Synthesis and cellular uptake of p-[123I]-phenyl-amino-thiazole (123I-PAT) as a potential agent for targeting tubulin polymerization in tumors

Article abstract of DOI:10.1002/jlcr.3178

The phenyl-amino-thiazole (PAT) templates of methoxylbenzoyl-aryl-thiazole are potent agents against cancer by inhibiting tubulin polymerization in the nanomolar range. Herein, a radioiodinated PAT, [¹²³I]-PAT 1, was prepared via a tributylstannyl precursor and [¹²³I]iodide through electrophilic aromatic radioiodination. Radiolabelling of [¹²³I]-PAT 1 was achieved in less than 15 min, with a radiochemical purity of over 99%. The accumulated radioactivity in tumor cellular uptake experiments suggested that [¹²³I]-PAT could serve as a potential radioprobe for targeting tumor cells. Copyright

Full text of DOI:10.1002/jlcr.3178

Research Article
Received 5 August 2013,
Revised 27 November 2013,
Accepted 30 November 2013
Published online 10 January 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3178
123
Synthesis and cellular uptake of p-[ I]-phenyl-
123
amino-thiazole ( I-PAT) as a potential agent
for targeting tubulin polymerization in tumors
Da-Ming Wang,a Jia-Wei Kuo,b Wei-Ti Kuo,b Cheng-Fang Hsu,a
b
a
b
b,c
*
*
Mei-Hui Wang, Yu Chang, Wuu-Jyh Lin, and Jen-Tsung Wang
The phenyl-amino-thiazole (PAT) templates of methoxylbenzoyl-aryl-thiazole are potent agents against cancer by inhibiting
tubulin polymerization in the nanomolar range. Herein, a radioiodinated PAT, [123I]-PAT 1, was prepared via a tributylstannyl
precursor and [123I]iodide through electrophilic aromatic radioiodination. Radiolabelling of [123I]-PAT 1 was achieved in less
than 15 min, with a radiochemical purity of over 99%. The accumulated radioactivity in tumor cellular uptake experiments
suggested that [123I]-PAT could serve as a potential radioprobe for targeting tumor cells.
Keywords: iodine-123; phenyl-amino-thiazoles; radioiodinated analog; tubulin polymerization
Therefore, it would have similar inhibitory activity with PAT.
Introduction
The cellular uptake of [123I]-PAT 1 with different types of tumor
Microtubules (MTs) including α-tubulin and β-tubulin dimers are the
cell lines was also studied.
major structural elements in the cytoskeleton. Their involvement
in motility and chromosome movement plays a crucial role in
organization of the cytoplasm.1 Because of their contribution to
many cellular functions and modulated dynamics, MTs are
recognized as an important target underlying the antitumor
mechanism of most antimitotic compounds.2,3
Results and discussion
Chemistry
The 123I was introduced through a radioiodination of the
stannylated precursor 5, outlined in Scheme 1. The commercial
2-bromophenylthiourea was reacted with ethyl 3-bromopyruvate
in ethanol, and the ethyl ester was hydrolyzed to afford 2-
(bromophenylamino)-thiazole-4-carboxylic acid 2. The acid was
coupled with N,O-dimethylhydroxylamine to provide Weinreb
amide 3 in 59% yield for the three steps.8 Concerning
tributylstannyl precursor 5, the C-ring was first installed followed
by introduction of the trimethoxyphenyl group and the
tributylstannyl group. However, no stannylated product 5 was
found, and the starting material was recovered. As a result, we
changed our reaction scheme to insert the tributyltin at A-ring
position of Weinreb amide 3 first by a modified Stille coupling,
which added lithium chloride as a promoter to afford the
stannylated compound 4 successfully.9 Then, the stannylated
Miller et al. has reported a novel series of 4-substituted
methoxybenzoyl-aryl-thiazole (SMART) compounds as shown in
Figure 1. Such molecules can disrupt tubulin polymerization,
therefore inhibit the production of functional MTs and cell
mitosis.4 Moreover; SMART compounds also overcome the
growth of overexpressed P-glycoprotein-mediated multidrug
resistance in vitro. Despite the high inhibitory activity in broad
cancer cell types, the use of SMART compounds was limited
because of their high lipophilicity. Although surfactant formulation
strategies were adopted to improve the solubility for in vivo tests,
the side effects and toxicity deserve careful attention when these
biologically active surfactants were used.5,6
A series of aryl-benzoyl-imidazoles7 and phenyl-amino-thiazoles
(PAT)8 derivatives have been reported to improve their water
solubility and pharmacokinetic parameters. The most potent PAT
derivatives (Figure1) inhibit cancer cells (PC-3, DU145, LNCaP,
PPC-1) with average IC50 values ranging from 36 to 70 nM. When
substituting smaller substituent at para position of the aromatic
A-ring of PAT skeleton, the electronic change did not alter
the bioactivity profile.8 Therefore, incorporation of radiotracer
at this position was appropriate for developing a potential
radiopharmaceutical for cancer diagnostic imaging.
