Preparation and Preliminary Biological Evaluation of Novel 99mTc-Labelled Thymidine Analogs as Tumor Imaging Agents

Article abstract of DOI:10.3390/molecules17078518

Two kinds of novel thymidine derivatives, N-thymidine-yl-N′-methyl- N′-{N′′- [2-sulfanyl-(ethylamino)acetyl]-2-aminoethylsulfanyl- 1-hexanamide}-ethanediamine (TMHEA) and N-thymidine-yl-N′-methyl-N′- {N′′-[2-sulfanyl-(ethylamino)acetyl]-2- aminoethylsulfanyl-1- hexanamide}-hexanediamine (TMHHA) were prepared and successfully labeled with ^99mTc in high labeling yields. The in vitro stability and in vivo biodistribution of ^99mTc-TMHEA and ^99mTc-TMHHA were investigated and compared. The biodistribution studies indicate that the radiotracer ^99mTc-TMHEA displays selective tumor uptake, suggesting it is a potential tumor imaging agent.

Full text of DOI:10.3390/molecules17078518

Molecules 2012, 17, 8518-8532; doi:10.3390/molecules17078518
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Preparation and Preliminary Biological Evaluation of Novel
99mTc-Labelled Thymidine Analogs as Tumor Imaging Agents
Chunxiong Lu 1,2,*, Quanfu Jiang 1, Cheng Tan 1, Jie Tang 1 and Jiankang Zhang 1
1
Key Laboratory of Nuclear Medicine, Ministry of Health-Jiangsu Key Laboratory of Molecular
Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China
2
School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China
* Author to whom correspondence should be addressed; E-Mail: luchunxiong@yahoo.com.cn;
Tel.: +86-0510-8551-4482; Fax: +86-0510-8551-3113.
Received: 18 May 2012; in revised form: 27 June 2012 / Accepted: 5 July 2012 /
Published: 16 July 2012
Abstract: Two kinds of novel thymidine derivatives, N-thymidine-yl-N′-methyl-N′-{N′′-
[2-sulfanyl-(ethylamino)acetyl]-2-aminoethylsulfanyl-1-hexanamide}-ethanediamine
(TMHEA)
and
N-thymidine-yl-N′-methyl-N′-{N′′-[2-sulfanyl-(ethylamino)acetyl]-2-
aminoethylsulfanyl-1-hexanamide}-hexanediamine (TMHHA) were prepared and
successfully labeled with 99mTc in high labeling yields. The in vitro stability and in vivo
biodistribution of 99mTc-TMHEA and 99mTc-TMHHA were investigated and compared.
The biodistribution studies indicate that the radiotracer 99mTc-TMHEA displays selective
tumor uptake, suggesting it is a potential tumor imaging agent.
Keywords: thymidine derivative; 99mTc-labelled; biodistribution; tumor imaging
1. Introduction
In clinical oncology, 2′-deoxy-2′-[18F]fluoro-D-glucose (18F-FDG), a glucose derivative, has been
widely used in recent years for tumor imaging with positron emission tomography (PET). 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
18F-FDG, many studies have focused on the development of a variety of DNA precursors [1,3–5].
Specifically, labeled thymidine analogs can target the proliferative activity of malignant lesions [6,7],
and several useful ligands, such as 11C-labeled nucleoside thymidine [1], 3′-deoxy-3′-[18F]fluoro

Molecules 2012, 17
8519
thymidine (18F-FLT) [1,3–5] and its analog 18F-FMAU [8] have demonstrated their good imaging
18
features. However, these tracers were labeled with either 11C or F, which are short half-life isotopes
produced by a cyclotron, with complicated radiochemical synthesis and the lower radiochemical yield
and high cost of PET examination, all of which limit 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, γ = 140 keV). Therefore it is important to develop a 99mTc labeled thymidine analog so
as to provide the ideal characteristics needed for routine clinical studies [9–11]. In the previous work
of our group, a series of technetium-99m labeled thymine derivatives have been prepared and their
in vivo biological properties were systematically investigated [12,13]. It was found that the uptake ratio
of tumor to muscle of 99mTc-NHT was higher than that of 99mTc-ANMdU, which means that uptake
ratio of tumor to muscle maybe increase with increasing carbon chain length between the thymidine
and N2S2 ligand. However, to the best of our knowledge, extension and optimization of the linker
chain between the thymidine and N2S2 ligand to develop novel tumor imaging agent has been
largely unexplored.
For the purpose of developing novel tumor imaging agents with excellent biological properties, we
have continued to extend the number of methylene units between the thymidine and N2S2 ligand.
In this work, two novel 99mTc-labeled thymidine derivatives were prepared and reported,
i.e., 99mTc-TMHEA and 99mTc-TMHHA (Figure 1). Their in vitro stability and in vivo biodistribution
were also studied.
Figure 1. Structures of TMHEA and TMHHA.
O
N
O
H
N
N
HN
N
SH
SH
n
O
HO
O
O
NH
n=2, TMHEA;
n=6, TMHHA
OH
2. Results and Discussion
2.1. Chemistry and Radiolabeling
TMHEA and TMHHA were synthesized by seven step reactions from the starting materials
ethanediamine and hexanediamine, respectively. The target compounds were identified by MS,
1H-NMR and 13C-NMR, and the results agreed well with the expected chemical structures.
99mTc-TMHEA and 99mTc-TMHHA were labeled with Na99mTcO4 by reduction with stannous chloride.
For TLC analysis, with the toluene/acetonitrile/methanol (v/v/v = 3/1/1) system, the Rf values of
99mTc-TMHEA and 99mTc-TMHHA were about 0.7–0.8, while 99mTc-colloidal impurities remain at 0–0.1.
HPLC analysis revealed the formation of free technetium (Na99mTcO4) that was eluted at a retention
time of 9.9 min, whereas 99mTc-TMHEA and 99mTc-TMHHA eluted at retention times of 13.3 min
and 12.8 min, respectively (Figure 2). For each radiolabeled complex, the single peak in the

Molecules 2012, 17
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HPLC-chromatogram clearly shows the formation of only one complex and excludes the possibility of
residual Na99mTcO4 or other components. That is, the chelation of technetium with the N2S2 is unique
and complete.
Figure 2. HPLC chromatograms (99mTc-TMHEA tR = 13.3 min, 99mTc-TMHHA
−
tR = 12.8 min and 99mTcO4 tR = 9.9 min).
According to the TLC and HPLC analysis, the radiochemical purities of 99mTc-TMHEA and
99mTc-TMHHA were all greater than 95%. The radiolabeled compounds were used immediately after
the formulation for both in vitro and in vivo studies.

