Synthesis and evaluation of radioiodinated 1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin-4-amine derivatives for platelet-derived growth factor receptor β (PDGFRβ) imaging

Article abstract of DOI:10.1016/j.bmc.2017.08.025

Platelet-derived growth factor receptor β (PDGFRβ) is a transmembrane tyrosine kinase receptor and it is upregulated in various malignant tumors. Radiolabeled PDGFRβ inhibitors can be a convenient tool for the imaging of tumors overexpressing PDGFRβ. In this study, [¹²⁵I]-1-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinoline-8-yl}piperidin-4-amine ([¹²⁵I]IIQP) and [¹²⁵I]-N-3-iodobenzoyl-1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}-piperidin-4-amine ([¹²⁵I]IB-IQP) were designed and synthesized, and their potential as PDGFRβ imaging agents was evaluated. In cellular uptake experiments, [¹²⁵I]IIQP and [¹²⁵I]IB-IQP showed higher uptake by PDGFRβ-positive cells than by PDGFRβ-negative cells, and the uptake in PDGFRβ-positive cells was inhibited by co-culture with PDGFRβ ligands. The biodistribution of both radiotracers in normal mice exhibited hepatobiliary excretion as the main route. In mice inoculated with BxPC3-luc (PDGFRβ-positive), the tumor uptake of radioactivity at 1 h after the injection of [¹²⁵I]IIQP was significantly higher than that after the injection of [¹²⁵I]IB-IQP. These results indicated that [¹²⁵I]IIQP can be a suitable PDGFRβ imaging agent. However, further modification of its structure will be required to obtain a more appropriate PDGFRβ-targeted imaging agent with a higher signal/noise ratio.

Full text of DOI:10.1016/j.bmc.2017.08.025

Accepted Manuscript
Synthesis and evaluation of radioiodinated 1-{2-[5-(2-methoxyethoxy)-1H-
benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin-4-amine derivatives for plate-
let-derived growth factor receptor β (PDGFRβ) imaging
Nurmaya Effendi, Kazuma Ogawa, Kenji Mishiro, Takeshi Takarada, Daisuke
Yamada, Yoji Kitamura, Kazuhiro Shiba, Takehiko Maeda, Akira Odani
PII:
S0968-0896(17)31225-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.025
BMC 13928
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 June 2017
10 August 2017
15 August 2017
Please cite this article as: Effendi, N., Ogawa, K., Mishiro, K., Takarada, T., Yamada, D., Kitamura, Y., Shiba, K.,
Maeda, T., Odani, A., Synthesis and evaluation of radioiodinated 1-{2-[5-(2-methoxyethoxy)-1H-
benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin-4-amine derivatives for platelet-derived growth factor receptor β
(PDGFRβ) imaging, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.08.025
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Synthesis and evaluation of radioiodinated
1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin-4-
amine derivatives for platelet-derived growth factor receptor β (PDGFRβ)
imaging
Nurmaya Effendia,b, Kazuma Ogawaa,c*, Kenji Mishiroc, Takeshi Takaradad, Daisuke
Yamadad,f, Yoji Kitamurae, Kazuhiro Shibae, Takehiko Maedaf, Akira Odania
aKanazawa University, Graduate School of Pharmaceutical Sciences,
Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan;
bUniversitas Muslim Indonesia, Faculty of Pharmacy, Urip Sumiharjo KM. 10,
Makassar 90-231, Indonesia;
cKanazawa University, Institute for Frontier Science Initiative, Kakuma-machi,
Kanazawa, Ishikawa 920-1192, Japan;
dOkayama University, Graduate School of Medicine, Department of Regenerative
Science, Dentistry and Pharmaceutical Sciences, Okayama, 700-8558, Japan;
eKanazawa University, Advanced Science Research Centre, Takara-machi,
Kanazawa, Ishikawa 920-8640, Japan;
fNiigata University of Pharmacy and Applied Sciences, Division of Pharmacology,
265-1 Higashijima, Akiha-ku, Niigata-shi, Niigata-ken, 956-8603, Japan.

*Corresponding Author
Kazuma Ogawa Tel.: 81-76-234-4460; fax: 81-76-234-4459;
E-mail: kogawa@p.kanazawa-u.ac.jp
Running Title: PDGFRβ imaging agent
Word Count of this manuscript: 5125 words
Conflict of interest: The authors have declared that no conflict of interest.
Financial support: This work was supported in part by Grants-in-Aid for Scientific
Research (16H01332, 16KT0192) from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.

Abstract
Platelet-derived growth factor receptor β (PDGFRβ) is a transmembrane tyrosine
kinase receptor and it is upregulated in various malignant tumors. Radiolabeled
PDGFRβ inhibitors can be a convenient tool for the imaging of tumors
overexpressing
[125I]-1-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl)quinoline-8-yl}
([125I]IIQP)
PDGFRβ.
In
this
study,
piperidin-4-amine
and
[125I]-N-3-iodobenzoyl-1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quin
olin-8-yl}-piperidin-4-amine ([125I]IB-IQP) were designed and synthesized, and
their potential as PDGFRβ imaging agents was evaluated. In cellular uptake
experiments, [125I]IIQP and [125I]IB-IQP showed higher uptake by PDGFRβ-positive
cells than by PDGFRβ-negative cells, and the uptake in PDGFRβ-positive cells was
inhibited by co-culture with PDGFRβ ligands. The biodistribution of both
radiotracers in normal mice exhibited hepatobiliary excretion as the main route. In
mice inoculated with BxPC3-luc (PDGFRβ-positive), the tumor uptake of
radioactivity at 1 h after the injection of [125I]IIQP was significantly higher than that
after the injection of [125I]IB-IQP. These results indicated that [125I]IIQP can be a
suitable PDGFRβ imaging agent. However, further modification of its structure will
be required to obtain a more appropriate PDGFRβ-targeted imaging agent with a
higher signal / noise ratio.
Keywords: radiotracer, molecular imaging, PDGFRβ, tyrosine kinase inhibitor