aChemistry Division, Institute of Nuclear Energy Research, Longtan, Taoyuan 325,
Taiwan
bIsotope Application Division, Institute of Nuclear Energy Research, Longtan,
Taoyuan 325, Taiwan
cDepartment of Specialty Chemical Division, Eternal Chemical Co., Ltd., Lu-Chu
district, Kaohsiung 821, Taiwan
Herein, we reported the synthesis of radioiodinated PAT, [123I]-
PAT 1. By introducing iodine similar to methyl group in size, we
supposed that the steric and electronic interferences at the para
position of the aromatic A-ring in [123I]-PAT 1 was minimized.
*Correspondence to: Wuu-Jyh Lin; Jen-Tsung Wang, Isotope Application Division,
Institute of Nuclear Energy Research, Longtan, Taoyuan 325, Taiwan.
E-mails: wjlin@iner.gov.tw; jentsung_wang@email.eternal-group.com
J. Label Compd. Radiopharm 2014, 57 132–135
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D.-M. Wang et al.
Figure 2. HPLC chromatograms of [123I]-PAT 1 were obtained by ultraviolet absorption at
292 nm and by radiodetector. The analysis was performed on a C-18 column (250 × 4.6 mm,
5 μm) by eluting with a gradient 40–90% CH3CN for 26 min at 1 mL/min. The peak at
16.9 min is [123I]-PAT 1. This figure is available at wileyonlinelibrary.com/journal/jlcr
around 16.9 min was also compared with cold standard by UV
absorption at 292 nm and a gamma detector.
Figure 1. Structure of 4-substituted methoxybenzoyl-aryl-thiazole (SMART) and
phenyl-amino-thiazole (PAT) derivatives.
In vitro cellular uptake studies
For cellular uptake experiment, the [123I]-PAT 1 was incubated
with various cancer cell lines including PC-3, HepG2, MCF-7,
and A549. The results in Figure 3 showed that the radioactivity
product 4 was reacted with 3,4,5-trimethoxyphenyllithium to
construct C-ring skeleton in the final step and produced the
desired tributylstannyl precursor 5 in 60% yield. We proposed
that the palladium catalyst during stannylation might be
poisoned on account of the activation of the sulfur atom on
triazole. The activation was mediated by the trimethyloxyphenyl
group that was introduced before this step.
