Radiosynthesis and evaluation of novel 99mTc(CO)3-labelled thymidine dithiocarbamate derivatives for tumor imaging with SPECT

Article abstract of DOI:10.1016/j.jorganchem.2018.05.009

A series of novel thymidine dithiocarbamate derivatives (DTC-TdR) were successfully synthesized and then radiolabelled using [^99mTc(CO)₃]⁺ core with high yields. The chemical characterizations of ^99mTc(CO)₃-labelled dithiocarbamate derivatives have been carried out by preparing their corresponding rhenium complexes. The radiotracers were stable in vitro, and the partition coefficient results indicated that they were lipophilic. The cell uptake studies showed the uptakes of these ^99mTc(CO)₃-labelled thymidine derivatives were mediated by nucleoside transporters. Biodistribution of the complexes in mice bearing tumor showed that they had high tumor uptake and good tumor/muscle ratio. A clear SPECT imaging of the tumor location was obtained in mice bearing S180 tumor with one of radiotracers, suggesting they would be potential tumor imaging agents.

Full text of DOI:10.1016/j.jorganchem.2018.05.009

Accepted Manuscript
99m
Tc(CO) -labelled thymidine
dithiocarbamate derivatives for tumor imaging with SPECT
Radiosynthesis and evaluation of novel
3
Xiaojiang Duan, Qing Ruan, Qianqian Gan, Xiaoqing Song, Si'an Fang, Xuran Zhang,
Junbo Zhang
PII:
S0022-328X(18)30334-6
10.1016/j.jorganchem.2018.05.009
JOM 20443
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 19 February 2018
Revised Date: 8 May 2018
Accepted Date: 9 May 2018
Please cite this article as: X. Duan, Q. Ruan, Q. Gan, X. Song, Si'. Fang, X. Zhang, J. Zhang,
99m
Radiosynthesis and evaluation of novel
for tumor imaging with SPECT, Journal of Organometallic Chemistry (2018), doi: 10.1016/
j.jorganchem.2018.05.009.
Tc(CO) -labelled thymidine dithiocarbamate derivatives
3
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ACCEPTED MANUSCRIPT
Radiosynthesis and evaluation of novel 99mTc(CO)3-labelled
thymidine dithiocarbamate derivatives for tumor imaging with
SPECT
Xiaojiang Duan a, Qing Ruan a, Qianqian Gan a, Xiaoqing Song a, Si’an Fang a, Xuran
Zhang a, b, and Junbo Zhang*
a Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of
Education, College of Chemistry, Beijing Normal University, Beijing 100875, China.
b Department of Isotopes, China Institute of Atomic Energy, P. O. Box 2108, Beijing
102413, China
*Corresponding Author:
For Junbo Zhang: phone +86-10-62208126, E-mail zhjunbo@bnu.edu.cn.
Abstract:
A series of novel thymidine dithiocarbamate derivatives (DTC-TdR) were
successfully synthesized and then radiolabelled using [99mTc(CO)3]+ core with high
yields. The chemical characterizations of 99mTc(CO)3-labelled dithiocarbamate
derivatives have been carried out by preparing their corresponding rhenium
complexes. The radiotracers were stable in vitro, and the partition coefficient results
indicated that they were lipophilic. The cell uptake studies showed the uptakes of
these 99mTc(CO)3-labelled thymidine derivatives were mediated by nucleoside
transporters. Biodistribution of the complexes in mice bearing tumor showed that they
had high tumor uptake and good tumor/muscle ratio. A clear SPECT imaging of the
tumor location was obtained in mice bearing S180 tumor with one of radiotracers,
suggesting they would be potential tumor imaging agents.
1. Introduction
As the most established radiotracer for positron emission tomography (PET), the
uptake of 18F-FDG (2-[18F]fluoro-2-deoxy-D-glucose) is strongly related with glucose
18
metabolism [1]. Due to the presence of the Warburg effect, the uptake of F-FDG in
tumor is usually significantly increased [2], making it the gold standard radiotracer for
tumor detection and clinical staging. While 18F-FDG also accumulates in brain,
inflammatory lesions and other metabolically active organs [3]. Sustained
proliferation is one of the hallmarks of cancer[4], and DNA synthesis is the most

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reliable marker for cell proliferation. For years, nucleoside and nucleoside analogs,
including some nucleoside complexes, have played an important role in the diagnosis
and treatment of cancer and viral diseases [5-9]. Thymine is the only base that
incorporates DNA rather than RNA, and the metabolic process of thymidine is the
most specific way to measure cell proliferation. To overcome the shortcomings of
18F-FDG, 18F-FLT (3’-deoxy-3’-[18F]fluorothymidine), has been proposed as a
radiotracer for cell proliferation imaging [10]. As an analog of thymidine deoxyribose
(TdR), 18F-FLT is taken up by cell via the same mechanisms as nucleoside thymidine,
and then phosphorylated by thymidine kinase 1 (TK1), resulting trapped in the cell
[11]. 18F-FLT is an effective tracer of tumor proliferation, and is a specific, prognostic
predictor in the clinical treatment of cancer [12]. Compared to the positron nuclide 18F,
generator produced isotope 99mTc can be obtained at a reasonable cost. In addition,
99mTc has suitable physical characteristics (Eγ = 140 keV, T1/2 = 6.02 h), versatile
chemistry due to its multi-oxidation states and the ability to produce a variety of
complexes, being the major advantages of 99mTc as a radionuclide for
radiopharmaceutical development. Thus, using 99mTc to label thymidine derivatives
has always attracted the attention of researchers.
To date, many 99mTc labelled thymidine derivatives have been prepared [13-22]. In
2003, R. Schibi et al. prepared six M(CO)3 (M= 99mTc, Re) labelled 5’-carboxamide
derivatives of 5’-aminothymidine and the affinity of the rhenium-tricarbonyl
compounds were evaluated in vitro with TK-1 [13]. M. Stichelberger et al.
synthesized several M(CO)3-complexes (M= 99mTc, Re), and evaluated them in vitro.
The mixed inhibition of TK-1 was observed with Ki values ranging from 4.4 to 334
ꢀM.
Then S. Celen et al. synthesized 99mTc-MAMA-propyl-thymidine
(3-N-[S-trityl-2-mercaptoethyl]-N-[NV-(S-trityl-2-mercaptoethyl)amidoacetyl]-amino
propyl-thymidine), a derivative at position N3 of thymidine. Unfortunately, unlike the
clear uptake of 18F-FLT can be visualized with ꢀPET, 99mTc-MAMA-propyl-thymidine
had no uptake in the tumor sites, probably due to the bulky 99mTc-MAMA ligand [15].
D. Desbouis et al. also prepared a series of thymidine derivatives labelled with the
M(CO)3 core (M= 99mTc, Re). The complexes carried different charges: neutral,
cationic and anionic. The neutral, lipophilic complexes with a log P value > 1 had a
higher phosphorylation rate in phosphorylation assay and a higher uptake in a human
neuroblastoma cell line (SKNMC) in vitro cell internalization experiments [18].
Similarly, H. Struthers et al. also found neutral or cationic compounds maintained