1. Introduction
PDGFRβ belongs to a subfamily of receptor tyrosine kinases (RTKs). It
possesses an outer membrane with a PDGF ligand binding site and an inner
membrane tyrosine kinase (TK) domain with an adenosine three phosphate (ATP)
binding site.1 Binding of PDGF ligands to the extracellular binding domain triggers
PDGFRβ dimerization, thus inducing phosphorylation of its intracellular regions
due to an activated ATP-binding domain. This is followed by the activation of
signaling pathways that regulate important cellular functions.2 The expression of
PDGFRβ is highly restricted in most normal cells; however, upregulation of
PDGFRβ in numerous human tumors and its relationship with tumor progression
features such as cell migration, metastasis, angiogenesis, and proliferation, have
been reported.3-5 Therefore, PDGFRβ can be an attarctive target not only for cancer
therapy but also for developing tumor-imaging agents.6-8
Nuclear medicine has been a revolutionary invention in the field of diagnostic
imaging because it is noninvasive and facilitates continous monitoring of disease
progression or treatment response.9,10 Moreover, the imaging modalities in nuclear
medicine, such as single photon emission computed tomography (SPECT) and
positron emission tomography (PET) can quantify receptor density and molecular
target expression in patients, thus becoming a useful tool in drug development.10-12
Previous studies have reported imaging agents, including radiolabeled protein,13,14
aptamer,15 affibody molecules,16,17 and peptide18,19 that target the extracellular parts
of PDGFRβ. Although the imaging of intracellular parts of PDGFRβ, particularly its
ATP-binding site, has hardly been reported,8 the ATP-binding is very important for

following signal transductions and growth of cancer cells. Therefore, the
ATP-binding site can be a target for TK inhibitors (TKIs). Meanwhile, mutations of
the ATP-binding site have frequently been found20,21. The mutation status often
affects therapeutic effects of TKIs. Thus, the information of the ATP-binding site,
containing the mutation status, is useful for the prediction of therapeutic effects of
TKIs. Other radiolabeled TK inhibitors (TKIs)22-26 and their potential advantages,
such as assessment of drug sensitivity20, mutation status21, and usefulness in drug
screening11,27,28 have been demonstrated.
1-{2-[5-(2-Methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin
-4-amine (CP-673451, IQP) (Figure 1) is a highly selective benzimidazole inhibitor
of PDGFRβ (IC50 = 1 nM) over PDGFRα (IC50 = 10 nM). Importantly, it is 1,000
times selective compared with many other RTKs. It competitively inhibits
ATP-binding to the ATP-binding site of PDGFRβ and blocks phosphorylation.29,30
High binding affinity and high selectivity to the molecular target are important for a
lead compound of imaging probes. IQP can be an attractive compound in an attempt
to image the ATP binding site of PDGFRβ. Therefore, we speculated that
radiolabeled IQP derivatives that bind the ATP-binding site may be useful imaging
probes.
To develop novel radiolabeled molecular probes for PDGFRβ imaging, we
chose radioiodine as a radionuclide in this study because 123I (t1/2 = 13.2 h) and 124I
(t1/2 = 4.2 d) are useful radionuclides for SPECT and PET, respectively. We did not
have sufficient information about the introduction of radioiodine into IQP structure
to retain high affinity for PDGFRβ. To evaluate the feasibility of IQP derivatives as

PDGFRβ imaging probes, we designed and synthesized two different types of iodine
labeled IQP,
1-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}piperid
in-4-amine (IIQP) and
N-3-iodobenzoyl-1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-quinolin-8
-yl}-piperidin-4-amine (IB-IQP). In IIQP, iodine was directly introduced to C-5 of
the quinoline group of IQP because it is a reactive site for halogenation of IQP. In
IB-IQP, iodine was indirectly introduced by conjugation of 3-iodo benzoyl group
using SIB (N-succinimidyl-3-iodobenzoate) with the primary amine group of IQP.
We then examined the binding affinities of two derivatives for PDGFRβs.
Subsequently, radiotracers [125I]IIQP and [125I]IB-IQP (Figure 1) were synthesized
and evaluated in vitro and in vivo to investigate their usefulness as PDGFRβ
imaging agents. Although we are interested in developing imaging probes for
SPECT and PET, 125I was used as an alternative radionuclide in these initial studies
because of its long half-life (t1/2 = 59.4 d).
Figure 1. Chemical structures of IQP, [125I]IIQP, and [125I]IB-IQP.

2. Material and methods
2.1. General
Commercial reagents and solvents were purchased from Sigma-Aldrich (St.
Louis, MO, USA), Wako Pure Chemical Industries (Osaka, Japan), Nacalai Tesque,
Inc., (Kyoto, Japan), Tokyo Chemical Industry, Co., Ltd., (Tokyo, Japan) and Kanto
Chemical, Co., Inc. (Tokyo, Japan) and used without further purification unless
otherwise stated.
[125I]Sodium iodide (644 GBq/mg) was purchased from Perkin Elmer
(Waltham, MA, USA). The radioactivity was measured by an Auto Gamma System
ARC-7010B (Hitachi, Ltd., Tokyo, Japan).
Bicinchoninic Acid (BCA) Protein Assay Kit and Cell Counting Kit (CCK)
were purchased from Nacalai Tesque, Inc. and Dojindo (Kumamoto, Japan),
respectively. Recombinant murine platelet-derived growth factor-BB (PDGF-BB)
was purchased from PeproTech (Rocky Hill, NJ, USA). TR-PCT1 rodent brain
pericyte cell line was a generous gift from Dr. Emi Nakashima (Keio University,
Tokyo, Japan),31 MCF7 breast cancer cell lines were purchased from DS Pharma
Biomedical (Osaka, Japan), and BxPC3-luc pancreatic cell line was purchased from
JCRB Cell Bank (Ibaraki, Japan).
Proton and carbon nuclear magnetic resonance (1H-NMR and 13C-NMR)
spectra were obtained with JEOL JNM-ECS400 (JEOL Ltd, Tokyo, Japan). Direct
analysis in real time mass spectra (DART-MS) and Electrospray ionization mass
spectra (ESI-MS) were obtained with JEOL JMS-T100TD (JEOL Ltd). Purification
were conducted using reversed-phase high-performance liquid chromatography