Molecules 2012, 17
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2.2. In Vitro Stability and Octanol-Water Partition Coefficient
The in vitro stabilities of 99mTc-TMHEA and 99mTc-TMHHA were performed in PBS (pH = 7.4) for
different time intervals (1, 2, 3, 4, 5, 6 h) at 37 °C. The stability was presented as RCP on the basis of
the HPLC analysis. After 6 h of incubation, more than 95% of 99mTc-TMHEA and 99mTc-TMHHA
remained intact in the PBS. The results indicate that the labeling efficiency of these complexes was
high and their stability duration was long enough to allow further biodistribution and imaging studies.
The octanol-water partition coefficients (logP) for 99mTc-TMHEA and 99mTc-TMHHA were 1.01,
0.99 and 1.06, 1.02 in PBS at two different pH values of 7.0 and 7.4, respectively (see Table 1), which
demonstrated that the longer the carbon chain, the smaller the logP, and the liposolubility at pH = 7.4
was higher than that at pH = 7.0. As well known, the logP value is a very useful parameter that can be
used to understand the behavior of a drug and predict its distribution in the organism in combination
with other parameters [14].
Table 1. Octanol-Water partition coefficient.
Constituent
99mTc-TMHEA
99mTc-TMHHA
pH = 7.0
1.01 ± 0.03
0.99 ± 0.01
pH = 7.4
1.06 ± 0.02
1.02 ± 0.03
2.3. Blood Kinetics Studies
Pharmacokinetic parameters are listed in Table 2. Figure 3 shows the blood clearance of
99mTc-TMHEA and 99mTc-TMHHA in the mice 3 h post injection. Pharmacokinetics of
99mTc-TMHEA and 99mTc-TMHHA comply with the two-compartment model with the
pharmacokinetic equations of C = 5.24e−0.11t + 1.14e−0.02t and C = 5.51e−0.21t + 2.44e−0.02t, respectively.
The values of total body clearance (CL) were 0.10 and 0.09 and the area under the curve (AUC) were
162 and 184 for 99mTc-TMHEA and 99mTc-TMHHA, respectively. In the early phase, the blood
clearance of 99mTc-TMHHA was slower than 99mTc-TMHEA. After 2 h, the radioactivity concentration
of two tracer agents in blood reaches an equilibrium which coincides with the pharmacokinetic
parameters CL, AUC and the pharmacokinetic curves.
Table 2. Pharmacokinetic parameters of the 99mTc-TMHEA and 99mTc-TMHHA in mice.
Parameters
K12 (min−1)
99mTc-TMHEA
0.033
99mTc-TMHHA
0.101
K21 (min−1)
0.035
0.032
0.082
0.035
Ke (min−1)
CL (%ID/g/min)
T1/2α (min)
0.098
6.785
0.086
3.234
T1/2β (min)
AUC (%ID/g/min)
36.029
162.658
33.448
186.894

Molecules 2012, 17
Figure 3. Pharmacokinetic curves in the mice for 99mTc-TMHEA and 99mTc-TMHHA.
8522
5
4
3
2
1
0
99mTc-TMHEA
99mTc-TMHHA
0
20
40
60
80
100
120
140
160
180
200
Time(min)
2.4. Biodistribution Studies
Biodistributions of 99mTc-TMHEA and 99mTc-TMHHA were determined in tumor-bearing mice, and
the data is shown in Table 3 as the percentage of administered activity (injected dose) per gram of
tissue (%ID/g). Inspecting Table 3, one can observe that 99mTc-TMHEA and 99mTc-TMHHA are
mainly accumulated in the kidney, bladder and liver, which means that the clearances of
99mTc-TMHEA and 99mTc-TMHHA are mainly through the renal pathway, and to a lesser extent,
through the hepatobiliary pathway.
At 5 min post injection, the tumor uptake was 2.51 ± 0.28 and 2.38 ± 0.41 %ID/g, the muscle
uptake was 1.93 ± 0.16 and 1.75 ± 0.21 %ID/g for 99mTc-TMHEA and 99mTc-TMHHA, respectively.
The tumor uptake value was higher than that of muscle, and the uptake ratio of tumor to muscle was
increased with time for 99mTc-TMHEA and 99mTc-TMHHA, respectively. In previous work of our
group [12,13], it was found that the uptake ratio of tumor to muscle increases with the increasing
carbon chain length between the thymidine and N2S2 ligand (i.e., from 99mTc-ANMdU to 99mTc-NHT).
However, in the present work the ratio of 99mTc-TMHHA was samller than that of 99mTc-TMHEA (see
Table 3), and the both ratios of 99mTc-TMHHA and 99mTc-TMHEA were smaller than that of
99mTc-NHT (4.41 ± 0.32, at 2 h post injection) [13]. This indicates that limitless extension of the
carbon chain is not always beneficial to improve the uptake ratio of tumor to muscle. The uptake ratio
of tumor to bone of 99mTc-TMHHA was decreased with time. The uptake ratio of tumor to blood was
increased with time for 99mTc-TMHEA and 99mTc-TMHHA, respectively.
In summary, 99mTc-TMHEA and 99mTc-TMHHA had similar biological behavior, however, the
uptake ratios of tumor to muscle, tumor to bone and tumor to blood of 99mTc-TMHEA and
99mTc-TMHHA were smaller than those of 99mTc-NHT, which suggests that limitless extension of the
carbon chain is not always beneficial to improve the uptake ratios of tumor to muscle, tumor to bone
and tumor to blood.

