperature. The mixture was stirred at room
temperature for 2 h and then quenched by saturated aqueous sodium bisulfite (NaHSO3).
The aqueous layer was extracted thrice with CHCl3, while the organic layer was

Ishikawa et al. Page 22/30
successively washed with saturated aqueous sodium bicarbonate (NaHCO3). The
organic layer was washed with brine and then dried in the presence of sodium sulfate
(Na2SO4). The crude product was chromatographed on silica gel with a 30:1 mixture of
1
CHCl3:MeOH to give a pale yellow powder of IPI-1 (14 mg, quant). H NMR
(CD3OD): δ 3.62(d, J= 17.3 Hz, 1 H), 3.96 (d, J= 17.1 Hz, 1 H), 6.04 (s, 2 H), 6.72 (d,
J= 8.1 Hz, 1 H), 6.89 (d, J=8.3 Hz, 1 H), 7.29(d, J= 1.2 Hz, 1 H), 7.56 (m, J= 16.8 Hz, 2
H), 7.59 (d, J= 1.48 Hz, 1 H). HRMS (FAB) m/z calcd for C17H12INO5[M+H]+,
437.9839; found, 437.9841.
4.2.5.
3-(2-(3,4-dimethoxyphenyl)-2-oxoethyl)-3-hydroxy-5-iodoindolin-2-one
(IPI-2)
A solution of 5-iodoisatin (136.5 mg, 0.5 mmol) in MeOH (2.0 mL) was added to a
solution of 3’,4’-(dimethoxy acetophenone) (180.2 mg, 1.0 mmol) in MeOH (2.0 mL).
Diethylamine was added dropwise to the solution mixture and the mixture was stirred at
room temperature for 31 h. The reaction mixture was filtrated and washed with MeOH.
The residue was chromatographed on silica gel with a 30:1 mixture of CHCl3:MeOH to
give a pale yellow solid of IPI-2 (24 mg, 58% yield).
1H NMR (CD3OD): δ 3.65(d, J= 17.2 Hz, 1 H), 3.82 (s, 3 H), 3.89 (s, 3 H), 3.99 (d,

Ishikawa et al. Page 23/30
J= 17.6 Hz, 1 H), 6.71 (d, J=8.4 Hz, 1 H), 7.01(d, J= 8.8 Hz, 1 H), 7.37 (d, J=2.0 Hz, 1
H), 7.54 (dd, J=2.0, 8.4 Hz, 1 H), 7.59 (d, J=1.6 Hz, 1 H), 7.63 (dd, J=2.0, 8.0 Hz, 1 H).
HRMS (FAB) m/z calcd for C18H16INO5 [M+H]+, 454.0152 ; found, 454.0153.
4.2.6. 3-(2-(benzo[d]oxazol-6-yl)-2-oxoethyl)-3-hydroxy-5-iodoindolin-2-one (IPI-3)
A solution of 5-iodoisatin (136.5 mg, 0.5 mmol) in MeOH (2.0 mL) was added to a
solution of 5-acetylbenzoxazole (161.2 mg, 1.0 mmol) in MeOH (2.0 mL).
Diethylamine was added dropwise to the solution mixture and the mixture stirred at
room temperature for 31 h. The reaction mixture was filtrated and washed with MeOH.
The residue was chromatographed on silica gel with a 20:1 mixture of CHCl3:MeOH to
give a pale reddish brown solid of IPI-2 (15 mg, 19% yield).
1H NMR (CD3OD): δ 3.74(d, J= 12 Hz, 1 H), 4.10 (d, J= 17.6 Hz, 1 H), 6.70 (d, 8.4,
1 H), 7.51 (d, J= 8.4 Hz, 1 H), 7.59 (s, 1 H), 7.69 (d, J= 8.8 Hz, 1 H), 8.00 (d, J= 8.4 Hz,
1 H), 8.31 (s, 1 H), 8.52 (s, 1 H). HRMS (FAB) m/z calcd for C17H11IN2O4 [M+H]+,
434.9842 ; found, 434.9839.
4.2.7. Radioiodination of [125I]IPI-1
Chloramine-T (20 µL, 15 mg/mL), [125I]NaI (3.7-7.4 MBq, specific activity 2200