(RP-HPLC) system. TLC analysis were performed with silica plates (Art 5553,
Merck, Darmstadt, Germany). Optical density in WST-8 assay was obtained using
Infinite® F200 Pro microplate reader (TECAN, Männedorf, Switzerland).
2.2. Synthesis of reference compounds and precursors
Intermediate and reference compounds, IQP, ATE, and SIB, were synthesized
according to the reported studies, with a slight modification.32-37 Detailed procedures
were shown in the Supporting Information.
2.2.1. 1-{5-Iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]qui
nolin-8-yl}piperidin-4-amine (IIQP) (13)
A mixture of N-chlorosuccinimide (NCS) (80 mg, 0.6 mmol, 1.2 eq.) and
sodium iodide (NaI) (90 mg, 0.6 mmol, 1.2 eq.) in acetic acid (5 mL) was added to
12 (209 mg, 0.5 mmol, 1.0 eq.) in acetic acid (5 mL) and the mixture was stirred at
50 °C for 18 h under nitrogen atmosphere. After adjusting pH to 9.0 using saturated
aqueous sodium bicarbonate (NaHCO3), the reaction mixture was extracted with
dichloromethane (3 x 30 mL). The organic layer was dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The crude product was purified
by column chromatography on silica gel (chloroform/methanol = 50/1) to afford 13
1
(217 mg, 80%) as a pale yellow solid. H NMR (400 MHz, CDCl3): δ 1.76 – 1.85
(2H, m), 2.01 – 2.10 (2H, m), 2.85 – 2.96 (3H, m), 3.50 (3H, s), 3.82 – 3.84 (2H,
m), 3.99 (2H, br d), 4.23 – 4.25 (2H, m), 7.00 (1H, d, J = 8.0 Hz), 7.26 (1H, dd, J =
11.2, 2.0 Hz), 7.38 (1H, d, J = 2.0 Hz), 7.74 (1H, d, J = 9.2 Hz), 7.98 (1H, d, J = 8.0

13
Hz), 8.39 (1H, d, J = 9.2 Hz), 8.56 (1H, d, J = 9.2 Hz), 8.68 (1H, s). C NMR (100
MHz, CDCl3): δ 156.05, 150.46, 147.08, 145.51, 144.70, 142.19, 141.34, 137.42,
129.16, 126.68, 119.56, 114.91, 114.35, 113.68, 103.52, 88.63, 71.08, 67.77, 59.26,
51.44 (2C), 48.73, 36.28 (2C). HRMS (ESI+) calcd for C24H27IN5O2 [M+H+]: m/z =
544.1204, found 544.1166.
Scheme 1. Synthesis of reference compound IQP (a) sodium tungstate dihydrate, hydrogen
peroxide, 75 °C, 5 h (b) acetic anhydrate, 100 °C, 7.5 h (c) HCl, 95 °C, 2 h (d) benzyl bromide,
potassium carbonate, reflux, 6 h (e) POCl3, reflux, 16 h (f) cesium carbonate, reflux, 3 d (g)
palladium acetate, DIPHOS, cesium carbonate, phenyl boronic acid, 115 °C, 3 d (h) (i) palladium
chloride, TEA, formic acid, 65 °C, 2 h (ii) formamidine acetate, reflux, overnight (i)
N-phenyl-bis(trifluoromethanesulfonimide), room temperature, 2 d (j) cesium carbonate,
rac-BINAP, tris(dibenzylideneacetone)dipalladium(0), reflux, 3 d (k) TFA, room temperature, 15
min.
2.2.2. tert-Butyl
(1-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-
8-yl}piperidin-4-yl)carbamate (14)
A mixture of 13 (100 mg, 0.2 mmol, 1.0 eq.), triethylamine (TEA) (56 µL, 0.4
mmol, 2.0 eq.) and di-tert-butyl dicarbonate (Boc2O) (109 mg, 0.5 mmol, 2.5 eq.) in
dry dichloromethane (2 mL) was stirred at room temperature for 3 days under

nitrogen atmosphere. The reaction mixture was diluted with dichloromethane and
washed successively with saturated aqueous NaHCO3 and brine. The organic layer
was dried over sodium sulfate, filtered, and concentrated under reduced pressure to
afford 14 (100 mg) as a pale yellow solid. The product was used in the following
reaction without further purification.
2.2.3. tert-Butyl
(1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-5-(tributylstann
yl)quinolin-8-yl}piperidin-4-yl)carbamate (tin-IQP) (15)
A mixture of 14 (47 mg, 72.3 µmol, 1.0 eq.), hexabutyldistannane (73 µL,
144.6 µmol, 2.0 eq.), and tetrakis(triphenylphosphine)palladium(0) Pd[P(C6H5)3]4 (4
mg, 3.6 µmol, 5% eq.) in dry toluene (2 mL) was refluxed for 24 h under nitrogen
atmosphere. After removing the catalyst by filtration through a pad of Celite® and
washing with toluene, the filtrate was concentrated under reduced pressure and
purified by HPLC with mobile phase system methanol (A) (with 0.05% TEA) and
water (B) (with 0.05% TEA), A : 97.5% - 100%, 20 min using 5C18 MSII (10ID x
250 mm) column, flow rate 4 mL/min, to afford 15 (28 mg, 48%) as a pale yellow
solid. 1H NMR (400 MHz, DMSO-d6): δ 0.82 – 0.86 (9H, m), 1.11 – 1.21 (6H, m),
1.26 – 1.35 (6H, m), 1.43 (9H, s), 1.47 – 1.59 (6 H, m), 1.83 – 1.88 (4H, m), 2.74 –
2.79 (2H, m), 3.35 (3H, s), 3.46 (1H, br s), 3.72 – 3.74 (2H, m), 3.78 (2H, br d),
4.19 – 4.21 (2H, m), 7.32 (1H, d, J = 7.2 Hz), 7.34 (1H, s), 7.38 (1H, d, J = 8.8 Hz),
7.57 (1H, d, J = 7.6 Hz), 8.17 – 8.23 (2H, m), 8.93 (1H, d, J = 8.8 Hz), 9.16 (1H, s).
13C NMR (100 MHz, CDCl3): δ 155.66, 154.99, 149.57, 146.49, 145.02, 142.18,