Molecules 2012, 17
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Table 3. Biodistribution of 99mTc-TMHEA and 99mTc-TMHHA in mice (mean ± SD, n = 5, %ID/g).
Time (min)
Tissue
5
10
15
30
60
120
180
99mTc-TMHEA
Brain
Heart
Liver
Spleen
Lung
0.27 ± 0.01
3.61 ± 0.14
5.71 ± 0.81
2.42 ± 0.03
5.59 ± 0.73
23.99 ± 0.79
2.51 ± 0.28
1.57 ± 0.58
3.82 ± 0.99
14.18 ± 0.87
1.93 ± 0.16
2.02 ± 0.14
3.86 ± 0.60
1.30 ± 0.04
1.24 ± 0.06
0.65 ± 0.03
0.24 ± 0.07
2.32 ± 0.63
4.83±0.87
0.20 ± 0.04
2.04 ± 0.11
5.22 ± 0.86
1.62 ± 0.09
4.53 ± 0.95
0.13 ± 0.07
1.01 ± 0.46
4.25 ± 0.36
1.06 ± 0.34
2.30 ± 0.66
0.04 ± 0.01
0.42±0.07
4.17 ± 0.19
0.79 ± 0.15
1.45 ± 0.20
0.02 ± 0.01
0.23 ± 0.04
2.72 ± 0.44
0.81 ± 0.30
1.50 ± 0.46
0.02 ± 0.00
0.18 ± 0.03
3.27 ± 0.18
0.79 ± 0.21
0.95 ± 0.22
9.32 ± 0.24
0.48 ± 0.07
1.17 ± 0.29
1.98 ± 0.28
1.17 ± 0.87
0.14 ± 0.05
0.33 ± 0.15
0.29 ± 0.06
2.98 ± 0.37
1.62 ± 0.18
1.80 ± 0.41
1.69 ± 0.59
4.15 ± 0.48
15.50 ± 0.42
2.00 ± 0.53
1.17 ± 0.37
3.16 ± 0.57
12.29 ± 0.52
1.31 ± 0.41
1.30 ± 0.48
2.77 ± 0.91
1.54 ± 0.08
1.59 ± 0.22
0.78 ± 0.12
Kidney
Tumor
Stomach
S. intestines
Bladder
Muscle
Bone
Blood
Tumor/Muscle
Tumor/Bone
Tumor/Blood
14.20 ± 0.50 12.45 ± 0.81 10.22 ± 0.74 8.87 ± 0.44
1.94 ± 0.31
1.35 ± 0.24
2.90 ± 0.10
10.51 ± 0.55
1.55 ± 0.56
1.57 ± 0.52
2.48 ± 0.73
1.56 ± 0.02
1.51 ± 0.03
0.93 ± 0.01
1.34 ± 0.24
0.73 ± 0.12
2.45 ± 0.36
7.09 ± 0.04
0.66 ± 0.15
0.87 ± 0.16
1.02 ± 0.25
1.98 ± 0.21
1.46 ± 0.17
1.21 ± 0.07
0.62 ± 0.12
0.47 ± 0.12
2.36 ± 0.41
4.24 ± 0.38
0.38 ± 0.06
0.63 ± 0.23
0.48 ± 0.09
1.85 ± 0.02
1.14 ± 0.12
1.27 ± 0.22
0.51 ± 0.05
1.82 ± 0.79
2.31 ± 0.03
1.56 ± 0.62
0.22 ± 0.08
0.51 ± 0.17
0.29 ± 0.05
2.84 ± 0.76
1.27 ± 0.20
1.87 ± 0.02
99mTc-TMHHA
Brain
Heart
Liver
Spleen
Lung
0.27 ± 0.02
3.01 ± 0.29
8.79 ± 0.60
2.41 ± 0.30
4.55 ± 0.77
15.38 ± 0.52
2.38 ± 0.41
1.55 ± 0.32
5.97 ± 0.79
16.05 ± 0.95
1.75 ± 0.21
2.27 ± 0.67
3.81 ± 0.47
1.36 ± 0.15
1.09 ± 0.20
0.72 ± 0.10
0.25 ± 0.03
2.44 ± 0.10
8.21 ± 0.30
2.13 ± 0.47
4.42 ± 0.04
14.14 ± 0.95
2.23 ± 0.60
1.52 ± 0.43
5.73 ± 0.01
14.47 ± 0.32
1.67 ± 0.43
2.20 ± 0.53
3.37 ± 0.14
1.35 ± 0.18
1.07 ± 0.12
0.74 ± 0.09
0.20 ± 0.04
1.79 ± 0.42
8.08 ± 0.66
1.84 ± 0.43
2.96 ± 0.43
9.46 ± 0.19
1.43 ± 0.20
0.81 ± 0.20
4.32 ± 0.42
8.66 ± 0.98
1.00 ± 0.23
1.71 ± 0.65
2.12 ± 0.52
1.34 ± 0.21
1.06 ± 0.11
0.67 ± 0.12
0.13 ± 0.04
0.11 ± 0.04
0.96 ± 0.09
5.39 ± 0.28
1.19 ± 0.20
1.76 ± 0.55
5.83 ± 0.45
0.88 ± 0.22
0.62 ± 0.18
2.06 ± 0.21
2.82 ± 0.56
0.49 ± 0.20
0.79 ± 0.14
0.83 ± 0.22
1.62 ± 0.12
0.98 ± 0.05
1.07 ± 0.05
0.03 ± 0.00
0.25 ± 0.05
4.43 ± 0.83
0.83 ± 0.17
0.86 ± 0.15
3.00 ± 0.74
0.42 ± 0.12
0.50 ± 0.20
0.89 ± 0.35
1.43 ± 0.35
0.17 ± 0.04
0.47 ± 0.14
0.31 ± 0.06
2.42 ± 0.33
0.90 ± 0.16
1.28 ± 0.22
0.03 ± 0.00
0.23 ± 0.03
4.13 ± 0.34
0.90 ± 0.13
0.75 ± 0.34
3.27 ± 0.36
0.41 ± 0.10
0.46 ± 0.08
0.85 ± 0.17
1.05 ± 0.06
0.14 ± 0.02
0.52 ± 0.05
0.27 ± 0.03
2.46 ± 0.24
0.81 ± 0.28
1.34 ± 0.08
1.10 ± 0.30
5.43 ± 0.01
1.42 ± 0.17
2.04 ± 0.48
7.65 ± 0.39
1.26 ± 0.12
0.80 ± 0.16
4.00 ± 0.19
4.95 ± 0.27
0.65 ± 0.17
0.99 ± 0.21
1.14 ± 0.31
1.60 ± 0.08
1.03 ± 0.06
0.96 ± 0.04
Kidney
Tumor
Stomach
S. intestines
Bladder
Muscle
Bone
Blood
Tumor/Muscle
Tumor/Bone
Tumor/Blood