Ishikawa et al. Page 24/30
Ci/mmol, 3 µL), and 1 M hydrochloric acid (HCl; 50 µL) were added to a solution of 2
(0.2 mg in ethanol 50 µL) in a sealed vial. The reaction was performed at room
temperature for 15 min and terminated by the addition of saturated aqueous NaHSO3
(100 µL). After alkalization with 100 µL of saturated NaHCO3 and extraction with
EtOAc, the extract was dried by passing it through an anhydrous Na2SO4 column and
evaporated to dryness. The crude products were purified by high-performance liquid
chromatography (HPLC) on Cosmosil C18 column (Nacalai Tesque, 5C18-AR-II, 4.6 ×
250 mm) with an isocratic solvent mixture of acetonitrile (CH3CN) and water (4:6) at a
flow rate of 1.0 mL/min. HPLC retention time of [125I]IPI-1 was 6.8 min, consistent
with that of the corresponding non-radioactive IPI-1.
4.3. Saturation binding assay of PI derivatives to recombinant survivin protein
Recombinant human survivin was expressed and purified as previously described
[13]. Kinetic analyses were performed with QCM system (Affinix Q, Initium Inc.,
Tokyo, Japan). The survivin protein (40 µg/mL) was immobilized on the gold electrode
of the sensor chip via amide bonds. The sensor chip was set on QCM apparatus and
soaked in a buffer. An 8 µL aliquot of 10–256 µM PI derivatives was sequentially
injected into the cuvette. The changes in the frequency were monitored over time. The

Ishikawa et al. Page 25/30
kinetic analysis was performed by AQUA ver. 1.3 software (Initium Inc.).
4.4. Cell culture
The breast cancer cell line MDA-MB-231 and a normal immortalized human
mammary epithelial cell line MCF-10A were obtained from the American Type Culture
Collection (ATCC; Manassas, VA, USA). MDA-MB-231 cells were grown in
Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum (FBS). MCF-10A
were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 supplemented with
5% horse serum, 20 ng/mL epidermal growth factor (EGF), 10 µg/mL insulin, and 0.5
µg/mL hydrocortisone. All media mentioned above were supplemented with 100 IU/mL
penicillin and 100 µg/mL streptomycin.
4.5. In vitro binding of [125I]IPI-1
MDA-MB-231 and MCF-10A cells were grown to confluence on six-well plates and
washed with phosphate-buffered saline (PBS). The cells were fixed with 3.7%
formaldehyde and permeabilized by 0.3% Triton X. [125I]IPI-1 in 1% dimethyl sulfoxide
(DMSO)/PBS (1.0 kBq/mL, 0.5 mL) with or without S12 (5 µM) was added to the cells
and incubated at 37 °C for 1 h. The cells were washed with 1% DMSO/PBS and PBS

Ishikawa et al. Page 26/30
(0.5 mL) and lysed using 0.5 M sodium hydroxide (NaOH; 200 µL) at 37 °C for 42 h.
The radioactivity of the lysate was measured with gamma counter and the protein
concentration was determined using the DC protein assay kit (Bio-Rad, Hercules,
USA).
4.6. In vitro uptake of [125I]IPI-1
MDA-MB-231 and MCF-10A cells were grown to confluence on six-well plates and
washed with PBS. [125I]IPI-1 dissolve in growing medium (1.0 kBq/mL, 0.5 mL) was
added to each cells and incubated at 37 °C for 2 h with or without S12 (0.5, 5, 50 µM).
Following incubation, cells were washed twice with 20 units heparin/PBS (0.5 mL) and
lysed using 0.5 M NaOH (200 µL) at 37 °C for 18 h. The radioactivity of the lysate was
measured with gamma counter and the protein concentration was determined using the
DC protein assay kit.
4.7. Tumor xenograft model
BALB/c nu/nu mice (female, 4 weeks old) were maintained in a room with a
constant ambient temperature and a 12 h light/dark cycle and had free access to food
and water. Mice were subcutaneously injected on their right shoulders with

Ishikawa et al. Page 27/30
approximately 1.0 × 107 MDA-MB-231 cells. The tumors were allowed to reach
300–500 mm3 (34 weeks after inoculation) before biodistribution studies were
performed.
4.8. Biodistribution of [125I]IPI-1 in tumor-bearing mice
[
125I]IPI-1 (10 kBq) was dissolved in 1 % DMSO saline (100 µL) and intravenously
injected into the mice via the tail vein. At the designated time intervals, mice were
sacrificed and their organs dissected. The tissues were weighed and the radioactivity
was measured with automated gamma counting (n = 3).
4.9. Statistical analysis
Statistical significance was determined at P < 0.01 using the one-way analysis of
variance (ANOVA) for comparison of more than two means, followed by the post hoc
tests using Bonferroni’s correction for in vitro experiments or unpaired t-test for in vivo
experiments.
Acknowledgments
Financial support was provided by a Grant-in-Aid for Young Scientists (B) (Grant

Ishikawa et al. Page 28/30
No. 21791180) and Grant-in-Aid for Scientific Research (C) from the Japan Society for
the Promotion of Science (JSPS). This work was supported partly by Takeda Science
Foundation, Konica Minolta Science and Technology Foundation, Mochida Memorial
Foundation for Medical and Pharmaceutical Research, and SGH Foundation.
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