141.49, 141.45, 135.50, 134.54, 132.47, 126.25, 118.10, 116.49, 114.49, 112.23,
102.50, 77.51, 70.53, 67.21, 58.19, 51.30 (2C), 47.61, 32.67 (2C), 28.69 (3C), 28.32
(3C), 26.66 (3C), 13.52 (3C), 10.01 (3C). HRMS (ESI+), calcd for C41H62N5O4Sn
[M+H+]: m/z = 808.3818, found 808.3846.
2.2.4. N-3-iodobenzoyl-1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazo
l-1-yl]-quinolin-8-yl}-piperidin-4-amine (IB-IQP) (18)
A mixture of 12 (12 mg, 30.0 µmol, 1.0 eq.), N,N-diisopropylethylamine
(DIPEA) (6 µL, 33.0 µmol, 1.1 eq.), and 17 (11 mg, 33.0 µmol, 1.1 eq.) in dry
dichloromethane (1 mL) was stirred at 50 °C for 2 h under nitrogen atmosphere.
After completion of the reaction, the reaction mixture was diluted with
dichloromethane and washed with water. The organic layer was dried over sodium
sulfate, filtered, and concentrated under reduced pressure. The crude product was
purified by column chromatography on silica gel (chloroform/methanol = 100/1) to
afford 18 (15 mg, 77%) as a colorless solid. 1H-NMR (400 MHz, DMSO-d6): δ 2.01
– 2.06 (4H, m), 2.86 – 2.91 (2H, m), 3.32 (3H, s), 3.71 – 3.73 (2H, m), 3.87 (2H, br
d), 4.02 (1H, br s), 4.19 – 4.21 (2H, m), 7.31 – 7.38 (4H, m), 7.52 (1H, t, J = 8.0
Hz), 7.65 (1H, d, J = 8.4 Hz), 7.92 (2H, t, J = 8.0 Hz), 8.19 (1H, d, J = 8.8 Hz), 8.24
(1H, s), 8.56 (1H, d, J = 8.4 Hz), 8.64 (1H, d, J = 8.0 Hz), 8.95 (1H, d, J = 8.8 Hz),
13
9.19 (1H, s). C NMR (100 MHz, CDCl3): δ 164.41, 155.56, 149.04, 146.97,
145.08, 142.42, 140.50, 140.34, 139.66, 136.95, 135.67, 130.48, 127.34, 126.97,
126.40, 126.31, 121.63, 118.35, 116.37, 114.21, 112.45, 102.76, 94.65, 70.52,

67.26, 58.22, 51.40 (2C), 47.18, 32.11 (2C). HRMS (ESI+), calcd for C31H31IN5O3
[M+H+]: m/z = 648.1466, found 648.1468.
2.2.5. N-3-(tributylstannyl)-benzoyl-1-{2-[5-(2-methoxyethoxy)-1H-ben
zo[d]imidazol-1-yl]-quinolin-8-yl}-piperidin-4-amine (ATE-IQP) (19)
The mixture of 12 (27 mg, 73.0 µmol, 1.0 eq.), DIPEA (15 µL, 80.0 µmol, 1.1
eq.), and 16 (40 mg, 80.0 µmol, 1.1 eq.) in dry dichloromethane (3 mL) was stirred
at 50 °C for 3 h under nitrogen atmosphere. After completion of the reaction, the
reaction mixture was diluted with dichloromethane and washed with water. The
organic layer was dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The crude product purified by column chromatography on silica
gel (chloroform/methanol = 100/1) to afford 19 (33 mg, 56%) as a pale yellow solid.
1H-NMR (400 MHz, DMSO-d6): δ 0.84 – 0.88 (9H, m), 1.01 – 1.18 (6H, m), 1.26 –
1.35 (6H, m), 1.45 – 1.60 (6H, m), 2.03 – 2.06 (4H, m), 2.86 – 2.91 (2H, m), 3.31
(3H, s), 3.66 – 3.72 (2H, m), 3.88 (2H, br d), 4.03 (1H, br s), 4.17 – 4.18 (2H, m),
7.30 – 7.38 (3H, m), 7.46 (1H, t, J = 7.2 Hz), 7.52 (1H, t, J = 8.0 Hz), 7.60 (1H, d, J
= 7.6 Hz), 7.65 (1H, d, J = 7.2 Hz), 7.81 (1H, d, J = 8.0 Hz), 7.91 (1H, s), 8.18 (1H,
d, J = 9.2 Hz), 8.51 (1H, d, J = 7.6 Hz), 8.56 (1H, d, J = 9.2 Hz), 8.92 (1H, d, J =
13
8.8 Hz), 9.19 (1H, s). C NMR (100 MHz, DMSO-d6): δ 166.56, 155.56, 149.08,
146.94, 145.12, 142.39, 141.39, 140.50, 140.31, 138.81, 135.13, 134.47, 127.60,
127.33, 126.94, 126.35, 126.29, 121.56, 118.27. 116.30, 114.26, 112.43, 102.68,
70.51, 67.21, 58.13, 51.40 (2C), 47.01, 32.21 (2C), 28.58 (3C), 26.68 (3C), 13.53