Radiochemistry
[
123I]-PAT
1
was obtained via electrophilic aromatic
radioiodination from 5 and [123I]iodide under 0.1 M Na2HPO4
(pH 4.5) and EtOH using chloroamine-T as the oxidant at room
temperature.10 The radiolabeling process was monitored by RP-
18 TLC with 80% acetonitrile as eluent and completed within less
than 15 min. The retardation factors of [123I]-PAT and [123I]iodide
radiotracer were 0.3 and 1.0, respectively. The labeling yield of
[
123I]-PAT 1 was achieved up to 95.3 1.5% (n = 9). After a simple
extraction, the radiochemical purity of [123I]-PAT 1 was recorded
over 99% by HPLC analysis with an ultraviolet (UV) detector and
a tandem radiodetector, shown in Figure 2. The retention time
Figure 3. In vitro cellular uptake of [123I]-PAT 1 (n = 5).
Scheme 1. (a) (i) Ethyl 3-bromopyruvate, EtOH, reflux, 3 h and (ii) NaOH, EtOH, reflux, 18 h; (b) N,O-dimethylhydroxyamine, EDCI, HOBt, NMM, CH2Cl2, rt, 24 h, three steps
59%; (c) Pd(PPh3)4, [(n-Bu)3Sn]2, LiCl, BHT, 1,4-dioxane, 100°C, 4 h, 45%; (d) 3,4,5-trimethoxy- phenylbromide, n-BuLi, THF, rt, 2 h, 60%; (e) NH1423I, chloramine-T, Na2HPO4,
EtOH, 95%.
J. Label Compd. Radiopharm 2014, 57 132–135
Copyright © 2014 John Wiley & Sons, Ltd.
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D.-M. Wang et al.
accumulation in tumor cells was about 6–9%/106 cells. The uptake Synthesis of N-methoxy-N-methyl-2-(4-tributylstannylphenylamino)
level at 4 h was similar to that at 30 min. The initial study of cellular thiazole-4-carboxamide (4)
uptake indicated its potential use for cancer imaging. In vivo
biodistribution and single-photon emission computed tomography
imaging studies will be carried out for further evaluations.
A mixture of tetrakis(triphenylphosphine)palladium (899 mg, 0.78 mmol),
lithium chloride (993 mg, 23.44 mmol), and BHT (89.8 mg, 0.41 mol) was
purged with nitrogen several times. Then, compound 3 (2.64 g, 7.74 mmol)
in anhydrous 1,4-dioxane (52 mL) and hexa n-butylditin (8.0 mL, 15.83 mmol)
was sequentially added to the mixture. The mixture was stirred at 100°C for
4 h. The cooled solution was filtered through a pad of Celite by eluting
with CH2Cl2, and the filtrate was evaporated. The crude product was
purified by silica gel column chromatography (EtOAc/hexanes = 1 : 1) to
afford compound 4 as a pale yellow oil (1.92 g, yield 45%). Rf 0.38
(EtOAc/hexane = 1 : 1); IR (KBr) 3277, 2955, 2925, 2853, 1627, 1590,
Conclusion
We have developed a synthetic method for the radiosynthesis of
[
123I]-PAT as
a
potential targeting probe for tubulin
polymerization. The radioiodination was completed within less
than 15 min and provided final radiochemical purity over 99%.
On the basis of the encouraging cellular uptake data observed
for [123I]-PAT 1, further in vivo investigation will be conducted
to confirm the usefulness of this agent for single-photon
emission computed tomography imaging in cancer diagnosis.
1534 cmÀ1 1H NMR (300MHz, CDCl3) δ 7.69 (br, 1H), 7.45–7.42 (m, 2H),
;
7.39 (s, 1H), 7.33–7.29 (m, 2H), 3.77 (s, 3H), 3.44 (s, 3H), 1.59–0.86 (m, 27H);
13C NMR (75 MHz, CDCl3) δ 163.7, 162.7, 144.2, 139.9, 137.5, 136.1, 118.0,
114.5, 61.4, 34.6, 29.1, 27.3, 13.7, 9.6; ESI-MS: m/z 554.19 (M+H)+.