Molecules 2012, 17
8524
2.5. Abnormal Toxicity Test
The abnormal toxicity test was evaluated by the death and 48-h survival of the mice, which were
injected with 0.2 mL 99mTc-TMHEA and 99mTc-TMHHA (3.7 MBq), respectively. Saline-injected
(of the same volume) mouse group was used as the control group. As expected, the mice showed no
signs of toxicity through the overall study period.
3. Experimental
3.1. General
All analytical chemical reagents employed were purchased from commercial sources and used
without further purification. Na99mTcO4 was supplied by Jiangsu Institute of Nuclear Medicine.
Electron spray ion (ESI) mass spectra were measured using a Waters Platform ZMD4000 LC/MS.
NMR spectra were obtained on a Bruker DRX-500 spectrometer, and the chemical shift value was
given relative to the internal tetramethylsilane (TMS). A Packard multi-prias γ Counter was used. The
animal experiments in this study were approved by the Animal Care and Ethnics Committee of Jiangsu
Institute of Nuclear Medicine.
3.2. Synthesis of 99mTc-TMHEA and 99mTc-TMHHA
99mTc-TMHEA and 99mTc-TMHHA were synthesized according to the synthetic route summarized
in Scheme 1.
3.2.1. General Procedure for the Preparation of Compounds 1a and 1b
The solution of corresponding diamine (0.5 mol) in methanol (200 mL) was cooled down to 0 °C
and t-butoxycarbonyl anhydride (t-BOC2O, 10.8 g, 50 mmol) in methanol (10 mL) was added
dropwise. The reaction mixture was stirred for 20 h at room temperature. The reaction mixture was
concentrated and diluted with water. The mixture was then extracted with CH2Cl2 (60 mL) for three
times. The organic layer was dried with anhydrous Na2SO4 and the solvent was evaporated to give
compound 1.
N-BOC-ethanediamine (1a): Yield: 67%. ESI-MS, m/z (%): 161 (100) = [M+H+]; 1H-NMR (CDCl3):
δ 3.23–2.26 (m, 2H), 2.90–2.92 (m, 2H), 2.10–2.15 (m, 1H), 1.39–1.41 (m, 9H); 13C-NMR (CDCl3): δ
(ppm) 156.0, 79.5, 43.3, 40.5, 28.5.
N-BOC-ethanediamine (1b): Yield: 85%. ESI-MS, m/z (%): 216 (100) = [M+H+]; 1H-NMR (CDCl3):
δ 2.90–3.02 (m, 2H), 2.60–2.70 (m, 2H), 1.98–2.02 (m, 2H), 1.53–1.57 (m, 4H), 1.39–1.41 (s, 9H),
1.28–1.31 (m, 4H); 13C-NMR (CDCl3): δ (ppm) 156.0, 79.5, 42.1, 41.9, 32.8, 30.0, 28.5, 26.5.

Molecules 2012, 17
8525
Scheme 1. Syntheses of 99mTc-TMHEA and 99mTc-TMHHA.
O
Br
H
N
S
S
t-BOC2O
NH2
NH2
H2N
N
K2CO3
BOC
O
O
+
n
n
O
NH
1a (n=2)
1b (n=6)
1
O
H
N
H
N
O
H
N
N
S
S
BOC
N
n
CH3I/K2CO3
N
S
S
BOC
n
O
O
O
NH
O
O
2
O
NH
O
O
N
2a (n=2)
2b (n=6)
O
N
Br
3
3a (n=2)
3b (n=6)
O
O
O
O
O
N
HCl
H2N
O
N
S
S
n
O
O
O
4
O
NH
4a (n=2)
4b (n=6)
O
N
O
O
N
O
N
O
O
N
HN
N
NH
N
N
NH
S
S
O
n
S
S
O
O
n
HO
O
O
K2CO3
O
O
O
O
O NH
O
NH
6
OH
5
O
O
O
5a (n=2)
5b (n=6)
6a (n=2)
6b (n=6)
O
N
O
O
O
HN
HN
N
NH
N
n
NH
Hg(AcO)2/anisole/TFA
O
99mTcO4-/SnCl2
O
O
N
SH
N
N
S
S
n
N
HO
HO
O
O
99mTc
O
NH SH
OH
OH
O
8, a hypothetical structure
7
8a (n=2) 99mTc-TMHEA
8b (n=6) 99mTc-TMHHA
7a (n=2) TMHEA
7b (n=6) TMHHA
3.2.2. General Procedure for the Preparation of Compounds 2a and 2b
Compound 1 (2.0 mmol), potassium carbonate (2 g, 14.49 mmol) and N-{[2-(2-(S-(4-
methoxybenzyl)sulfanyl)ethylamino)acetyl]-S-(4-methoxybenzyl)-2-aminoethylsulfanyl}-6-bromo-1-
hexanamide (1 g, 1.6 mmol) were dissolved in an acetone/DMF (1/1, v/v) mixed solvent (100 mL),
and the mixture was heated at 80 °C and stirred for 20 h under nitrogen atmosphere. Then the solvent
was evaporated under reduced pressure and the desired product (compound 2) was purified by silica
gel column chromatography using ethyl acetate/methanol = 4/1 (v/v).
N-BOC-N′-{N′′-[2-(2-(S-(4-Methoxybenzyl)sulfanyl)ethylamino)acetyl]-S-(4-methoxybenzyl)-2-
aminoethylsulfanyl-1-hexanamide}-ethanediamine (2a): Yield: 93%. ESI-MS, m/z (%): 691 (70) = [M+H]+,
591 (100) = [M+H−BOC]+, 1H-NMR (CDCl3): δ 7.18–7.24 (m, 4H), 6.81–6.88 (m, 4H), 5.02 (s, 1H),
3.85–3.89 (d, 2H, J = 12 Hz), 3.78 (s, 6H), 3.67–3.72 (d, 2H, J = 16 Hz), 3.64 (s, 2H), 3.32–3.42
(m, 4H), 3.13–3.21 (m, 2H), 2.70 (t, 2H), 2.55–2.59 (m, 4H), 2.48–2.54 (m, 2H), 2.20–2.29 (m, 2H),
1.42–1.50 (m, 16H), 1.28–1.34 (m, 2H); 13C-NMR (CDCl3): δ (ppm) 173.8, 173.6, 169.2, 168.1, 158.9,