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substrate activity towards the TK-1 [19, 20] for the N3-functionalized derivatives. In
view of the fact that 99mTc-tricarbonyl complexes tend to be rather lipophilic in
comparison to other 99mTc complexes, the 99mTc tricarbonyl complexes of thymidine
analogs are more likely to be the imaging agent we are striving for. In the previous
work of our group, a series of 99mTc(CO)3-labelled macrocylic triamine-derivatised
thymidine analogs have been prepared and their biodistributions were systematically
evaluated [23]. However, due to the fact that the ligand moiety was too bulky, the
biological properties of those complexes were not satisfactory.
Dithiocarbamate (DTC) is a kind of ligand with low charge and relatively small pitch,
which can be strongly complexed with metal ions. Based on our previous reported
work [24-32], thymidine dithiocarbamate analog can be assumed to form stable
99mTc-tricarbonyl complex on the basis of efficient binding of the sulfur atoms. In this
study, novel thymidine dithiocarbamate analogs functionalized at position N3 of
thymidine were successfully synthesized, and then labelled by M(CO)3 core (M=
99mTc, Re) to prepare the corresponding complexes. The partition coefficients,
stability in vitro, in vitro cell internalization experiments, biodistribution and
SPECT/CT imaging in mice bearing tumor were also evaluated.
2. Materials and methods
2.1. Materials and measurements
All A.R. grade chemical reagents used in synthesis were purchased from
commercial sources and were used without any further purification. The
99Mo/99mTc generator was obtained from Beijing Atomic High-Tech Co. The 1H
NMR spectra and 13C NMR spectra were obtain at 400 MHz and 100MHz on a
Bruker spectrometer, respectively (Bruker, Billerica, MA, USA). Mass
spectrometry were performance on the LC-MS Shimadzu 2010 series (Shimadzu,
Kyoto, Japan). The HRMS spectra were acquired from AB SCIEX TripleTOFTM 5600
(AB Sciex, Concord, Canada). IR spectrum was obtained with an AVATAR 360 FT-IR
spectrometer using KBr pellets. Radiochemical purity was determined by HPLC
performed on a Waters 600 binary HPLC pump and a Waters 2489 UV absorbance
dual detector (Waters, Milford, USA) with a reversed-phase C-18 column (Kromasil
C18, 4.6 mm 250 mm). The murine sarcoma S180 cell line was purchased from
Peking University Health Science Center (Beijing, China). HCT116 cell line was
obtained from China Infrastructure of Cell Line Resource (Beijing, China).

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2.2. Methods
2.2.1. Synthesizes of thymidine dithiocarbamates
Compound 1-4 were prepared by multi-step reactions from initial material thymidine.
The reaction route of 1-4 were shown in Scheme 1. Compound 5 was synthesized
according to the literature reported by Tronchet et al [33].
2.2.1.1. General procedure for the preparation of 6-9
Compound 6-9 was prepared according the literature reported by Cheng L et.al [34]
and with some modifications. Namely, phthalimide (2.9 g, 20 mmol) and the
appropriate dibromoalkanes (80 mmol) were added into MeCN (50 mL). After an
addition of K2CO3 (11 g, 80 mmol), the mixture was refluxed for 24 h and followed
by TLC (Thin Layer Chromatography). The solvent was evaporated under reduced
pressure, and then the residuum was dissolved in ethyl acetate (200 mL) and washed
by water (3×100 mL). The organic phase was dried by MgSO4, and then was
evaporated. The crude product was purified by column chromatography (PE/EtOAc =
10:1, Rf = 0.4) to give 6-9.
Compound 6 (white solid, 3.6 g, yield 70.9%). 1H-NMR (400 MHz, Chloroform-d) δ
7.86 – 7.80 (m, 2H), 7.74 – 7.68 (m, 2H), 3.71 (t, J = 6.7 Hz, 2H), 3.43 (t, J = 6.3 Hz,
2H). 13C NMR (100 MHz, Chloroform-d) δ 167.91, 134.35, 131.97, 123.63, 39.42,
28.29. MS (m/z): 254.1 (calc. 254.0 [C10H8BrNO2]H+).
1
Compound 7 (white solid, 3.7 g, yield 69.0%). H NMR (400 MHz, Chloroform-d) δ
7.90 – 7.79 (m, 2H), 7.73 (m, 2H), 3.84 (q, J = 6.7 Hz, 2H), 3.42 (q, J = 6.4 Hz, 2H),
2.26 (dq, J = 13.2, 6.5, 5.5 Hz, 2H). MS (m/z): 268.0 (calc. 268.0 [C11H10BrNO2]H+).
1
Compound 8 (white solid, 4.7 g, yield 83%). H NMR (400 MHz, Chloroform-d) δ
7.87 – 7.78 (m, 2H), 7.75 – 7.66 (m, 2H), 3.71 (t, J = 6.7 Hz, 2H), 3.43 (t, J = 6.4 Hz,
2H), 1.95 – 1.78 (m, 4H). 13C NMR (100 MHz, Chloroform-d) δ 167.94, 133.74,
131.77, 122.93, 36.69, 32.80, 29.66, 27.02. MS (m/z): 282.0 (calc. 282.0
[C12H12BrNO2]H+).
Compound 9 (colorless oil, 4.8 g, yield 80.4%).1H NMR (400 MHz, Chloroform-d) δ
7.73 (dt, J = 8.2, 4.1 Hz, 1H), 7.63 (dt, J = 6.9, 3.4 Hz, 1H), 3.60 (t, J = 7.2 Hz, 1H),
3.31 (t, J = 6.8 Hz, 1H), 1.82 (p, J = 6.8 Hz, 1H), 1.62 (q, J = 7.2 Hz, 1H), 1.45 – 1.35
(m, 1H). 13C NMR (100 MHz, Chloroform-d) δ 168.21, 133.85, 132.03, 123.09, 37.56,
33.38, 32.13, 27.65, 25.33.MS (m/z): 296.2 (calc. 296.0 [C13H14BrNO2]H+).

