Synthesis and preliminary biological evaluation of a technetium-99m labeled thymidine analog

Article abstract of DOI:10.1016/j.cclet.2011.06.012

The synthesis and labeling of ^99mTc-N³-{N′-[2- sulfanyl-ethylamino)acetyl]-2-aminoethyl-sulfanyl-1-hexanamide}thymidine ( ^99mTc-NHT) were studied. In the presence of sodium glucoheptonate (GH) and ethylene diamine tetraace

Full text of DOI:10.1016/j.cclet.2011.06.012

Available online at www.sciencedirect.com
Chinese Chemical Letters 22 (2011) 1309–1312
www.elsevier.com/locate/cclet
Synthesis and preliminary biological evaluation of a
technetium-99m labeled thymidine analog
b,c
a
a
a
Chun Xiong Lu a,b, , Zheng Wu Wang , Quan Fu Jiang , Jie Tang , Cheng Tan ,
*
Jian Kang Zhang a
a Key Laboratory of Nuclear Medicine, Ministry of Health/Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear
Medicine, Wuxi 214063, China
b School of Chemical & Material Engineering, Jiangnan University, Wuxi 214122, China
c Department of Food Science & Technology, School of Agriculture and Biology Shanghai Jiao Tong University, Shanghai 200240, China
Received 2 April 2011
Available online 28 July 2011
Abstract
The synthesis and labeling of 99mTc-N3-{N0-[2-sulfanyl-ethylamino)acetyl]-2-aminoethyl-sulfanyl-1-hexanamide}thymidine
(
99mTc-NHT) were studied. In the presence of sodium glucoheptonate (GH) and ethylene diamine tetraacetic acid (EDTA), 99mTc-
NHT was obtained by using bisaminoethanethiol (N2S2) as a bifunctional coupling agent. The radiochemical purity of the 99mTc-
NHT was over 95%. Biodistribution of 99mTc-NHTwas performed in hepatoma HepA tumor-bearing mice. At 2 h p.i., the ratios of
tumor-to-muscle, tumor-to-bone and tumor-to-blood were 4.41 ꢀ 0.32, 2.45 ꢀ 0.24 and 1.51 ꢀ 0.18, respectively.
# 2011 Chun Xiong Lu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Synthesis; 99mTc-NHT; Biodistribution; Thymidine analog; Tumor imaging
In clinical oncology, 20-deoxy-20-[18F]fluoro-D-glucose (18F-FDG), a glucose derivative, has been widely used for
tumor imaging with positron emission tomography (PET) in recent years. However, 18F-FDG is a non-specific tracer
for tumor imaging since glucose is highly utilized by many other cells, such as macrophages found in inflammatory
lesions [1,2].
To overcome this inconvenience of FDG, many studies have been focus on the development of a variety of DNA
precursors [1,3–5]. Specifically, the labeled thymidine analog can target the proliferative activity of malignant lesions
[6,7], several useful ligands, such as 11C-labeled nucleoside thymidine [1], 30-deoxy-30-[18F]fluoro thymidine ([18F]-
FLT) [1,3–5] and its analog 18F-FMAU [8] have been demonstrated their good imaging features. However, these
tracers labeled with either 11C or 18F, which were short half-life isotopes produced by a cyclotron, with complicated
radiochemical synthesis and the lower radiochemical yield and high cost of PET examination, all these limited their
use as tracers in routine clinical studies.
Technetium-99m (99mTc), the most commonly used radioisotope in SPECT, is continuously available at a
reasonable cost in many hospitals and has ideal nuclear properties for imaging (T1/2 = 6.02 h, g = 140 keV). Therefore
* Corresponding author.
E-mail address: luchunxiong@yahoo.com.cn (C.X. Lu).
1001-8417/$ – see front matter # 2011 Chun Xiong Lu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2011.06.012

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C.X. Lu et al. / Chinese Chemical Letters 22 (2011) 1309–1312
O
N
NH
O
O
N
O
NH
O
O
a
O
O
+
Cl
HO
O
O
O
OH
1
O
Br
HN
NH
S
S
N
S
O
OCH
3
b
OCH
3
+
Br
Cl
O
S
NH
O
OCH
3
OCH
3
O
N
O
2
N
N
S
OCH
3
O
d
c
O
S
NH
O
O
1
2
+
O
OCH
3
O
3
O
O
N
O
N
O
O
N
N
S
S
N
N
SH
SH
OCH
3
e
O
HO
O
NH
O
O
HO
NH
OCH
O
OH
O
3
OH
4
5, NHT
Scheme 1. Reagents and conditions: (a) DMAP, CH2Cl2, ref; (b) DMAP, CH2Cl2, 0 8C; (c) K2CO3, DMF/acetone (1/1, v/v), 50 8C; (d) CH3ONa,
methanol, rt; (e) CF3COOH, Hg(CH3COO)2, H2S, 0 8C.
it is important to develop a 99mTc labeled thymidine analog so as to provide the ideal characteristics needed for routine
clinical studies [9–12].
In this communication, we report the synthesis of a thymidine analog, N3-{N0- [2-sulfanylethylamino)acetyl]-2-
amino-ethylsulfanyl-1-hexanamide}thymidine (NHT), which could be labeled easily by technetium-99m and
explored the primary labeling conditions. Biodistribution of 99mTc-NHT was performed in hepatoma HepA tumor-
bearing mice. The purpose of this study is to conjugate thymidine analog with chelating agent and evaluate the
feasibility of technetium-99m-labeled thymidine analog as candidate for tumor imaging agent.
The labeled precursor NHTwas synthesized through a multiple-step reaction using thymidine as a starting material
and the total yield was 34.68%. The synthesis procedure is outlined in Scheme 1. Thymidine was protected at the 30,50-
O-position with p-toluoyl chloride in CH2Cl2 to give compound 1, and 6-bromohexanoyl chloride was coupled with N-
[2-((2-S-(4-methoxybenzyl)sulfanyl)ethyl)amino]acetyl-S-(4-methoxybenzyl)-2-aminoethanethiol (N2S2) in CH2Cl2
to give compound 2. The subsequent synthesis of compound 3 was through substitution reaction using compound 1
O
O
O
N
O
N
N
N
SH
SH
N
S
99mTc
O
N
O
HO
HO
NH
O
O
S
N
O
O
OH
OH
99mTc-NHT
5, NHT
Scheme 2. Reagents and conditions: SnCl2, GH, EDTA, Na99mTcO4, 100 8C, 30 min.