Synthesis of (2-(4-tributylstannylphenylamino)thiazol-4-yl) (3,4,5-
trimethoxyphenyl)methanone (5)
Experimental
To a stirring solution of 5-bromo-1,2,3-trimethoxybenzene (473 mg, 1.92 mmol)
in anhydrous tetrahydrofuran (11 mL) was added n-BuLi (0.9 mL, 2.25 mmol,
2.5 M solution in hexane) dropwise at À78°C under nitrogen atmosphere.
The reaction mixture was stirred for 10 min at À78°C, and compound 4
(347 mg, 1.01 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise.
The solution was warmed to room temperature for 2.5 h. The reaction was
quenched with saturated NH4Cl (16 mL) and extracted with ether (16 mL)
twice. The combined organic layers were dried over Na2SO4, filtered, and
concentrated in vacuo. The crude product was purified by silica gel column
chromatography (EtOAc/hexanes = 15 : 85 to 3 : 7) to afford compound 5 as a
yellow oil (214 mg, yield 60%). Rf 0.28 (EtOAc/hexane= 2 :8); IR (KBr) 3322,
General
Reagents and solvents were purchased from J. T. Baker, Alfa Aesar, CHEM-
IMPEX, Sigma-Aldrich, Merck, and used without further purification unless
otherwise specified. All moisture-sensitive or air-sensitive reactions were
carried out with continuous stirring under a nitrogen atmosphere. Thin-
layer chromatography was carried out on Merck silica gel 60 F254 plates
and visualized by UV light. Column chromatography was performed with
silica gel 60 (63–200 μm particle sizes). Infrared (IR) spectra were recorded
on Varian FTS-800 spectrometers. 1H NMR spectra were recorded on a
Varian Gemini 2000/BB 300 MHz Fourier transform nuclear magnetic
resonance spectrometer using tetramethylsilane as an internal standard.
Chemical shifts (δ) are given in parts per million (ppm) relative to
tetramethylsilane (δ = 0), and splitting patterns are reported as s (singlet),
d (double), t (triplet), m (multiple), and br (broad). Mass spectra were
taken on a LTQ Orbitrap XL mass spectrometer. Radioactivity was
measured by Caointec CRC-15R isotope dose calibrator (Capintec Inc., USA).
The retention factor of RP-TLC was obtained by Bioscan AR-2000 radio-TLC
imaging scanner (Bioscan, Inc., Washington, DC, USA).
1
2955, 2925, 2852, 1637, 1584, 1534cmÀ1; H NMR (300MHz, CDCl3) δ 7.64
(br, 1H), 7.54 (s, 1H), 7.51 (s, 2H), 7.46–7.43 (m, 2H), 7.38–7.35 (m, 2H), 3.94
(s, 3H), 3.91 (s, 6H), 1.57–1.49 (m, 6H), 1.39–1.32 (m, 6H), 1.08–1.05 (m, 6H),
0.89 (t, J=7.2Hz, 9H); 13C NMR (75MHz, CDCl3) δ 185.9, 164.5, 152.8, 150.5,
142.3, 139.7, 137.5, 136.5, 132.6, 118.2, 118.1, 107.9, 60.9, 56.3, 29.1, 27.3, 13.7,
9.6; ESI-MS: m/z 661.23 (M+H)+; HRMS: found 661.2065, calcu. 661.2122.
Radiochemical synthesis
Preparation of (2-(4-[123I]iodophenylamino)thiazol-4-yl)(3,4,5-
trimethoxyphenyl)methanone (123I-PAT, 1)
Chemical synthesis
To a reaction vial were added 0.1 M Na2HPO4 (pH 4.5, 60 μL), EtOH (20 μL)
containing Bu3Sn-PAT 5 (50 μg), freshly prepared chloramine-T solution
(10 mg/mL water, 1 μL), and NH4123I (~220 MBq). The reaction mixture
was vortexed briefly and kept at room temperature for 15 min. The
reaction was quenched with Na2S2O3 (1.0 M, 2 μL). Purification was carried
out by solvent extraction. The CH2Cl2 (400 μL) was added, and organic
phase was washed with water (150 μL). The organic phase was collected
and evaporated in vacuo to afford 123I-PAT 1. The radiochemical purity
of 123I-PAT 1 confirmed by HPLC was over 99% (Figure 2). For cellular
uptake assay, 123I-PAT 1 was suspended in phosphate buffered saline.