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2.2.1.2. General procedure for the preparation of 10-13
Compound 5 (5.67 g, 12 mmol) was added into MeCN (50 mL), and then Compound
6-9 (14.2 mmol) were added, respectively. After an addition of K2CO3 (5.9 g, 42.6
mmol), the mixture was refluxed for 24 h and followed by TLC. After the solvent was
removed by distillation under reduced pressure, the residue was dissolved in EtOAc
(200 mL), and washed by water (3×50 mL). The organic phase was dried by MgSO4,
and then was evaporated. The crude product was purified by column chromatography
(DCM/EtOAc = 20:1, Rf = 0.7) to give 10-13 as white solid.
Compound 10 (2.0 g, yield 26%). 1H NMR (400 MHz, Chloroform-d) δ 7.73 (dd, J =
5.4, 3.1 Hz, 2H), 7.61 (dd, J = 5.5, 3.1 Hz, 2H), 7.38 (d, J = 1.4 Hz, 1H), 6.09 (dd, J =
7.5, 5.9 Hz, 1H), 4.30 (dq, J = 6.1, 3.0 Hz, 1H), 4.27 – 4.16 (m, 2H), 3.95 (ddd, J =
6.4, 4.6, 2.0 Hz, 2H), 3.84 (q, J = 2.7 Hz, 1H), 3.74 (ddd, J = 41.4, 11.3, 2.7 Hz, 2H),
2.05 (ddd, J = 13.2, 5.9, 2.9 Hz, 1H), 1.88 (ddd, J = 13.4, 7.6, 6.1 Hz, 1H), 1.76 (d, J
= 1.2 Hz, 3H), 0.88 (s, 9H), 0.82 (s, 9H), 0.06 (s, 6H), 0.01 (s, 3H), 0.00 (s, 3H). 13C
NMR (100 MHz, Chloroform-d) δ 168.31, 163.56, 150.96, 133.74, 133.65, 132.21,
123.10, 109.61, 87.83, 85.75, 72.02, 62.86, 41.45, 39.84, 36.12, 25.99, 25.78, 18.44,
18.01, 13.23, -4.58, -4.80, -5.32, -5.39. MS (m/z): 644.4 (calc.
644.3[C32H49N3O7Si2]H+).
Compound 11 (5.9 g, yield 68.2%). 1H NMR (400 MHz, Chloroform-d) δ 7.82 (dd, J
= 5.4, 3.0 Hz, 2H), 7.73 – 7.65 (m, 2H), 7.44 (d, J = 1.1 Hz, 1H), 6.32 (dd, J = 7.9, 5.8
Hz, 1H), 4.39 (dt, J = 5.6, 2.6 Hz, 1H), 4.04 (dd, J = 7.3, 2.6 Hz, 2H), 3.92 (q, J = 2.5
Hz, 1H), 3.89 – 3.70 (m, 4H), 2.25 (ddd, J = 13.1, 5.8, 2.6 Hz, 1H), 2.11 – 1.95 (m,
3H), 1.89 (d, J = 1.0 Hz, 3H), 0.92 (s, 9H), 0.89 (s, 9H), 0.11 (s, 6H), 0.08 (s, 3H),
13
0.07 (s, 3H). C NMR (100 MHz, Chloroform-d) δ 168.42, 163.47, 150.91, 133.97,
133.64, 132.31, 123.35, 110.10, 87.89, 85.59, 72.38, 63.11, 41.56, 39.26, 36.08, 27.07,
26.09, 25.89, 18.54, 18.13, 13.40, -4.49, -4.69, -5.22, -5.30.
1
Compound 12 (4.7 g, yield 55%). H NMR (400 MHz, Chloroform-d) δ 7.73 (tt, J =
5.0, 2.4 Hz, 2H), 7.62 (td, J = 5.2, 2.1 Hz, 2H), 7.36 (d, J = 1.1 Hz, 1H), 6.26 (dd, J =
7.9, 5.8 Hz, 1H), 4.33 (dt, J = 5.4, 2.5 Hz, 1H), 3.94 – 3.83 (m, 3H), 3.74 (ddd, J =
39.1, 11.4, 2.6 Hz, 2H), 3.63 (t, J = 6.5 Hz, 2H), 2.18 (ddd, J = 13.1, 5.8, 2.6 Hz, 1H),
1.98 – 1.89 (m, 1H), 1.82 (d, J = 1.0 Hz, 3H), 1.71 – 1.49 (m, 4H), 0.85 (s, 9H), 0.81
(s, 9H), 0.04 (s, 6H), 0.0025 (s, 3H), -0.0032 (s, 3H). 13C NMR (100 MHz,
Chloroform-d) δ 168.25, 163.32, 150.78, 133.79, 133.39, 132.15, 123.10, 109.91,
87.70, 85.41, 72.25, 62.96, 41.40, 40.73, 37.62, 26.17, 25.93, 25.74, 25.10, 18.37,

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17.96, 13.24, -4.64, -4.84, -5.37, -5.46. MS (m/z): 672.6 (calc. 672.4
[C34H53N3O7Si2]H+).
Compound 13 (colorless oil, 7.1 g, yield 86%). 1H NMR (400 MHz, Chloroform-d) δ
7.78 (tt, J = 5.0, 2.4 Hz, 2H), 7.72 – 7.61 (m, 2H), 7.39 (d, J = 1.1 Hz, 1H), 6.30 (dd,
J = 7.8, 5.8 Hz, 1H), 4.36 (dt, J = 5.5, 2.5 Hz, 1H), 3.89 (tt, J = 7.7, 4.3 Hz, 3H), 3.77
(ddd, J = 39.5, 11.4, 2.6 Hz, 2H), 3.64 (t, J = 7.2 Hz, 2H), 2.22 (ddd, J = 13.1, 5.8, 2.6
Hz, 1H), 1.96 (ddd, J = 13.4, 8.0, 6.2 Hz, 1H), 1.86(d, J = 1.0 Hz, 3H), 1.64 (ddd, J =
18.8, 15.2, 7.5 Hz, 4H), 1.36 (d, J = 8.1 Hz, 2H), 1.30 – 1.15 (m, 2H), 0.89 (s, 9H),
0.85 (s, 9H), 0.07 (s, 6H), 0.04 (s, 3H), 0.03 (s, 3H). 13C NMR (100 MHz,
Chloroform-d) δ 168.41, 163.46, 150.91, 133.85, 133.40, 132.31, 123.21, 110.04,
87.80, 85.52, 72.34, 63.06, 41.51, 41.10, 37.89, 28.37, 27.26, 26.99, 26.02, 25.83,
24.34, 18.47, 18.06, 13.32, -4.55, -4.75, -5.29, -5.37. MS (m/z): 686.5 (calc. 686.4
[C35H55N3O7Si2]H+).
2.2.1.3. General procedure for the preparation of 14-17
Compound 10-13 (6.5 mmol) was dissolved in ethanol (50 mL), respectively. Then
the hydranzine hydrate (80%) (2.0 mL) was added. The reaction mixture was stirred
over night at room temperature. In the course of reaction, a white precipitate formed
which was removed by filtration after the completion of the reaction. The solvent and
excess H2N·NH2 were evaporated under reduced pressure. The crude mixture was
purified by column chromatography (DCM/MeOH = 10:1, Rf = 0.4) to give 14-17.
1
Compound 14 (white soild, 2.1 g, yield 63%). H NMR (400 MHz, Chloroform-d) δ
7.29 (s, 1H), 6.18 (t, J = 6.6 Hz, 1H), 4.24 (dt, J = 5.9, 2.7 Hz, 1H), 3.85 (t, J = 5.9 Hz,
2H), 3.76 (s, 1H), 3.72 – 3.55 (m, 2H), 2.77 (t, J = 6.1 Hz, 2H), 2.13 – 2.03 (m, 1H),
1.84 (dt, J = 13.1, 6.8 Hz, 1H), 1.75 (t, J = 2.1 Hz, 3H), 1.41 (s, 2H), 0.76 (s, 9H),
0.72 (s, 9H), -0.06 (s, 6H), -0.08 (s, 3H), -0.09 (s, 3H). 13C NMR (100 MHz,
Chloroform-d) δ 163.39, 150.83, 133.32, 109.66, 87.47, 85.22, 72.00, 62.73, 43.70,
41.16, 39.92, 25.73, 25.53, 18.15, 17.74, 13.06, -4.85, -5.05, -5.59, -5.66. MS (m/z):
514.4 (calc. 514.3 [C24H47N3O5Si2]H+).
Compound 15 (white soild, 2.3 g, yield 69.2%). 1H NMR (400 MHz, Chloroform-d) δ
8.47 (s, 2H), 7.51 (s, 1H), 6.38 – 6.22 (m, 1H), 4.38 (dt, J = 5.7, 2.7 Hz, 1H), 4.10 (t,
J = 6.2 Hz, 2H), 3.92 (q, J = 2.5 Hz, 1H), 3.80 (ddd, J = 44.7, 11.4, 2.5 Hz, 2H), 3.00
(t, J = 6.3 Hz, 2H), 2.25 (ddt, J = 20.7, 12.7, 6.1 Hz, 3H), 1.98 (dt, J = 13.5, 6.8 Hz,
1H), 1.90 (s, 3H), 0.91 (s, 9H), 0.88 (s, 9H), 0.09 (s, 6H), 0.07 (s, 3H), 0.06 (s, 3H).
13C NMR (100 MHz, Chloroform-d) δ 164.41, 151.08, 134.51, 110.12, 88.03, 85.81,