C.X. Lu et al. / Chinese Chemical Letters 22 (2011) 1309–1312
1311
30
25
20
15
10
5
5min
30min
60min
120min
180min
0
Brain
Heart
Liver
Spleen
Lung
Kidney
Tumor
Stomach S.intestines Bladder
Muscle
Bone
Blood
Fig. 1. Biodistribution of 99mTc-NHT in tumor-bearing mice (x ꢀ s, n = 5, %ID/g).
7
6
5
4
3
2
1
0
Tumor/Muscle
Tumor/Bone
Tumor/Blood
5min
30min
60min
120min
180min
Fig. 2. Ratios of tumor-to-muscle, tumor-to-bone and tumor-to-blood.
and compound 2 in an acetone/DMF mixed solvent. The compound 4 was obtained by removing the toluoyl protecting
groups of compound 3 with sodium methoxide in methanol. The thiol protecting groups, 4-methoxybenzyl, of
compound 4 were removed with Hg(OAc)2 in trifluoroacetic acid to give compound 5 (NHT) [13].
Using SnCl2 as reducing agent, and in the presence of sodium glucoheptonate (GH) and ethylene diamine
tetraacetic acid (EDTA), a series of studies were performed to optimize labeling efficiency of 99mTc-NHT, as show in
Scheme 2. When the reaction temperature was set at 100 8C and kept for 30 min, the labeled yield and radiochemical
purity (RCP) were over 95%, which determined by thin layer chromatography (TLC) and high performance liquid
chromatography (HPLC) [14].
RCP of freshly prepared 99mTc-NHT was evaluated every hour at room temperature to determine whether it was
stable within 6 h. The results showed that the 99mTc-NHT had good stability in vitro.
Biodistribution of 99mTc-NHT was performed in hepatoma HepA tumor-bearing mice showed that the high uptake
of 99mTc-NHT in liver and kidney, which means that the clearance of 99mTc-NHT was mainly through the
hepatobiliary pathway and the renal pathway, as show in Fig. 1. At 2 h post injection, the ratios of tumor-to-muscle,
tumor-to-bone and tumor-to-blood were 4.41 ꢀ 0.32, 2.45 ꢀ 0.24 and 1.51 ꢀ 0.18, respectively, as show in Fig. 2.
In summary, we have synthesized a novel thymidine analog NHT, which can be easily labeled with 99mTc.
Biodistribution of 99mTc-NHT in tumor-bearing mice showed that the high uptake of 99mTc-NHT in liver and kidney.
At 2 h p.i., the ratios of tumor-to-muscle, tumor-to-bone and tumor-to-blood were 4.41 ꢀ 0.32, 2.45 ꢀ 0.24 and
1.51 ꢀ 0.18, respectively. This indicated that 99mTc-NHT might be a potential SPECT imaging agent for tumor study.
Acknowledgments
Financial support was provided by Natural Science Foundation of Jiangsu Province (no. BK2008112), and
Department of Health of Jiangsu Province (no. H200624).

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C.X. Lu et al. / Chinese Chemical Letters 22 (2011) 1309–1312
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[13] Analytical data for NHT. IR (film, cmꢁ1): (3360, 2926, 2835, 1680, 1663, 1629, 1556, 1470, 1363, 1271; 1H NMR (400 MHz, CD3OD): d 8.25
(s, 1H), 7.82 (m, 1H), 6.31 (t, 1H, J = 6.6 Hz), 4.37–4.42 (m, 1H), 4.16 (s, 1H), 3.97 (m, 2H), 3.92 (m, 2H), 3.73–3.80 (m, 2H), 3.61 (m, 2H),
3.50 (m, 1H), 3.33–3.43 (m, 4H), 3.22 (m, 1H), 2.70–2.74 (t, 1H, J = 7.2 Hz), 2.59–2.69 (m, 3H), 2.51–2.56 (t, 1H, J = 7.6 Hz), 2.41–2.44 (d,
1H, J = 6 Hz), 2.17–2.33 (m, 2H), 1.91 (s, 3H), 1.58–1.74 (m, 4H), 1.35–1.42 (m, 2H); 13C NMR (400 MHz, CD3OD): d 175.1, 170.0, 164.5,
151.5, 135.4, 110.2, 87.8, 86.5, 78.0, 71.2, 61.8, 49.0, 41.5, 41.0, 33.1, 31.2, 29.5, 26.8, 26.5, 26.0, 25.0, 13.2; ESI–MS (m/z): 533.1.([M+H]+).
[14] HPLC of 99mTc-NHT: Lichrospher C18 column (150 mm ꢂ 4.6 mm, 5 mm), eluted with water/methanol (85/15, v/v); flow rate: 1.0 mL/min,
retention time: 6.70 min.