Molecules 2012, 17
8526
158.7, 155.9, 130.1, 129.7, 114.1, 113.8, 79.2, 77.3, 76.9, 76.0, 55.0, 52.5, 50.9, 49.5, 49.2, 46.5, 41.5,
40.5, 37.8, 35.8, 35.0, 33.1, 32.7, 30.8, 29.8, 29.3, 28.8, 28.0, 26.8, 25.0.
N-BOC-N′-{N′′-[2-(2-(S-(4-Methoxybenzyl)sulfanyl)ethylamino)acetyl]-S-(4-methoxybenzyl)-2-
aminoethylsulfanyl-1-hexanamide}-hexamethylene diamine (2b): Yield: 84%. ESI-MS, m/z (%):
1
747 (100) = [M+H+], H-NMR (CDCl3): δ 7.17–7.22 (m, 4H), 6.80–6.88 (m, 4H), 6.65–6.70 (s, 1H),
4.50 (s, 1H), 3.82–3.90 (d, 2H, J = 12 Hz), 3.78 (s, 6H), 3.72(s, 2H), 3.62–3.69 (d, 2H, J = 8 Hz),
3.50–3.55 (m, 1H), 3.32–3.46 (m, 4H), 3.08–3.12 (d, 2H, J = 8 Hz), 2.55–2.62 (m, 6H), 2.48–2.52 (m,
2H), 2.20–2.30 (m, 2H), 1.55–1.68 (m, 4H), 1.39–1.50 (m, 13H), 1.26–1.36 (s, 6H); 13C-NMR
(CDCl3): δ (ppm) 173.8, 169.2, 168.8, 164.2, 158.9, 158.7, 156.1, 129.2, 128.8, 113.8, 113.6, 77.1,
76.8, 55.2, 52.8, 51.5, 50.5, 50.1, 49.5, 46.8, 40.5, 37.8, 37.6, 36.1, 35.1, 34.9, 33.0, 32.7, 30.8, 30.0,
29.8, 29.8, 28.8, 27.1, 26.9, 26.8, 25.0, 24.8.
3.2.3. General Procedure for the Preparation of Compounds 3a and 3b
A mixture of compound 2 (1.5 mmol) and potassium carbonate in acetone (30 mL), and
iodomethane (95 μL, 1.5 mmol) in acetone (20 mL) was added. The reaction mixture was stirred for 2 h,
then the solvent was evaporated under reduced pressure. The desired product (compound 3) was
purified by silica gel column chromatography using ethyl acetate/methanol/triethylamine = 19/2/1 (v/v).
N-BOC-N′-Methyl-N′-{N′′-[2-(2-(S-(4-methoxybenzyl)sulfanyl)ethylamino)acetyl]-S-(4methoxybenzyl)-
2-aminoethylsulfanyl-1-hexanamide}-ethanediamine (3a): Yield: 86%. ESI-MS, m/z (%):
1
705 (100) = [M+H+], H-NMR (CDCl3): δ 7.19–7.25 (m, 4H), 6.82–6.88 (m, 4H), 3.87–3.92 (d, 2H,
J = 16 Hz), 3.78 (s, 6H), 3.67–3.73 (d, 2H, J = 16 Hz), 3.65 (s, 2H), 3.48–3.52 (m, 1H), 3.32–3.45 (m,
4H), 3.12–3.21 (m, 2H), 2.57–2.61 (m, 2H), 2.48–2.54 (m, 2H), 2.40–2.45 (m, 2H), 2.28–2.34 (m,
2H), 2.22–2.28 (m, 2H), 2.18 (s, 3H), 1.51–1.52 (m, 5H), 1.49 (s, 11H), 1.47–1.48 (m, 2H); 13C-NMR
(CDCl3): δ (ppm) 173.7, 173.5, 169.2, 168.1, 158.9, 158.7, 156.0, 130.2, 129.4, 114.1, 113.8, 78.9,
77.4, 76.9, 76.1, 57.3, 56.5, 55.0, 52.1, 50.7, 49.1, 47.1, 41.8, 37.8, 36.1, 35.3, 35.1, 33.1, 32.7, 30.9,
29.2, 28.8, 28.0, 26.9, 25.0, 24.8.
N-BOC-N′-Methyl-N′-{N′′-[2-(2-(S-(4-methoxybenzyl)sulfanyl)ethylamino)acetyl]-S-(4-methoxybenzyl)-2-
aminoethylsulfanyl-1-hexanamide}-hexamethylenediamine (3b): Yield: 82%. ESI-MS, m/z (%):
1
761 (100) = [M+H+], H-NMR (CDCl3): δ 7.19–7.24 (m, 4H), 6.82–6.88 (m, 4H), 3.87–3.91 (d, 2H,
J = 12 Hz), 3.78 (s, 6H), 37.0–37.3 (s, 2H), 3.64–3.69 (d, 2H, J = 8 Hz), 3.51–3.56 (m, 1H), 3.32–3.45
(m, 4H), 3.03–3.13 (d, 2H, J = 8 Hz), 2.54–2.62 (m, 2H), 2.48–2.53 (m, 2H), 2.22–2.34 (m, 6H), 2.18
(s, 3H), 1.58–1.68 (m, 4H), 1.42–1.50 (s, 13H), 1.34–1.38 (m, 6H); 13C-NMR (CDCl3): δ (ppm) 174.0,
169.2, 168.1, 159.0, 158.8, 156.0, 130.0, 129.4, 114.0, 113.8, 78.9, 77.4, 76.9, 76.1, 57.6, 57.4, 55.5,
52.2, 51.1, 49.6, 46.9, 41.8,40.5, 37.8, 37.6, 36.0, 35.3, 35.1, 33.1, 32.7, 31.0, 29.9, 29.5, 29.2, 28.7,
28.1, 27.0, 26.3, 25.1, 25.0.
3.2.4. General Procedure for the Preparation of Compounds 4a and 4b
The solution of compound 3 (1.3 mmol) in methanol (50 mL), and concentrated hydrochloric acid
(5 mL) was added. The reaction mixture was stirred for 4 h at 45 °C, then the solvent was evaporated