(3C), 9.21 (3C). HRMS (ESI+) calcd for C43H58N5O3Sn [M+H+]: m/z = 812.3556,
found 812.3573).
2.3. Cell viability assays
TR-PCT1 cells were seeded on 96-well culture plates (5 x 103 cells/well) and
cultured in DMEM medium containing 2% FBS and 20 ng/mL PDGF-BB at 33 °C
in a humidified 5% CO2 atmosphere. Cells were treated with each compound for 72
h and cell viability was assessed using the Cell Counting Kit following the
manufacturer's instructions. Briefly, cells were incubated with WST-8 at 33 °C in a
humidified 5% CO2 atmosphere for 30 min, followed by measuring optical density
at 450 nm.
2.4. Radiolabeling
Radiotracers, [125I]IIQP and [125I]IB-IQP, were synthesized by an
iododestannylation reaction of the corresponding tributylstannyl precursors (15 or
19) using NCS as an oxidizing agent. The radiotracers were isolated by RP-HPLC
using a Cosmosil 5C18MSII column (4.6 ID × 150 mm) at the flow rate of 1 mL/min
with a gradient mobile phase of 70% methanol in water with 0.05% TEA to 90%
methanol in water with 0.05% TEA for 20 min. The column temperature was
maintained at 40 °C. Radiochemical yield and purity were determined by an auto
well gamma counter.
2.4.1. Synthesis of [125I]IIQP (20)

A solution of [125I]NaI in 0.1 M NaOH aqueous solution (non-carrier added,
370 kBq, 2 µL) was charged into a sealed vial containing 5 (1 mg/mL, 5 µL), acetic
acid (5%, 30 µL), acetonitrile (55 µL), and NCS (5 mg/mL, 10 µL). The mixture
was shaken at room temperature for 15 min, quenched by addition of sodium
hydrogensulfite (5 mg/mL, 10 µL) and the solvent was removed by nitrogen
gassing. TFA was added to the residue and the shaking was allowed to continue for
30 min. After removing TFA by nitrogen gassing, the residue was mixed with initial
mobile phase of HPLC. The reaction mixture was shaken for some minutes, filtered,
and analyzed by HPLC.
Scheme 2. Synthesis of reference compound, precursor, and radioiodinated compound
[
125I]IIQP (a) NCS, NaI, 50 °C, 18 h (b) Boc2O, TEA, room temperature, 3 d (c)
hexabutyldistannane, Pd[P(C6H5)3]4, reflux, 24 h (d) [125I]NaI, NCS, acetic acid, room
temperature, 15 min (e) TFA, room temperature, 30 min.
2.4.2. Synthesis of [125I]IB-IQP (21)
A solution of [125I]NaI in 0.1 M NaOH aqueous solution (non-carrier added,
370 kBq, 2 µL) was added in a sealed vial containing 19 (1 mg/mL, 5 µL), acetic
acid (5%, 10 µL), acetonitrile (10 µL), and NCS (5 mg/mL, 15 µL). The mixture

was shaken at room temperature for 15 min, quenched by addition of sodium
hydrogensulfite (5 mg/mL, 15 µL) and purified by HPLC.
Scheme 3. Synthesis of reference compound, precursor, and radioiodinated compound
[
125I]IB-IQP (a) 50 °C, 2 h; (b) 50 °C, 3 h; (c) [125I]NaI, NCS, acetic acid, room temperature, 15
min.
2.5. Determination of partition coefficients
Partition coefficients of [125I]IIQP and [125I]IB-IQP into n-octanol and 0.1 M
phosphate buffer (PB) pH 7.4 were measured as described previously with a slight
modification.38 Briefly, [125I]IIQP or [125I]IB-IQP was added to the mixture of
n-octanol (3.0 mL) and PB (3.0 mL) in a test tube. The test tube was vortexed (1
min), left at room temperature (10 min), and centrifuged (5 min, 3,060g, 4 °C).
n-Octanol layer (2.0 mL) was transferred into new test tube followed by addition of
fresh n-octanol (1.0 mL) and PB (3.0 mL). After repeating vortex, standing, and

centrifuge, radioactivity of each layer, n-octanol (1.0 mL) and PB (1.0 mL), was
counted using an auto well gamma counter (n = 4).
2.6. In vitro stability assay
The stability of radiotracers, [125I]IIQP and [125I]IB-IQP, were analyzed as
described previously with a slight modification.39 Briefly, [125I]IIQP or [125I]IB-IQP
solution (50 µL) in sealed tube containing 0.1 M phosphate buffered saline (PBS)
pH 7.4 (450 µL) was incubated at 37 °C for 1, 3, 6, and 24 h. After incubation, the
purities of radiotracers were analyzed by TLC using chloroform/methanol = 5/1 and
20/1 for [125I]IIQP and [125I]IB-IQP, respectively as a developing solvent and the
results were confirmed by HPLC.
2.7. Cellular uptake study
BxPC3-luc or MCF7 cells were cultured in RPMI 1640 medium containing
10% FBS and penicillin (100 IU/mL)-streptomycin (100 µg/mL) on 6-well culture
plates (containing 2 x 105 cells/well) for 24 h using a humidified atmosphere (5%
CO2) incubator at 37 °C. After removal of medium, a solution of [125I]IIQP or
[125I]IB-IQP (3.7 kBq/well) in medium without FBS was added. After incubation for
0.5, 1, 2, and 4 h, the medium from each well was removed and the cells were
washed once with ice-cold PBS (1 mL). The cells were dissolved using 1 M NaOH
aqueous solution (0.5 mL) and wells were washed with 1 M NaOH aqueous solution
(0.5 mL). The radioactivity of pooled basic fractions was determined using an auto
well gamma counter. The total cell protein was quantified using a BCA Protein