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72.27, 63.03, 41.70, 38.06, 37.04, 26.10, 25.91, 25.47, 18.56, 18.15, 13.38, -4.45,
-4.69, -5.20, -5.27. MS (m/z): 528.3 (calc. 528.3 [C25H49N3O5Si2]H+).
Compound 16 (light yellow oil, 2.95 g, yield 84%). 1H NMR (400 MHz,
Chloroform-d) δ 7.43 – 7.34 (m, 1H), 6.29 (dd, J = 7.9, 5.8 Hz, 1H), 4.33 (dt, J = 5.4,
2.5 Hz, 1H), 3.94 – 3.82 (m, J = 7.3 Hz, 3H), 3.74 (ddd, J = 39.5, 11.4, 2.6 Hz, 2H),
2.65 (t, J = 7.0 Hz, 2H), 2.19 (ddd, J = 13.1, 5.8, 2.6 Hz, 1H), 1.98 – 1.88 (m, 1H),
1.88 – 1.80 (m, 3H), 1.59 (p, J = 7.5 Hz, 2H), 1.42 (p, J = 7.4 Hz, 4H), 0.85 (s, 9H),
0.82 (s, 9H), 0.04 (s, 6H), 0.01 (s, 3H), 0.00 (s, 3H). 13C NMR (100 MHz,
Chloroform-d) δ 163.45, 150.87, 133.38, 110.01, 87.76, 85.45, 72.30, 63.01, 41.80,
41.44, 41.05, 30.99, 25.96, 25.77, 24.90, 18.41, 18.00, 13.30, -4.61, -4.81, -5.34, -5.43.
MS (m/z): 542.4 (calc. 542.3 [C26H51N3O5Si2]H+).
1
Compound 17 (white solid 1.8 g, yield 40%). H NMR (400 MHz, Chloroform-d) δ
7.44 (d, J = 1.2 Hz, 1H), 6.36 (dd, J = 7.9, 5.8 Hz, 1H), 4.40 (dt, J = 5.5, 2.5 Hz, 1H),
4.00 – 3.89 (m, 3H), 3.81 (ddd, J = 40.3, 11.3, 2.6 Hz, 2H), 2.73 (t, J = 7.0 Hz, 2H),
2.26 (ddd, J = 13.1, 5.8, 2.6 Hz, 1H), 2.17 (s, 2H), 1.99 (ddd, J = 13.3, 7.9, 6.0 Hz,
1H), 1.92 (d, J = 1.1 Hz, 3H), 1.64 (p, J = 7.5 Hz, 2H), 1.53 (p, J = 7.3 Hz, 2H), 1.44
– 1.34 (m, 2H), 0.92 (s, 9H), 0.89 (s, 9H), 0.11 (s, 6H), 0.08 (s, 3H), 0.07 (s, 3H). 13C
NMR (100 MHz, Chloroform-d) δ 163.56, 150.57, 133.64, 109.74, 87.59, 85.34,
72.01, 62.78, 41.31, 40.87, 39.89, 26.75, 25.84, 25.64, 23.73, 18.28, 17.86, 13.25,
-4.71, -4.95, -5.46, -5.54. HRMS (m/z): 556.3596 (calc. 556.3596
[C27H53N3O5Si2]H+).
2.2.1.4. General procedure for the preparation of 18-21
Compound 14-17 was dissolved into ethanol (70 mL), respectively. After an addition
of concentrated hydrochloric acid (2.0 mL), the mixture was stirred for 12 h at room
temperature and followed by TLC. After completing the reaction, the solvent was
removed under reduced pressure and the crude product was purified by column
chromatography (DCM/MeOH = 4:1, Rf = 0.4) to give 18-21.
Compound 18 (white solid, yield 97%). 1H NMR (400 MHz, Deuterium Oxide) δ 7.67
(s, 1H), 6.27 (t, J = 6.6 Hz, 1H), 4.45 (dt, J = 6.3, 4.1 Hz, 1H), 4.13 – 3.95 (m, 3H),
3.80 (ddd, J = 40.3, 11.3, 2.6 Hz, 2H), 2.92 (t, J = 6.4 Hz, 2H), 2.47 – 2.25 (m, 2H),
1.90 (s, 3H). 13C NMR (100 MHz, Deuterium Oxide) δ 165.38, 151.67, 135.62,
110.44, 86.64, 85.88, 70.40, 61.17, 42.51, 38.83, 38.48, 12.38. MS (m/z): 286.2 (calc.
286.1 [C12H19N3O5]H+).
Compound 19 (white solid, yield 92%). 1H NMR (400 MHz, Deuterium Oxide) δ 7.64