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under reduced pressure. The residue was dissolved in water (30 mL) and the mixture was adjusted to
pH 10 by aqueous sodium hydroxide solution (2 mol/L). The mixture was then extracted with ethyl
acetate (30 mL) for three times. The organic layer was dried with anhydrous Na2SO4 and the solvent
was evaporated under reduced pressure. The desired product (compound 4) was purified by silica gel
column chromatography using ethyl acetate/methanol/triethylamine = 8/8/1 (v/v).
N-Methyl-N-{N′-[2-(2-(S-(4-methoxybenzyl)sulfanyl)ethylamino)acetyl]-S-(4-methoxybenzyl)-2-
aminoethylsulfany-1-hexanamide}-ethanediamine (4a): Yield: 84%. ESI-MS, m/z (%):
1
605 (100) = [M+H+], H-NMR (CDCl3): δ 7.17–7.23 (m, 4H), 6.78–6.85 (m, 4H), 5.28 (s, 1H),
3.87–3.92 (d, 2H, J = 16 Hz), 3.78 (s, 6H), 3.62–3.70 (m, 4H), 3.51–3.57 (m, 1H), 3.31–3.45 (m, 4H),
2.71–2.80 (m, 3H), 2.50–2.61 (m, 4H), 2.38–2.42 (m, 2H), 2.30–2.38 (m, 2H), 2.20–2.28 (m, 2H),
13
2.15–2.18 (d, 3H, J = 4 Hz), 1.57–1.63 (m, 2H), 1.40–1.49 (m, 2H), 1.25–1.32 (m, 2H); C-NMR
(CDCl3): δ (ppm) 173.1, 168.1, 158.9, 158.7, 129.5, 113.1, 77.1, 76.9, 76.0, 56.0, 55.0, 53.5, 50.5,
49.2, 46.7, 41.5, 41.0, 37.8, 37.0, 35.5, 34.8, 34.2, 32.0, 30.5, 29.0, 28.8, 26.7, 26.2, 23.8, 23.5.
N-Methyl-N-{N′-[2-(2-(S-(4-methoxybenzyl)sulfanyl)ethylamino)acetyl]-S-(4-methoxybenzyl)-2-
aminoethylsulfanyl-1-hexanamide}-hexamethylenediamine (4b): Yield: 89%. ESI-MS, m/z (%):
1
661 (100) = [M+H+], H-NMR (CDCl3): δ 7.19–7.25 (m, 4H), 6.81–6.88 (m, 4H), 3.87–3.92 (d, 2H,
J = 16 Hz), 3.78 (s, 6H), 3.64–3.72 (m, 4H), 3.51–3.57 (m, 1H), 3.29–3.45 (m, 4H), 2.88–2.94 (s, 3H),
2.68–2.72 (m, 2H), 2.48–2.60 (m, 4H), 2.33–2.38 (m, 4H), 2.24–2.30 (m, 2H), 2.22 (s, 3H), 1.94
(s, 1H), 1.58–1.66 (m, 2H), 1.42–1.52 (m, 4H), 1.28–1.38 (m, 6H); 13C-NMR (CDCl3): δ (ppm) 178.1,
173.1, 169.1, 168.2, 158.9, 158.7, 129.6, 113.8, 77.5, 76.9, 76.0, 57.2, 57.0, 55.2, 53.3, 52.2, 50.8,
49.8, 46.7, 41.8, 41.2, 37.8, 35.8, 35.3, 35.1, 32.8, 32.5, 31.5, 30.5, 29.5, 28.8, 27.0, 26.2, 25.0, 24.0.
3.2.5. General Procedure for the preparation of compounds 5a and 5b
The solution of compound 4 (0.96 mmol) and triethylamine (2 mL) in CH2Cl2 (80 mL) was cooled
down to 0 °C and 3′,5′-diacetyl-5-bromo-N-BOC-thymidine (0.44 g, 0.86 mmol) in CH2Cl2 (20 mL)
was added. The reaction mixture was stirred for 4 h at room temperature. The mixture was washed
with aqueous NaHCO3 (75 mL × 2) and H2O (50 mL), respectively. The organic layer was dried
with anhydrous Na2SO4 and the solvent was evaporated. The residue was purified by column
chromatography using ethyl acetate/ methanol/triethylamine (19/1/1, v/v) to give compound 5.
N-(3′,5′-Diacetyl-N′-BOC-thymidinyl)-N′′-methyl-N′′-{N′′′-[2-(2-(S-(4-methoxybenzyl)sulfanyl)ethyl amino)
acetyl]-S-(4-methoxybenzyl)-2-aminoethylsulfanyl-1-hexanamide}-ethanediamine (5a): Yield: 51%.
1
ESI-MS, m/z (%): 1029 (100) = [M+H+], H-NMR (CDCl3): δ 7.55 (s, 1H), 7.17–7.23 (m, 4H),
6.81–6.87 (m, 4H), 6.28 (m, 1H), 5.19 (m, 1H), 4.28–4.40 (m, 2H), 4.21 (s, 1H), 3.85–3.91 (d, 2H,
J = 16 Hz), 3.78 (s, 6H), 3.61–3.71 (m, 4H), 3.55 (s, 2H), 3.30–3.50 (m, 4H), 2.65–2.70 (s, 2H),
2.55–2.60 (m, 2H), 2.45–2.51 (m, 2H), 2.30–2.35 (m, 2H), 2.20–2.30 (m, 4H), 2.18 (s, 3H), 2.02–2.10
(d, 3H, J = 16 Hz), 1.72–1.88 (m, 6H), 1.60 (s, 9H), 1.38–1.47 (m, 4H), 1.25–1.30 (m, 2H); 13C-NMR
(CDCl3): δ (ppm) 171.4, 170.8, 170.3, 170.2, 170.1, 162.3, 159.1, 154.3, 149.7, 136.5, 130.0, 129.8,
129.8, 129.7, 129.3, 114.3, 114.2, 114.1, 109.9, 84.9, 83.3, 78.8, 74.4, 62.2, 55.9, 52.9, 49.7, 48.8,
46.6, 43.4, 39.9, 39.1, 38.7, 36.8, 33.6, 31.7, 28.9, 28.7, 28.3, 27.4, 26.4, 25.7, 21.0, 20.7.