Assay Kit following the manufacturer's recommendations and bovine serum
albumin as a protein standard. All data were expressed as percent dose per
microgram protein (%dose/µg protein).
In blocking experiments, inhibitors (IQP, IIQP, or IB-IQP with final
concentration 10 µM) in 1 mL of medium without FBS were added to wells
containing 2 x 105 cells/well. After incubation for 10 min, [125I]IIQP or [125I]IB-IQP
(3.7 kBq/well) in 1 mL of medium without FBS was added to each well.
Radioactivity and protein concentration in cells were determined using the same
method above-mentioned.
2.8. Competitive binding assay using BxPC3-luc cells
BxPC3-luc cells in RPMI 1640 medium containing 10% FBS and penicillin
(100 IU/mL)-streptomycin (100 µg/mL) were seeded on 96-well plates (containing
5,000 cells/wells) and incubated for 24 h using a humidified atmosphere (5% CO2)
at 37 °C. [125I]IIQP and nine concentrations (ranging from 1 pM to 1 mM) of
displacing nonradiolabeled ligands (IQP, IIQP and IB-IQP) in assay buffer (25 mM
HEPES, pH adjusted to 7.4) were added. After incubation at 37 °C for 90 min, the
cells were washed twice with ice-cold PBS (150 µL) to remove the unbound
radioligand. The cells were dissolved using 1 M NaOH aqueous solution (150 µL)
and wells were washed with 1 M NaOH aqueous solution (150 µL). The bound
radioactivity was determined using an auto well gamma counter. IC50 values were
calculated by GraphPad Prism 5.0 software (La Jolla, CA, USA).

2.9. Animals
All animal handling procedures were approved by the Kanazawa University
Animal Care Committee (Permit Number: AP-132633). Experiments with animals
were conducted in accordance with the Guidelines for the Care and Use of
Laboratory Animals of Kanazawa University. The animals were housed with free
access to food and water at 23 °C with a 12 h light/dark schedule. Six week-old
male ddY (27 – 30 g) and four week-old female BALB/c nu/nu mice (12 – 17 g)
were purchased from Japan SLC Inc. (Hamamatsu, Japan). For preparing
tumor-bearing model, 5 x 106 BxPC3-luc cells were subcutaneously inoculated into
left shoulder of BALB/c nu/nu mice. The tumor reached palpable size after 2 weeks
of the inoculation.
2.9.1. Biodistribution study
A saline solution of [125I]IIQP or [125I]IB-IQP (74 kBq, 100 µL) containing 1%
tween-80 and 10% ethanol was injected intravenously into the tail of the mice. The
ddY mice were sacrificed at 2, 10 min, 1, 4, and 24 h, meanwhile the tumor-bearing
mice were sacrificed at 1 h and 4 h ([125I]IIQP ) postinjection.
For in vivo blocking studies, female BALB/c nu/nu mice were intraperitoneally
injected with IQP (40 mg/kg) as a blocking agent 1 h prior to intravenously injection
of [125I]IIQP (37 kBq, 100 µL). The mice were sacrificed at 1 h after administration
of radiotracer.
Tissues of interest were removed and weighed. The radioactivity of the tissues
was determined using an auto well gamma counter, and counts were corrected for

background radiation. The data were expressed as percent injected dose per gram
tissue (%ID/g).
2.10. Statistical analysis
All data were analyzed using GraphPad Prism 5.0 software and displayed as
mean
standard deviation (SD). Significance for in vitro blocking studies was
calculated using a one-way analysis of variance (ANOVA) followed by Dunnett's
post hoc test compared to the control group. Significance in cell viability assays was
determined using ANOVA followed by Tukey’s post hoc test. Significant
differences of biodistribution in normal mice and tumor-bearing mice between
[125I]IIQP and [125I]IB-IQP groups were determined using Student's t-test (unpaired,
two-tailed). Significance for in vivo blocking studies between control and blocking
groups were calculated using Student's t-test (unpaired, two-tailed). Results were
considered statistically significant at p < 0.05.
3. Results
3.1. Synthesis of reference compounds and precursors
Lead compound 12 (Scheme 1) and the non-radioactive iodinated reference
compounds, 13 and 18, were easily obtained. Iodine was incorporated to the C-5 of
quinoline core of IQP using mixture reagents of NCS and NaI with stirring at 50 °C
for 18 h in acetic acid to obtain compound 13 (Scheme 2). On the other hand,
compound 18 was synthesized by stirring the mixture of synthesized SIB and IQP at
50 °C for 2 h in a basic solution (Scheme 3). Retention time for compounds 13 and

18 on RP-HPLC were 11.7 and 12.5 min, respectively (Supporting Information). For
synthesis of tin precursor 15, utilizing the Pd(0) catalyst, tri-butyl tin group was
introduced into IQP instead of iodine in 14 to obtain compound 15 in 48% yield
after purification using RP-HPLC. Meanwhile, compound 19 was obtained by the
reaction between ATE and IQP with 56% yield.
3.2. Cell viability assays
To assess the binding potential of IIQP and IB-IQP to the ATP-binding site of
PDGFRβ, TR-PCT1 cells were cultured in the presence of various concentration of
IQP, IIQP, or IB-IQP (1 – 1000 nM). As displayed in Figure 2, the effects of IB-IQP
were similar to those of IQP; however, IIQP was more effective than IQP in
reducing the viability of TR-PCT1 cells.
Figure 2. Cell viability after exposure to IQP, IIQP, and IB-IQP by WST-8 assay. Data are
presented as mean SD for three samples. Significance was determined using a one-way ANOVA
followed by Tukey's post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001).
3.3. Radiolabeling