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(s, 1H), 6.25 (t, J = 6.7 Hz, 1H), 4.47 – 4.38 (m, 1H), 3.98 (t, J = 6.6 Hz, 3H), 3.77
(ddd, J = 40.3, 11.3, 2.6 Hz, 2H), 3.00 (t, J = 7.4 Hz, 2H), 2.41 – 2.25 (m, 2H), 1.98
13
(p, J = 6.9 Hz, 2H), 1.87 (s, 3H). C NMR (100 MHz, Deuterium Oxide) δ 165.49,
151.64, 135.79, 110.57, 86.60, 85.93, 70.44, 61.24, 38.71, 38.39, 37.12, 25.09, 12.37.
MS (m/z): 300.1 (calc. 300.2 [C13H21N3O5]H+). HRMS (m/z): 322.1375 (calc.
322.1373 [C13H21N3O5]Na+).
1
Compound 20 (white solid, 3.5 g, yield 97.8%). H NMR (400 MHz, Methanol-d4) δ
7.87 (d, J = 1.2 Hz, 1H), 6.31 (t, J = 7.2 Hz, 1H), 4.41 (dt, J = 6.4, 3.4 Hz, 1H), 3.97
(td, J = 6.7, 3.3 Hz, 2H), 3.93 (p, J = 3.4, 3.0 Hz, 1H), 3.77 (ddd, J = 40.3, 11.3, 2.6
Hz, 2H), 2.99 (t, J = 7.4 Hz 2H), 2.27 (ddd, J = 13.5, 6.2, 3.5 Hz, 1H), 2.22 (m, 1H),
1.91 (d, J = 1.2 Hz 3H), 1.69 (m, 4H). 13C NMR (100 MHz, Deuterium Oxide) δ
164.99, 151.29, 135.49, 110.31, 86.62, 85.83, 70.46, 61.25, 40.71, 39.11, 38.85, 24.21,
23.90, 12.42. MS (m/z): 314.3 (calc. 314.2 [C14H23N3O5]H+).
1
Compound 21 (white solid, yield 100%). H NMR (400 MHz, Deuterium Oxide) δ
7.67 (s, 1H), 6.25 (t, J = 6.7 Hz, 1H), 4.46 (dt, J = 7.8, 4.0 Hz, 1H), 4.02 (d, J = 4.2
Hz, 1H), 3.91 – 3.72 (m, 4H), 3.03 (t, J = 7.6 Hz 2H), 2.44 – 2.26 (m, 2H), 1.89 (s,
3H), 1.72 (p, J = 7.6 Hz, 2H), 1.60 (p, J = 7.5 Hz, 2H), 1.40 (p, J = 7.6 Hz, 2H). 13C
NMR (100 MHz, Deuterium Oxide) δ 165.13, 151.40, 148.40, 135.49, 110.43, 86.64,
85.90, 70.52, 61.32, 41.24, 39.43, 38.94, 26.39, 23.10, 12.52. MS (m/z): 328.2 (calc.
328.2 [C15H25N3O5]H+).
2.2.1.5. General procedure for the preparation of thymidine dithiocarbamates1-4
Carbon disulfide (0.5 mL) was added into water (5 mL) containing aminothymidine
with different carbon chain lengths (1.0 g), and then, 1.1 equivalents of NaOH or
KOH was added. The reaction mixture was stirred for 3 h in an ice-water bath. The
solvent removed under reduced pressure and the residue was recrystallized using
isopropanol to give 1-4 as white solid.
1
Compound 1 (400 mg, 30%). H NMR (400 MHz, Methanol-d4) δ 7.83 (s, 1H), 6.28
(t, J = 6.5 Hz, 1H), 4.41 (dt, J = 5.9, 4.0 Hz, 1H), 4.23 – 4.13 (m, 2H), 3.93 – 3.89 (m,
13
1H), 3.86 – 3.70 (m, 4H), 3.35 (s, 1H), 2.36 – 2.15 (m, 2H), 1.92 (s, 3H). C NMR
(100 MHz, Methanol-d4) δ 215.61, 165.68, 152.60, 136.57, 110.75, 88.73, 87.31,
71.83, 62.67, 47.14, 41.35, 40.98, 13.23. IR (KBr)/cm-1: 3331.21, 2920.35, 1693.57,
1662.71, 1631.85, 1473.68, 1265.36, 1089.83, 1057.04. HRMS (m/z): 384.0662 (calc.
384.0658 [C13H18N3O5S2Na]H+).
Compound 2 (450 mg, 32.5%). 1H NMR (400 MHz, Methanol-d4) δ 7.84 (s, 1H), 6.31

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(t, J = 6.6 Hz, 1H), 4.45 (dt, J = 7.3, 3.8 Hz, 1H), 4.00 (t, J = 6.6 Hz, 2H), 3.95 (q, J =
4.0, 3.5 Hz, 1H), 3.79 (ddd, J = 40.3, 11.3, 2.6 Hz, 2H), 3.55 (t, J = 6.7 Hz, 2H), 2.37
13
– 2.21 (m, 2H), 1.97 – 1.84 (m, 5H). C NMR (100 MHz, Methanol-d4) δ 214.43,
165.65, 152.57, 136.72, 110.87, 88.82, 87.25, 72.12, 62.90, 45.94, 41.24, 40.17, 28.03,
13.43. MS (m/z): 373.9 (calc. 374.1 [C14H20N3O5S2]-). IR (KBr)/cm-1: 3321.56,
2926.14, 1693.57, 1664.64, 1631.85, 1473.68, 1280.79, 1093.69, 1055.11. HRMS
(m/z): 374.0848 (calc. 374.0849 [C14H20N3O5S2]-).
Compound 3 (467mg, 34.2%). 1H NMR (400 MHz, Methanol-d4) δ 7.83 (s, 1H), 6.30
(t, J = 6.1 Hz, 1H), 4.40 (m, 1H), 4.05 – 3.88 (m, 4H), 3.86 – 3.70 (m, 2H), 3.58 (t, J
= 6.4 Hz, 1H), 2.36 – 2.15 (m, 2H), 1.91 (s, 3H), 1.76 – 1.55 (m, 4H). 13C NMR (100
MHz, Methanol-d4) δ 214.17, 165.35, 152.27, 136.42, 110.66, 110.65, 88.78, 87.08,
72.03, 62.75, 42.00, 41.28, 27.16, 26.21, 13.25. IR (KBr)/cm-1: 3346.64, 2922.28,
1691.64, 1664.64, 1629.92, 1469.82, 1280.79, 1076.33. HRMS (m/z): 388.1002 (calc.
388.1006 [C15H22N3O5S2]-)
Compound 4 (470mg, 36.0%). 1H NMR (400 MHz, Methanol-d4) δ 7.82 (s, 1H), 6.31
(t, J = 6.6 Hz, 1H), 4.43 (dt, J = 6.8, 3.7 Hz, 1H), 3.92 (m, 3H), 3.78 (m, 2H), 3.54 (t,
J = 7.1 Hz, 2H), 2.36 – 2.18 (m, 2H), 1.91 (s, 3H), 1.70 – 1.56 (m, 4H), 1.43 – 1.29
(m, 2H). 13C NMR (100 MHz, Methanol-d4) δ 214.03, 165.31, 152.29, 136.37, 110.69,
88.74, 87.06, 72.02, 62.77, 48.61, 42.15, 41.18, 29.38, 28.35, 25.35, 13.25. IR
(KBr)/cm-1: 3319.63, 2933.85, 1693.57, 1664.64, 1631.85, 1471.75, 1269.22, 1097.54,
1057.04. HRMS (m/z): 402.1169 (calc. 402.1162 [C16H24N3O5S2]-).
2.2.2. Radiolabelling and quality control techniques
99mTc(CO)3-DTC were prepared according to the previous reported methods [23, 35].
Namely, potassium sodium tartrate (15.0 mg), sodium carbonate (5.0 mg), sodium
borohydride (10.0 mg) were added to a 10 mL glass vial. After sealing, the vial was
flushed with CO for 15 min, followed by the addition of 1 mL of saline containing
-
99mTcO4 (370 MBq). The mixture was heated at 80 oC for 30 min. After being cooled
to the room temperature, the pH of fresh [99mTc(CO)3(H2O)3]+ was adjusted to
approximately 7-8 using 1.0 mol/L HCl. Then, 1 mL of saline containing 1.0 mg of
DTC and 0.5 mL of [99mTc(CO)3(H2O)3]+ precursor were added to a glass vial, and the
mixture was incubated at room temperature for 20 min (Scheme 2). The
radiochemical purities were determined by HPLC (High performance Liquid
Chromatography). The elution conditions of HPLC were as follows. Water
(containing 0.1 % TFA) (A) and acetonitrile (containing 0.1% TFA) (B) were used as