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N-(3′,5′-Diacetyl-N′-BOC-thymidinyl)-N′′-methyl-N′′-{N′′′-[2-(2-(S-(4-methoxybenzyl)sulfanyl)ethyl amino)
acetyl]-S-(4-methoxybenzyl)-2-aminoethylsulfanyl-1-hexanamide}-hexamethylenediamine (5b): Yield:
1
48%. ESI-MS, m/z (%): 1085 (100) = [M+H+], H-NMR (CDCl3): δ 7.54 (s, 1H), 7.18–7.24 (m, 4H),
6.80–6.87 (m, 4H), 6.29 (m, 1H), 5.20 (m, 1H), 4.27–4.38 (m, 2H), 4.20 (s, 1H), 3.83–3.90 (d, 2H,
J = 16 Hz), 3.78 (s, 6H), 3.60–3.71 (m, 4H), 3.55 (s, 2H), 3.30–3.50 (m, 4H), 2.65–2.70 (s, 2H),
2.55–2.60 (m, 2H), 2.45–2.51 (m, 2H), 2.30–2.35 (m, 2H), 2.20–2.30 (m, 4H), 2.18 (s, 3H), 2.02–2.10
(d, 3H, J = 16 Hz), 1.72–1.88 (m, 6H), 1.60 (s, 9H), 1.39–1.48 (m, 8H), 1.26–1.30 (m, 6H); 13C-NMR
(CDCl3): δ (ppm) 171.4, 170.8, 170.3, 170.2, 170.1, 162.3, 159.1, 154.3, 149.7, 136.5, 130.0, 129.8,
129.8, 129.7, 129.3, 114.3, 114.2, 114.1, 109.9, 84.9, 83.3, 78.8, 74.4, 62.2, 56.9, 55.9, 52.9, 49.7,
49.6, 48.8, 46.6, 43.4, 39.9, 39.1, 38.7, 36.8, 33.6, 31.7, 30.7, 28.9, 28.7, 28.3, 28.1, 27.4, 27.1, 26.8,
26.5, 25.5, 20.8, 20.5.
3.2.6. General Procedure for the Preparation of Compounds 6a and 6b
The mixture of compound 5 (0.45 mmol) and potassium carbonate (1 g, 7.25 mmol) in methanol
(50 mL) was heated to reflux for 4 h. Then the solvent was evaporated under reduced pressure and the
residue was dissolved in chloroform (50 mL). The organic layer was washed with H2O (50 mL) and
dried with anhydrous Na2SO4. The compound 6 was purified by silica gel column chromatography
using chloroform /methanol/triethylamine (5/2/0.1, v/v).
N-Thymidinyl-N′-methyl-N′-{N′′-[2-(2-(S-(4-methoxybenzyl)sulfanyl)ethylamino)acetyl]-S-(4-methoxy-
benzyl)-2-aminoethylsulfanyl-1-hexanamide}-ethanediamine (6a): Yield: 68%. ESI-MS, m/z (%):
1
845 (100) = [M+H+], H-NMR (CDCl3): δ 8.05 (d, 1H, J = 4 Hz), 7.18–7.25 (m, 4H), 6.80–6.87 (m,
4H), 6.19–6.24 (m, 1H), 4.45 (s, 1H), 4.00–4.20 (s, 5H), 3.90–4.00 (d, 2H, J = 12 Hz), 3.82–3.88 (d,
2H, J = 8 Hz), 3.78 (s, 6H), 3.60–3.70 (m, 4H), 3.28–3.50 (m, 6H), 2.65–2.72 (d, 2H, J = 8 Hz),
2.40–2.60 (m, 5H), 2.20–2.35 (m, 5H), 2.15 (s, 6H), 1.51–1.60 (m, 2H), 1.40–1.49 (m, 2H), 1.21–1.32
13
(m, 2H); C-NMR (CDCl3): δ (ppm) 207.1, 174.6, 174.4, 169.0, 168.5, 164.0, 158.3, 150.8, 138.2,
130.3, 114.0, 111.8, 87.1, 85.5, 77.0, 76.8, 76.5, 69.8, 61.2, 57.9, 56.8, 55.5, 52.9, 51.0, 49.5, 47.0,
46.0, 45.1, 42.0, 40.7, 37.9, 35.9, 35.0, 33.1, 32.8, 31.0, 29.3, 28.5, 27.0, 26.5, 24.5.
N-Thymidinyl-N′-methyl-N′-{N′′-[2-(2-(S-(4-methoxybenzyl)sulfanyl)ethylamino)acetyl]-S-(4-methoxy-
benzyl)-2-aminoethylsulfanyl-1-hexanamide}-hexamethylenediamine (6b): Yield: 67%. ESI-MS,
1
m/z (%): 901 (100) = [M+H+], H-NMR (CDCl3): δ 8.42 (s, 1H), 7.19–7.24 (d, 4H, J = 8 Hz),
6.79–6.86 (m, 4H), 5.51 (s, 2H), 4.45 (s, 1H), 3.90–4.02 (m, 4H), 3.78 (s, 6H), 3.65–3.72 (m, 4H),
3.50–3.61 (m, 7H), 3.40–3.45 (m, 2H), 3.32–3.37 (m, 1H), 2.95–3.07 (m, 11H), 2.75 (s, 2H),
2.51–2.60 (m, 3H), 2.25–2.38 (m, 2H), 1.58–1.62 (m, 2H), 1.32–1.48 (m, 12H); 13C-NMR (CDCl3): δ
(ppm) 205.1, 173.6, 173.4, 168.8, 168.3, 163.8, 158.1 150.6, 138.1, 130.1, 113.9, 111.6, 87.1, 85.3,
77.2, 76.9, 76.7, 57.0, 56.8, 55.2, 53.3, 52.2, 50.8, 49.8, 46.5, 41.8, 41.2, 37.8, 35.8, 35.3, 35.0, 32.8,
32.5, 31.5, 30.5, 29.5, 28.8, 26.8, 26.2, 24.8, 23.9.

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3.2.7. General Procedure for the Preparation of Compounds 7a and 7b
Compound 6 (0.17 mmol) was dissolved in trifluoroacetic acid (5 mL) and cooled in ice bath to
0 °C. Anisole (0.26 mL) and Hg(AcO)2 (0.17 g, 0.53 mmol) were added. The reaction mixture was
stirred for 30 min at room temperature and then concentrated in vacuum to obtain viscous brown oil
that was dried in vacuum for 30 min. Dry diethyl ether (15 mL) was added to the oil and the resultant
suspension stirred for about 10min. The ether was decanted and the precipitate washed again with
another 15 mL of ether. The colorless solid was collected by suction filtration, dried in vacuum and
dissolved again in absolute ethanol (10 mL). H2S gas was passed through the solution for 20 min. The
reaction mixture was filtered and the filtrate concentrated under vacuum to afford compound 7 as a
colorless oil.
N-Thymidinyl-N′-methyl-N′-{N′′-[2-sulfanylethylamino)acetyl]-2-aminoethylsulfanyl-1-hexanamide}-
ethanediamine (7a,TMHEA): Yield: 70%. ESI-MS, m/z (%): 605 (100) = [M+H+], 1H-NMR (D2O):
δ 8.18 (s, 1H), 6.24 (m, 1H), 4.49 (m, 1H), 4.07 (m, 1H), 3.98 (d, 2H, J = 12 Hz), 3.82–3.88 (m, 1H),
3.72–3.79 (m, 2H), 3.61–3.67 (m, 2H), 3.45–3.52 (m, 2H), 3.29–3.36 (m, 2H), 3.15–3.23 (m, 6H),
3.03–3.10 (m, 7H), 2.82 (m, 3H), 2.70–2.72 (m, 2H), 2.32–2.48 (m, 3H), 1.60–1.82 (m, 4H), 1.38–1.42
13
(m, 2H), 1.13–1.15 (t, 1H), 1.06–1.11 (t, 1H); C-NMR (D2O): δ (ppm) 164.8, 151.8, 145.0, 143.5,
104.8, 104.0, 87.0, 85.9, 70.4, 62.8, 61.1, 57.7, 55.9, 47.0, 43.9, 39.9, 39.1, 33.8, 25.7, 25.5, 24.4, 23.5,
23.1, 16.8, 10.0.
N-Thymidinyl-N′-methyl-N′-{N′′-[2-sulfanylethylamino)acetyl]-2-aminoethylsulfanyl-1-hexanamide}-
1
hexamethylenediamine (7b,TMHHA): Yield: 75%. ESI-MS, m/z (%): 661 = [M+H+], H-NMR
(D2O): δ 8.20 (s, 1H), 6.22–6.35 (m, 1H), 4.49 (m, 1H), 4.03–4.18 (m, 4H), 3.78–3.92 (m, 2H),
3.45–3.67 (m, 2H), 3.10–3.38 (m, 6H), 2.93–3.02 (s, 6H), 2.70–2.90 (m, 5H), 2.32–2.48 (m, 3H), 2.22
(s, 3H), 1.95–2.05 (m, 2H), 1.61–1.85 (m, 10H), 1.38–1.45 (m, 4H), 1.23–1.25 (t, 1H), 1.16–1.21
(t, 1H); 13C-NMR (D2O): δ (ppm) 165.9, 164.5, 151.0, 144.1, 104.6, 87.0, 86.5, 70.3, 62.5, 61.0, 56.8,
51.0, 48.0, 47.0, 44.7, 43.0, 41.1, 40.1, 39.2, 37.0, 33.9, 33.2, 30.0, 29.2, 25.0, 24.1, 23.5, 23.0, 22.0.
3.3. Radiochemical Syntheses of 99mTc-TMHEA (8a) and 99mTc-TMHHA (8b)
A solution of compound 7 (50 μL, 2 mg of compound 7 dissolved in 2 mL ethanol) was added to a
mixture of sodium glucoheptonate (0.8 mL, 10 mg/mL), freshly prepared solution of stannous chloride
dehydrate (20 μL, 1.0 mg SnCl2.2H2O dissolved in 1 mL 0.1 mol/L hydrochloric acid solution), and
pertechnetate eluate (50 μL, 37 MBq). The reaction mixture was vortexed adequately and reacted at
100 °C for 30 min.
3.4. Quality Control of 99mTc-TMHEA and 99mTc-TMHHA
The radiochemical purity (RCP) and radiolabeling yield (RLY) of 99mTc-TMHEA and
99mTc-TMHHA was determined by TLC and HPLC.