Two novel 125I labeled IQP, [125I]IIQP and [125I]IB-IQP, were synthesized by
an iododestannylation reaction with the corresponding tributyltin precursors, as
outlined in Schemes 2 and 3, respectively. Both these radiotracers were synthesized
using NCS as an oxidizing agent in an acidic solution at room temperature in high
radiochemical yield (95 and 85%, respectively). After purification using RP-HPLC,
the radiochemical purities were over 99%.
3.4. Partition coefficient
The n-octanol/PB partition coefficients for [125I]IIQP and [125I]IB-IQP were
2.71 0.03 and 3.19 0.17, respectively. Lipophilicity of [125I]IB-IQP was higher
than that of [125I]IIQP.
3.5. In vitro stability assay
The stability of radiotracers in 0.1 M PBS (pH 7.4) was evaluated using TLC
analysis and confirmed using HPLC. After incubation for 24 h at 37 °C, the purity
of the radiotracers remained high, and 93.9 0.4% of [125I]IIQP and 94.1 0.7% of
[125I]IB-IQP remained intact.
3.6. Cell uptake study
The in vitro cell uptake experiments of [125I]IIQP and [125I]IB-IQP were
performed using two types of cell lines: PDGFRβ-positive BxPC3-luc cells and
PDGFRβ-negative MCF7 cells.15,18,40 As shown in Figure 3, BxPC3-luc cells
showed higher uptake of [125I]IIQP and [125I]IB-IQP then that by MCF7 cells. The

uptake of [125I]IIQP in BxPC3-luc cells reached 2.34 %dose/µg protein after 4 h
incubation, whereas that of [125I]IB-IQP reached 0.95 %dose/µg protein.
Figure 3. Cellular uptake study. Time-dependent accumulation of (a) [125I]IIQP and (b)
[
125I]IB-IQP in BxPC3-luc (closed circles) and MCF7 (open circles) cells. Data were presented as
mean SD for three samples.
In the blocking studies, the uptakes of [125I]IIQP and [125I]IB-IQP in
BxPC3-luc cells were significantly reduced by the pretreatment of excess amount of
a PDGFRβ ligand, IQP, or non-radiolabeled compounds, IIQP or IB-IQP (Figure 4).

Figure 4. In vitro blocking study of (a) [125I]IIQP and (b) [125I]IB-IQP in BxPC3-luc cells.
Data were presented as mean SD for three samples. Significance was determined using a
one-way ANOVA followed by Dunnett's post hoc test (*p < 0.001, vs control [125I]IIQP or
125I]IB-IQP).
[
3.7. Competitive binding assay using BxPC3-luc cells
The competitive binding of [125I]IIQP to BxPC3-luc cells was investigated in
the presence of PDGFRβ ligand, IQP, IIQP, and IB-IQP. The IC50 values were
found to be 311 82 nM for IQP, 44 17 nM for IIQP, and 422 54 nM for
IB-IQP.
3.8. Biodistribution studies
Biodistribution of [125I]IIQP and [125I]IB-IQP in normal mice is summarized in
Table 1. High accumulation of radioactivity after administration in liver, small

intestine, and large intestine were shown. It was excreted into feces from these
tissues during 24 h. It indicated that main excretion route of both [125I]IIQP and
[125I]IB-IQP was hepatobiliary. It was confirmed that the radioactivity in feces
excreted during 24 h after injection of [125I]IIQP and [125I]IB-IQP (75.93 and 68.75
%ID, respectively) were much higher than the radioactivity in urine (3.79 and 13.48
%ID, respectively). Additionally, radioactivity was hardly observed in any tissues
after 24 h evaluation for both radiotracers.
Biodistribution of [125I]IIQP and [125I]IB-IQP in BxPC3-luc tumor-bearing
mice is summarized in Table 2. Accumulation of radioactivity in tumor at 1 h
postinjection of [125I]IIQP (1.29 %ID/gram) was significantly higher than that of
[125I]IB-IQP (0.52 %ID/gram) in biodistribution experiments using BxPC3-luc
tumor-bearing mice. Accumulation of radioactivity in tumor at 4 h postinjection of
[125I]IIQP was 0.45 %ID/gram, which was lower than that at 1 h postinjection.
In in vivo blocking studies, the effect of IQP, a high affinity ligand for
PDGFRβ, on tumor uptake of [125I]IIQP at 1 h after administration is shown in
Figure 5. In this case, the radioactivity level in the blood after injection of [125I]IIQP
was significantly increased by pretreatment of IQP. The values of % injected dose
per gram of control group and IQP pretreatment group were 0.69 0.10 and 3.16
0.15, respectively. Thus, the figures are shown as not only % injected dose per gram
but also as tumor / blood ratio. The result demonstrated that pretreatment of an
excess amount of IQP resulted in a significant decrease in tumor uptake and the
uptake ratio of tumor to blood at 1 h postinjection of [125I]IIQP.

Figure 5. Tumor uptake and tumor/blood uptake ratios of [125I]IIQP at 1 h postinjection with
IQP (40 mg/kg) (blocking group) or control group. Data were presented as means SD for three
or four mice. Significance was determined using an unpaired Student's t-test (*p < 0.05, **p <
0.001).
4. Discussion
To develop probes for PDGFRβ imaging, the affinity of newly synthesized IQP
analogs, which contain iodine molecules, for PDGFRβ, was evaluated using WST-8
assay. WST-8 assay determines cell viability; therefore, it cannot directly be used for
affinity studies. A negative correlation is speculated between the PDGFRβ-analog
affinity and TR-PCT1 cell viability because TR-PCT1 cells express PDGFRβ, and
they die when the receptor signaling pathway is interrupted.41,42 Therefore, WST-8
assay was used as an index to evaluate affinity changes between the ligand and