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mobile phase. The gradient elution technique was adopted: 0 min 10% B, 2 min 10%
B, 10 min 90% B, 18 min 90% B, 25 min 10% B.
2.2.3. Preparation of Re(CO)3 complexes
To confirm the speculative structure of 99mTc(CO)3-labelled thymidine analogs, their
corresponding
rhenium-tricarbonyl
complexes
were
also
synthesized.
Bromopentacarbonylrhenium (I) ([Re(CO)5] Br) (20 mg) and water (about 5 mL)
were added to a 10 mL pear-shaped flask, and then the mixture was heated to reflux
for 24 h [36, 37]. Successively, 250 ꢀL of the above solution (about 1.0 mg
intermediate) was transferred into another 5 mL pear-shaped flask and 250 ꢀL of
o
dithiocarbamate derivatives (20.0 mg/mL) was added, and then was heated at 90 C
for 3 h (Scheme 2). After the completion of the reaction, the mixture solvent was
purified by HPLC to obtain the Re(CO)3 complexes.
1b: 1H NMR (400 MHz, Methanol-d4) δ 7.82 (s, 1H), 6.46 – 6.07 (m, 1H), 4.48 – 4.25
(m, 3H), 4.11 – 3.87 (m, 3H), 3.86 – 3.63 (m, 2H), 3.48 (s, 1H), 2.30 – 2.28 (m, 2H),
1.93 (s, 3H). IR (KBr)/cm-1: 3414.15, 2015.70, 1907.68, 1701.29, 1635.71, 1099.47,
1058.97, 769.63. HRMS (m/z): 632.0167 (calc. 632.0171 [C16H20N3O9S2Re-H2O]H+).
1
2b: H NMR (400 MHz, Methanol-d4) δ 7.87 (s, 1H), 6.31 (t, J = 6.6 Hz, 1H), 4.40
(dt, J = 6.4, 3.4 Hz, 1H), 4.10 – 3.94 (m, 2H), 3.92 (q, J = 3.2 Hz, 1H), 3.84 – 3.69
(m, 2H), 3.48 (dq, J = 11.9, 7.0, 5.7 Hz, 2H), 2.35 – 2.16 (m, 2H), 2.03 – 1.80 (m,
5H). IR (KBr)/cm-1: 2015.70, 1901.89, 1689.72, 1621.13, 1473.68, 1082.11. HRMS
(m/z): 646.0317 (calc. 646.0319 [C17H22N3O9S2Re-H2O]H+).
1
3b: H NMR (400 MHz, Methanol-d4) δ 7.85 (s, 1H), 6.31 (t, J = 6.6 Hz, 1H), 4.40
(dt, J = 6.6, 3.4 Hz, 1H), 4.07 – 3.85 (m, 3H), 3.84 – 3.69 (m, 2H), 3.65 – 3.38 (m,
2H), 2.35 – 2.12 (m, 2H), 1.91 (s, 3H), 1.84 – 1.56 (m, 4H). IR (KBr)/cm-1: 3396.79,
2930.00, 2013.77, 1901.89, 1695.50, 1664.64, 1627.99, 1467.89,1093.69, 1047.39.
HRMS (m/z): 660.0473 (calc. 660.0476 [C18H24N3O9S2Re-H2O]H+).
1
4b: H NMR (400 MHz, Methanol-d4) δ 7.85 (s, 1H), 6.31 (t, J = 6.6 Hz, 1H), 4.40
(dt, J = 6.7, 3.5 Hz, 1H), 4.02 – 3.86 (m, 2H), 3.77 (ddd, J = 30.1, 12.0, 3.4 Hz, 2H),
3.52 – 3.39 (m, 1H), 2.34 – 2.13 (m, 2H), 2.09 – 1.98 (m, 1H), 1.91 (s, 3H), 1.67 (dp,
J = 16.2, 8.1, 7.7 Hz, 3H), 1.49 – 1.21 (m, 6H). IR (KBr)/cm-1: 2920.35, 2011.84,
1896.11, 1629.92, 1103.33, 1058.97. HRMS (m/z): 674.0638 (calc. 674.0632
[C19H26N3O9S2Re-H2O]H+);

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2.2.4. In vitro stability study
The complexes were incubated in the labelling saline at room temperature for 6 h, and
then the stability of complex was determined by HPLC. On the other hand, 0.1 mL
(3.7-7.4 MBq) of 1a, 2a, 3a, and 4a were added to 0.1 mL of mouse serum and
o
incubated at 37 C for 6 h, respectively. After removal of the proteins using
acetonitrile, the RCP (Radiochemical purities) of the complexes were measured by
HPLC.
2.2.5. Determination of the partition coefficient
In order to evaluate the complex’s lipophilicity, the partition coefficient of the
complex between phosphate buffer (0.025 mol/L, pH = 7.4) and 1-octanol was
measured. In a 2 mL centrifuge tube, 0.1 mL of the labeled complex solution was
added, followed by an addtion of 0.9 mL of phosphate buffer (0.025 mol/L, pH =
7.4) and 1.0 mL of 1-octanol. The centrifuge tube was shacked on a Vortex mixer for
30 seconds and then centrifuged at 5000 rpm for another 5 min. Three samples (100
ꢀL) from each layer in triplets were measured by gamma counter. The partition
coefficient was calculated as follow: P = (counters per minute in 1-octanol/counters
per minute in phosphate buffer). Usually, log P was the final expression of the
partition coefficient. Samples from the 1-octanol layer were redistributed until
consistent distribution coefficient values were obtained.
2.2.6. In vitro cell uptake experiments
HCT116 cell line was obtained from China Infrastructure of Cell Line Resource and
was maintained as manufacturer’s recommendations. Cells suspended in 1 mL of
culture medium were seeded into 24-well plates (0.1×106 cells/well). The plates were
o
incubated for 48 h in a 5% CO2-humidified atmosphere at 37 C. The medium was
removed and washed with 0.5 mL fresh culture medium. 0.1 mL medium containing
74 kBq technetium-99m complex and 0.4 mL fresh medium or 0.4 mL fresh medium
containing 2.0 mg thymidine were added into the well. After incubated for 2 h at 37
oC or 4 °C, the medium was removed and the cells were washed with 2 × 0.5 mL cold
PBS (4 oC, 0.01 M, pH = 7.4). After detached by addition of 0.5 mL 1M NaOH , the
cells were collected and counted in a gamma counter (Wizard 2480, PerkinElmer).
The protein content was measured spectrophotometrically using a Micro BCA protein
assay reagent (Macgene, Beijing, China).The results were expressed as a ratio of the
percentage of the initial activity to the protein mass (% ID/mg protein). Each