Synthesis of 2-(bromophenylamino)thiazole-4-carboxylic acid (2)
A suspension of 2-bromophenylthiourea (4.62 g, 20.0 mmol) and ethyl 3-
bromopyruvate (3.1 mL, 22.2 mmol) in ethanol (40 mL) was refluxed for
3 h. The mixture was cooled at À20°C for 3 days, and a pale yellow solid
was precipitated and collected by filtration. After drying in vacuo, the
pale yellow solid was dissolved in ethanol (40.0 mL), and NaOH (1.00 g,
25.0 mmol) was added. The mixture was heated to reflux for 18 h and
concentrated in vacuo to afford compound 2 as a white solid without
further purification.
Synthesis of 2-(4-bromophenylamino)-N-methoxy-N-methylthiazole-
4-carboxamide (3)
In vitro cellular uptake studies
A mixture of compound 2, EDCI (4.61 g, 24.0 mmol), HOBt (3.38 g,
Cells, 2 × 105 for each cancer cell lines PC-3 (prostate cancer), HepG2
22.1 mmol), N,O-dimethylhydroxylamine hydrochloride (2.07 g, 21.2mmol), (hepatocellular carcinoma), MCF-7 (breast cancer), and A549 (lung
and N-methylmorpholine (4.80mL, 43.7mmol) in CH2Cl2 (280 mL) was adenocarcinoma) were respectively seeded in six well culture plates in
stirred at room temperature for 24h. The mixture was sequentially washed 2 mL of growth medium. After 48 h, the medium was refreshed by using
with water (280 mL), saturated NaHCO3 (280mL), and NaCl (280mL). The a volume of 1 mL in each well. Then [123I]-PAT 1 (37 kBq) was added to
organic phase was dried over Na2SO4, filtered, and evaporated in vacuo. medium and further incubated at 37°C and 5% CO2 for 30 min and 4 h.
The crude product was purified by silica gel column chromatography Following incubation, the medium was collected in a counting tube.
(EtOAc/hexanes = 1: 1) to afford compound 3 as a brown foam (4.01 g, The cells were rinsed with ice-cold PBS twice and harvested using
three steps yield 59%). Rf 0.25 (EtOAc/hexane = 1:1); IR (KBr) 2925, 2854, trypsin-ethylenediaminetetraacetic acid. The radioactivity associated
1622, 1593, 1537 cmÀ1; 1H NMR (300 MHz, CDCl3) δ 7.42–7.31 (m, 5H), 3.76 with each collection was measured with a gamma counter. The amount
(s, 3H), 3.43 (s, 3H); 13C NMR (75MHz, CDCl3) δ 163.4, 162.6, 144.1, 139.4, of radioactivity associated with the cells was normalized to cell number,
132.2, 120.0, 115.3, 115.0, 61.5, 34.5; ESI-MS: m/z 342.05 (M+H)+.
and the results were expressed as the percentage uptake per 106 cells.
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D.-M. Wang et al.
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Acknowledgements
We thank Professor Chung-Shan Yu at the Department of Nuclear
Science, National Tsing-Hua University and Dr. Hung-Wen Yu for
reading and correcting the manuscript. We appreciated the
financial support from Executive Yuan in Atomic Energy Council
of Taiwan (101-2001-01-D-11). Authors also thank the Radiation
Applications and Molecular Imaging Platform, AM3 Resource
Center, and National Research Program for Biopharmaceuticals
for the related technical assistance in radiation applications.
Conflict of Interest
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The authors did not report any conflict of interest.
Supporting Information
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Additional supporting information may be found in the online
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