PDGFRβ by introducing an iodine molecule in IQP. IB-IQP and IQP displayed
similar affinity for PDGFRβ as a molecular target, whereas IIQP displayed a higher
affinity than that of IQP (Figure 2). A competitive binding assay was also performed
to evaluate the affinity of ligands for PDGFRβ. The competitive binding of
[125I]IIQP to BxPC3-luc cells was investigated in the presence of PDGFRβ ligands,
IQP, IIQP, and IB-IQP. In this assay, [125I]IIQP do not always bind to only PDGFRβ
in tumor cells. Thus, the IC50 values of the experiments may not indicate the affinity
for PDGFRβ completely. However, the IC50 values should be an index of the affinity.
As the results, the IC50 value of IIQP (44 nM) was lower than those of IQP (311 nM)
and IB-IQP (422 nM), indicating that IIQP possesses higher affinity than IQP and
IB-IQP. It was consistent with WST-8 assay. Generally, the introduction of certain
molecules, functional groups or elements into lead compounds can reduce their
affinity for target molecules. However, in this study, the introduction of iodine into
IQP increased the affinity of IIQP for PDGFRβ. This result is extremely favorable
for developing molecular probes for PDGFRβ imaging.
Iodogen, chloramine-T (CAT), NCS, and peracetic acid (PAA) are commonly
used as oxidizing agents for the preparation of radioiodinated compounds. [125I]IIQP
and [125I]IB-IQP were synthesized using an iododestannylation technique without
the addition of a carrier and using NCS as an oxidizing agent with excellent
radiochemical yields. We also tried to synthesize [125I]IIQP and [125I]IB-IQP using
CAT and PAA as oxidizing agents; however, the radiochemical yields were <50%.
These results indicated that NCS is the most suitable oxidizing agent for compounds
with oligo arene, such as IIQP and IB-IQP.

In cellular uptake experiments for [125I]IIQP and [125I]IB-IQP, these radiotracers
showed higher accumulation in PDGFRβ-positive tumor cells (BxPC3-luc) than in
PDGFRβ-negative tumor cells (MCF7). [125I]IIQP, which had the highest affinity
for PDGFRβ in the competitive binding assay and WST-8 assay, showed higher
accumulation in PDGFRβ-positive tumor cells than that of [125I]IB-IQP (Figure 3).
In in vivo experiments, [125I]IIQP showed significantly higher accumulation in
BxPC3-luc tumor than that of [125I]IB-IQP (Table 2). Moreover, the uptake of
[125I]IIQP in PDGFRβ-positive tumor cells was inhibited by treatment with an
excess amount of PDGFRβ ligand both in the cellular uptake study (Figure 4) and in
the in vivo blocking experiments using tumor-bearing mice (Figure 5). These results
indicated that the uptake of [125I]IIQP in tumors is PDGFRβ-specific, and it must
bind the ATP-binding site of PDGFRβ in the tumor cell.
Small molecule TKIs, including [125I]IIQP and [125I]IB-IQP, target the
intracellular domain of RTKs, and for this binding, they should diffuse through the
cellular membrane. The log P values of the compounds are an important factor to
penetrate the cellular membrane. It has been shown that a log P value of 1 to 3.5 is
required to passively penetrate cellular membrane.43-46 In this study, Log P values of
[125I]IIQP and [125I]IB-IQP were 2.71 and 3.19, respectively. This suggested that the
lipophilicity of both the radiotracers was suitable for them to be used as PDGFRβ
imaging probes.
In the biodistribution experiments, [125I]IIQP and [125I]IB-IQP were mainly
excreted through the hepatobiliary pathway, which is often observed for small
molecule radiotracers directed to the ATP-binding site of TKs.20,23,47,48 This

excretion route may be due to the moderately high lipophilicities of these
radiotracers. Meanwhile, it has been known that free iodine accumulates in the
stomach and thyroid; this is used as an index of deiodination of radioiodine-labeled
tracers in vivo. In this study, the accumulation in the stomach was not high,
suggesting that both [125I]IIQP and [125I]IB-IQP are highly stable not only in vitro
but also in vivo. In the case of [125I]IIQP, the biodistribution study in tumor-bearing
mice at 4 h postinjection was performed because we expected more effective tumor
detection derived from higher accumulation ratios of tumor / non-target tissues. The
results showed that the tumor accumulation decreased; however, tumor to blood and
tumor to muscle ratios at 4 h postinjection were higher than those at 1 h
postinjection because radioactivity in the blood and muscle was prominently
decreased. Many studies have reported that P glycoprotein (P-gp), breast cancer
resistant protein (BCRP), and/or other drug efflux transporters can hinder tumor
uptake of anticancer drugs.49,50 Planar structure, high lipophilicity, and neutral or
positive charges are some of requirements for P-gp substrates.51 Because [125I]IIQP
possesses these characteristics, it can be a substrate for P-gp and therefore, its
accumulation may have decreased in tumor cells. However, further research is
warranted to prove whether [125I]IIQP is a P-gp substrate. Another possibility is the
presence of radioactive metabolites that have less or no affinity to the molecular
target. Rapid metabolism leads to the loss of affinity for target molecules and
resulting low tumor uptake.52 Although [125I]IIQP showed high stability both in vitro
and in vivo, this possibility cannot be completely excluded because we did not
perform a metabolic study.

5. Conclusion
In this study, we synthesized the radiolabeled compounds [125I]IIQP and
[125I]IB-IQP using an iododestannylation reaction under non-carrier added condition
with high radiochemical yields and high purities. This method can also be used to
synthesize other radioiodinated compounds containing quinoline-benzimidazole
derivatives. The present study showed that radioiodinated IIQP could be a promising
imaging agent compared with radioiodinated IB-IQP. However, modification of its
structure may be needed to obtain appropriate PDGFRβ-targeted imaging agents,
which have higher tumor uptake and tumor to background ratios.
6. Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific Research
(16H01332, 16KT0192) from the Ministry of Education, Culture, Sports, Science
and Technology, Japan.