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experiment was performed three times. In the FU pretreatment assay, twenty-four
hours after being seeded, the exponentially cells were exposed to 100 ꢀM 5-FU for
another 24 h.
2.2.7. Biodistribution study
All the biodistribution studies were carried out in compliance with the national laws
related to the ethics during experimentation. Approximate 2 × 106 S180 cells were
implanted in the left front left armpit of male mice by subcutaneous injection. After
one week, tumors grew to diameters of 7-10 mm. 0.1 mL of 99mTc(CO)3-labelled
radiotracer (37 kBq) was injected into the mice bearing S180 tumor via a tail vein.
The mice were sacrificed by neck dislocation at 0.5 h, 2 h, and 4 h post-injection. The
tumors, interesting organs and blood were collected, weighed and measured for
radioactive counts. The results were expressed as the percent uptake of injected dose
per gram of tissue (% ID/g).
2.2.8. SPECT imaging study
A Triumph SPECT/CT scanner (TriFoil Imaging, California, USA), a dual-modality
scanner supporting CT and SPECT imaging acquisition, was used for SPECT/CT
imaging studies. 0.1 mL of 1a (11.1 MBq) was injected intravenously through tail
vein into a Kunming mouse bearing S180 tumor. After anesthetized with 1.5%
isoflurane in air, the mouse was fixed near the center of the SPECT/CT scanner. Static
imaging was obtained at 120 min after injection.
3. Results and discussion
3.1. Synthesis and radiolabelling
Compound 1-4, dithiocarbamate derivatives of TdR functionalized at position N3,
were synthesized via five steps from thymidine, and the reaction equations were
1
shown in Scheme 1. Intermediates and the final ligands were identified by H-NMR,
13C-NMR, MS or HRMS, and the results showed that the target ligands were
successfully synthesized.
The H2O in the fac-[99mTc(CO)3(H2O)3]+ precursor was easily substituted by sulfur
atoms in the dithiocarbamate [32, 38], and then 99mTc(CO)3-dithiocarbamates (1a-4a)
were prepared (Scheme 2). In order to study the structure of 1a-4a, the corresponding
rhenium complexes (1b-4b) were synthesized according to the previous literature

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[36]. When 99mTc and Re complexes were co-injected in HPLC, the retention time of
radioactivity and UV detectors provided strong evidence for confirmation (Figure 1).
Radiochemical purities (RCP) of the complexes were measured by HPLC. The
-
retention times of the precursor fac-[99mTc(CO)3(H2O)3]+ and 99mTcO4 were 12.17
min and 3.93 min, respectively. While the retention times of 1a-4a were 13.52 min,
13.85 min, 13.93 min and 14.18 min, respectively. The results of HPLC analysis
showed that the RCP of the radiotracers were more than 90%.
3.2．In vitro stability study and partition coefficients
The complexes were stable over 4 h under different conditions, including in the
o
reaction mixture at room temperature, and also in the mouse serum at 37 C,
suggesting that they had a high stability in vitro.
The log P values of 1a-4a were 0.65 ± 0.07, 0.82 ± 0.06, 0.93 ± 0.05 and 1.08 ± 0.07,
respectively. The results suggested that they were lipophilic. Moreover, the log P
values of the corresponding 99mTc(CO)3 complexes are in accordance with the
structure of the ligands, since incorporation of more CH2 group increases the
lipophilicity.
3.3．Cell uptake experiments
The thymidine kinase 1 is located in the cytosol, and the potential imaging complex
has to pass through the cell membrane before it can be phosphorylated. If this
transport is not achieved actively via nucleoside transporters, then it must occur by
passive diffusion [39]. In order to investigate the major cell internalization routes of
the complex 1a-4a, the incorporations of these complexes were evaluated in HCT116
colorectal carcinoma cells. The HCT116 cell line was chosen for its TK1
overexpression and prominent selectivity of the enzyme for thymidine and thymidine
analogs [40, 41]. Internalization was measured with and without pretreatment with
5-FU (5-fluorouracil) [20, 41], and with excessive thymidine. 5-FU, a thymidylate
synthase inhibitor, is known to increase the uptake of 5-iododeoxyuridine or 18F-FLT,
via inhibiting endogenous thymidine synthesis, increasing the levels of TK1 and
inducing re-distribution of nucleoside transporters [41-44]. The results of
internalization experiments were presented in Figure 2.
In the previous literatures, researchers proposed that uptakes of N3 functionalized
thymidine complexes occurred via passive diffusion and endocytosis [20, 45, 46]. In

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the present work, pretreatment with 5-FU and blocking with thymidine, had an effect
on the uptake of 1a-4a on tumor cells. For 1a and 2a, lower temperature could
significantly reduce the tumor uptake of the radioactivity, but as the carbon chain
lengthened, the effect of temperature on cell uptake disappeared. The above
experiment showed that the uptakes of 99mTc(CO)3-labelled thymiding
dithiocarbamate derivatives were mediated, at least in part, by nucleoside transporters,
especially the uptakes of 1a and 2a.
3.4． Biodistribution study
The results of biodistributions of 1a-4a in mice bearing S180 tuomor were shown in
Table 1 and Table 2. These four radiotracers had similar biological properties. The
tumor uptakes of those complexes were quite high and the retentions of them were
perfect. Taking 1a as an example, the tumor uptake of 1a was 3.28 ± 1.10% ID/g at
30 min post-injection, and the tumor uptake was still 2.95 ± 0.73% ID/g at 240 min
post-injection, suggesting that the complex had a good retention in tumor. At the same
time, the complex had a good tumor/muscle ratio. In other rapidly growing organs,
such as the spleen and intestine, the uptakes were 2.2-fold and 7.7-fold higher than
that of muscle at 120 min post-injection. Interestingly, the uptake of the complex in
the liver gradually increased with time, and the trend of kidney uptake was consistent.
Like many radionuclide labelled thymidine or tymidine derivatives [47-50], activity
accumulation in the kidney and liver showed that the major route of excretion was
renal and hepatobiliary. The tumor uptake values of complex 4a were significantly
lower than those of the other three complexes, indicating that the elongation of the
carbon chain didn’t cause an increase in the tumor uptake. Low uptake in the thyroid
-
suggested that there was no 99mTcO4 derived from the decompostion of the complex
in vivo.
Table 1. Biodistrbutions of 1a and 2a in mice bearing S180 tumor (mean ± SD, n = 5, % ID/g).
1a (99mTc(CO)3-DTC-2-TdR)
2a (99mTc(CO)3-DTC-3-TdR)
% ID/g
Heart
30 min
120 min
240 min
30 min
4.04±0.75
120 min
240 min
3.08±0.45 2.02±0.18 1.59±0.27
15.28±2.59 19.48±3.94 24.91±2.90
6.74±1.57 3.45±0.69 3.70±0.85
14.89±2.70 16.43±2.24 18.55±2.77
1.75±0.31 1.38±0.30 1.11±0.16
2.83±0.30 1.63±0.32
Liver
33.79±3.36 46.71±3.42 46.40±5.03
11.70±3.52 6.34±0.77 4.99±0.89
15.21±2.35 18.27±2.29 15.77±1.96
5.26±1.87 3.24±1.78 2.76±1.65
Lung
Kidney
Spleen