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3.4.1. TLC
About 3 μL 99mTc-TMHEA and 99mTc-TMHHA solutions were spotted with a glass capillary
at 1.2 cm from the bottom of polyamide layer strips. The polyamide layer strips were eluted by
ascending chromatography with toluene/acetonitrle/methanol (3/1/1, v/v). The 99mTc-colloidal
impurities remain at the bottom on polyamide layer strip, while 8a and 8b both migrate with the
solvent front. The strips were cut into pieces of 1 cm and the activity of these pieces was counted to
determine the RCP value on a well-type γ counter.
3.4.2. HPLC
The RCP of 99mTc-TMHEA and 99mTc-TMHHA were determined by HPLC using a Waters
600-type instrument. The sample was carefully passed through a Millipore filter and injected into the
HPLC column (SunFireTM C18, PN: 186002559, 4.6 mm × 150 mm × 5 μm, Waters, Milford, MA,
USA). Radioanalysis of the labeled compound was conducted using a Cd (Te) detector. The flow rate
was adjusted to 1.0 mL/min and the isocratic mobile phase was 68% water and 32% methanol.
3.5. In Vitro Stability of 99mTc-TMHEA and 99mTc-TMHHA
The in vitro stabilities of 99mTc-TMHEA and 99mTc-TMHHA were studied in PBS (pH = 7.4) after
different interval (1, 2, 3, 4, 5 and 6 h) at physiological temperature of 37 °C. The RCP values were
evaluated by HPLC at different time points to determine whether they were stable in vitro.
3.6. Octanol-Water Partition Coefficients of 99mTc-TMHEA and 99mTc-TMHHA
The partition coefficients (logP) of 99mTc-TMHEA and 99mTc-TMHHA were determined in
n-octanol and two kinds of phosphate buffered saline (PBS, pH 7.0 and pH 7.4, respectively). For each
pH, a sample of radiolabeled compound 99mTc-TMHEA and 99mTc-TMHHA (20 μL, 0.74 MBq) was
added to the two-phase system of 3.0 mL n-octanol and 3.0 mL PBS, respectively. The mixture was
vortexed for 1 min × 3 and centrifuged for 5 min at 4,000 r/min to ensure complete separation of
layers, and then 1.0 mL n-octanol and 1.0 mL PBS were taken out and counted with a γ-counter.
Afterwards, 1.0 mL n-octanol was transferred to another tube containing 3.0 mL PBS and 2.0 mL
n-octanol. The above procedure was repeated for six times. LogP values were calculated using the
formula of logP = log[counts(n-octanol)/counts(PBS)]．
3.7. Tumor Models
The mouse hepatoma HepA ascites tumor cells were maintained in ICR mice by weekly intraperi-
toneal transplantation into fresh ICR mice and were collected for transplantation under sterile
conditions. Tumor xenografts were established in 5- to 7-week-old ICR mice (18–20 g) by injection of
approximately 2 × 106 HepA cells in the right shoulder area. When the tumors were about 0.8 cm in
diameter (about 7 days), the mice were used for biodistribution as described below.

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3.8. Biodistribution in Tumor-Bearing Mice of 99mTc-TMHEA and 99mTc-TMHHA
Thirty-five tumor-bearing mice (18 male and 17 female) were randomly divided into seven groups
and injected via the tail vein with the test agent (99mTc-TMHEA and 99mTc-TMHHA) in a volume of
0.2 mL and activity of approximately 3.7 MBq. Groups of mice were sacrificed by decapitation at 5,
10, 15, 30, 60, 120 and 180 min after injection. The organs of interest (heart, muscle, lung, kidney,
spleen, liver and tumor etc.) were dissected and weighed, as well as 100 μL blood were taken from
carotid artery. The activity for each sample was determined by a γ counter. Distribution of the
radioactivity in different tissues and organs was calculated and expressed as percentage of injection
dose per gram (%ID/g).
4. Conclusions
TMHEA and TMHHA, two kinds of novel thymidine derivative, have been prepared and
successfully labeled with 99mTc in a high labeling yield and good in vitro stability. 99mTc-TMHEA and
99mTc-TMHHA had similar biological behavior, however, the uptake ratios of tumor to muscle, tumor
to bone and tumor to blood of 99mTc-TMHEA and 99mTc-TMHHA were smaller than those of
99mTc-NHT, which means limitless extension of the carbon chain is not always beneficial to improve
the uptake ratios.
Acknowledgments
The authors are very grateful to Ministry of Health Foundation of China (W201207) and the Natural
Science Foundation of Jiangsu Province (BK2008112) for their financial support.
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