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Stomach
Bone
1.77±0.78 1.98±0.70 2.40±0.59
0.63±0.22 0.64±0.31 0.60±0.14
2.67±0.40 1.73±0.30 0.98±0.20
0.91±0.18 0.78±0.12 0.61±0.21
6.08±1.00 3.82±0.52 2.67±0.40
3.98±0.91 3.99±0.58 4.29±0.88
20.62±3.18 9.93±1.66 5.67±1.65
0.05±0.03 0.04±0.02 0.02±0.01
1.58±0.25 1.16±0.12 1.01±0.15
0.87±0.22 0.63±0.34 0.66±0.12
8.42±2.74 4.88±2.60 4.04±0.82
3.28±1.10 3.35±0.51 2.95±0.73
13.33±1.56 7.75±0.51 5.54±0.27
0.06±0.03 0.06±0.02 0.05±0.01
Muscle
Intestine
Tumor
Blood
Thyroid a
T/B
0.25
3.77
0.43
5.29
0.53
4.50
0.19
4.35
0.40
5.09
0.76
7.03
T/M
a Expressed as % injected dose per organ (% ID)
T/B = tumor-to-blood ratio, T/M = tumor-to-muscle ratio
Table 2. Biodistrbutions of 3a and 4a in mice bearing S180 tumor (mean ± SD, n = 5, % ID/g).
3a (99mTc(CO)3-DTC-4-TdR)
4a (99mTc(CO)3-DTC-5-TdR)
% ID/g
Heart
30 min
4.43±0.56
120 min
240 min
2.06±0.30
30 min
1.61±0.24
120 min
240 min
1.11±0.19
2.80±0.40
1.29±0.17
Liver
25.28±1.57 32.76±3.47 36.34±2.82
9.48±1.81 6.00±0.78 4.60±0.36
14.77±1.36 15.21±0.63 15.42±1.89
37.93±3.93 40.76±5.77 38.64±7.04
4.00±0.72 2.97±0.35 2.63±0.59
10.79±1.60 12.59±0.93 11.47±1.28
Lung
Kidney
Spleen
Stomach
Bone
5.70±2.88
0.95±0.11
2.57±0.33
1.01±0.10
4.49±1.03
3.75±1.36
3.41±1.93
1.00±0.25
1.72±0.17
0.99±0.25
3.32±0.38
3.80±0.91
2.29±0.41
1.05±0.28
1.18±0.15
0.67±0.10
2.72±0.17
3.60±0.27
5.92±0.65
0.05±0.01
0.60
1.57±0.25
0.77±0.37
1.35±0.45
0.46±0.12
3.87±0.98
1.84±0.54
6.04±0.93
0.03±0.02
0.31
1.22±0.22
1.11±0.42
0.99±0.21
0.52±0.14
2.86±0.94
1.87±0.52
3.68±0.15
0.03±0.01
0.51
1.11±0.26
1.06±0.22
0.97±0.18
0.43±0.08
2.38±1.36
1.88±0.36
2.99±0.95
0.02±0.01
0.63
Muscle
Intestine
Tumor
Blood
Thyroid a
T/B
18.21±2.15 9.22±2.05
0.09±0.05
0.21
0.07±0.02
0.41
T/M
3.73
3.85
5.41
3.97
3.57
4.36
a Expressed as % injected dose per organ (% ID)
T/B = tumor-to-blood ratio, T/M = tumor-to-muscle ratio
Table 3. showed some results of biodistribution of previously reported radionuclide
labelled thymidine derivatives. When compared to the complexes
99mTc(CO)3-12N3-5-TdR (functionalized at N3 position) [23] and 99mTc-TMHEA
(functionalized at C5 position) [22], 1a exhibited a much higher tumor uptake. The
tumor uptake of 1a (3.35±0.51% ID/g) was more than five times higher than that of

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99mTc(CO)3-12N3-5-TdR (0.63±0.14% ID/g), and more than six times higher than that
of 99mTc-TMHEA ( 0.51±0.05% ID/g) at 2 h post-injection. As for the
tumor-to-muscle ratio, 1a was also higher than the other two complexes.
Table 3. Comparison of biodistribution of some reported 99mTc labelled thymidine derivatives.
complex
Time p.i (h)
1a
99mTc(CO)3-12N3-5-TdR 99mTc-TMHEA
2
2
2
Tumor uptake (%
ID/g)
3.35±0.51
0.63±0.14
0.51±0.05
Tumor/Muscle
Labelling core
Tumor models
Reference
5.29
99mTc(CO)3
S180
2.88
99mTc(CO)3
S180
2.84
99mTcO
HepA
22
Present study
23
3.5．SPECT imaging studies
Because 1a (99mTc(CO)3-DTC-2-TdR) showed a high tumor uptake and a lower liver
uptake, it was selected as a promising candidate for imaging studies. The solid tumor
could be clearly visualized with high tumor-to-background contrast (Figure 3),
suggesting 1a exhibited its potential as a tumor imaging agent. However, the liver
uptake of 1a was appreciable, thus making it unsuitable to localize tumor in liver. The
imaging findings were similar to the biodistribution results in mice. Low uptake in the
thyroid suggested good in vivo stability of 1a.
4. Conclusion
In the present study, we synthesized four novel thymidine derivatives, functionalized
at position N3. Their corresponding 99mTc(CO)3-labelled complexes were successfully
prepared in high yields via a ligand-exchange reaction. The complexes were lipophilic
and stable in vitro. Cell uptake studies showed that they were transported via

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nucleoside transporters. The biodistributions and SPECT imaging studies in mice
bearing S180 tumor showed that they had an obvious tumor uptake and high
tumor/muscle ratio. In this case, 1a revealed promising biological features as a
potential tracer for tumor proliferation imaging, justifying further investigations.
Acknowledgements
The work was financially supported, in part, by the National Natural Science
Foundation of China (21771023), the foundation of Key Laboratory of
Radiopharmaceuticals (Beijing Normal University), Ministry of Education.
Conflicts of interest
There are no conflicts to declare
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Scheme 1. The synthesis of thymidine dithiocarbamates. Reagents and conditions: (a)
dibromoalkanes, MeCN, K2CO3, reflux, 24 h; (b) EtOH, hydranzine hydrate, r.t., 12 h; (c) EtOH,
conc.HCl, 12 h; (d) CS2, H2O, NaOH/KOH, 0 oC, 3 h.

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Scheme 2. Preparations procedures and speculative structures of M(CO)3-labelled dithiocarbamate
derivatives (M=99mTc, Re). Reagents and conditions: (a) M = 99mTc, NaCO3, NaBH4, potassium
o
o
sodium tartrate, CO, 30 min, 80 C; (b) M = Re, H2O, 100 C, 24 h; (c) M = 99mTc, DTC, pH =
7-8, 10-20 min, r.t.; (d) M = Re, DTC-TdR, H2O, 90 oC, 3 h.

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Figure 1. HPLC profiles of the 99mTc(CO)3-labelled radiotracers (red) and the corresponding
Re(CO)3 complexes (black).

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Figure 2. Cell uptakes on the human colorectal carcinoma HCT116 cell line over a period of 2 h at
37 oC, with and without pretreatment with 5-FU, and blocking experiments with 2.0 mg thymidine，
and at 4 °C.
Statistical significance was determined using a bilateral t test of equal variance.
Statistical significance is reported for *p < 0.05, **p < 0.01 and ***p < 0.001.

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Figure 3. SPECT/CT image of 1a in S180 tumor-bearing mouse at 2 h post injection (left: visual
image; middle: sagittal image; right: transversal image).

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Highlights
ꢀ
A series of novel M(CO)3-labelled thymidine dithiocarbamate derivatives were prepared (M
99mTc, Re).
=
ꢀ
ꢀ
Some of the complexes entered the cell via the transport of the nucleoside transporters.
99mTc(CO)3-labelled complexes had high tumor uptake and good tumor/muscle ratio and the
SPECT imaging studies in mice bearing tumor showed that one of the complexes had an
obvious tumor uptake and high tumor-to-background ratio.