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

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.


Introduction
In clinical oncology, 2′-deoxy-2′-[ 18 F]fluoro-D-glucose ( 18 F-FDG), a glucose derivative, has been widely used in recent years for tumor imaging with positron emission tomography (PET). However, 18 F-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 18 F-FDG, many studies have focused on the development of a variety of DNA precursors [1,[3][4][5].
Specifically, labeled thymidine analogs can target the proliferative activity of malignant lesions [6,7], and several useful ligands, such as 11 C-labeled nucleoside thymidine [1], 3′-deoxy-3′-[ 18 F]fluoro OPEN ACCESS thymidine ( 18 F-FLT) [1,[3][4][5] and its analog 18 F-FMAU [8] have demonstrated their good imaging features. However, these tracers were labeled with either 11 C or 18 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 ( 99m Tc), the most commonly used radioisotope in SPECT, is continuously available at a reasonable cost in many hospitals and has ideal nuclear properties for imaging (T 1/2 = 6.02 h, γ = 140 keV). Therefore it is important to develop a 99m Tc labeled thymidine analog so as to provide the ideal characteristics needed for routine clinical studies [9][10][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 99m Tc-NHT was higher than that of 99m Tc-ANMdU, which means that uptake ratio of tumor to muscle maybe increase with increasing carbon chain length between the thymidine and N 2 S 2 ligand. However, to the best of our knowledge, extension and optimization of the linker chain between the thymidine and N 2 S 2 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 N 2 S 2 ligand. In this work, two novel 99m Tc-labeled thymidine derivatives were prepared and reported, i.e., 99m Tc-TMHEA and 99m Tc-TMHHA ( Figure 1). Their in vitro stability and in vivo biodistribution were also studied.

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, 1 H-NMR and 13 C-NMR, and the results agreed well with the expected chemical structures. 99m Tc-TMHEA and 99m Tc-TMHHA were labeled with Na 99m TcO 4 by reduction with stannous chloride. For TLC analysis, with the toluene/acetonitrile/methanol (v/v/v = 3/1/1) system, the R f values of 99m Tc-TMHEA and 99m Tc-TMHHA were about 0.7-0.8, while 99m Tc-colloidal impurities remain at 0-0.1. HPLC analysis revealed the formation of free technetium (Na 99m TcO 4 ) that was eluted at a retention time of 9.9 min, whereas 99m Tc-TMHEA and 99m Tc-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 HPLC-chromatogram clearly shows the formation of only one complex and excludes the possibility of residual Na 99m TcO 4 or other components. That is, the chelation of technetium with the N 2 S 2 is unique and complete. Figure 2. HPLC chromatograms ( 99m Tc-TMHEA t R = 13.3 min, 99m Tc-TMHHA t R = 12.8 min and 99m TcO 4 − t R = 9.9 min).
According to the TLC and HPLC analysis, the radiochemical purities of 99m Tc-TMHEA and   99m Tc-TMHHA were all greater than 95%. The radiolabeled compounds were used immediately after the formulation for both in vitro and in vivo studies.

In Vitro Stability and Octanol-Water Partition Coefficient
The in vitro stabilities of 99m Tc-TMHEA and 99m Tc-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 99m Tc-TMHEA and 99m Tc-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 99m Tc-TMHEA and 99m Tc-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].

Blood Kinetics Studies
Pharmacokinetic parameters are listed in Table 2. Figure 3 shows the blood clearance of Tc-TMHEA and 99m Tc-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 99m Tc-TMHEA and 99m Tc-TMHHA, respectively. In the early phase, the blood clearance of 99m Tc-TMHHA was slower than 99m Tc-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.

Biodistribution Studies
Biodistributions of 99m Tc-TMHEA and 99m Tc-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 99m Tc-TMHEA and 99m Tc-TMHHA are mainly accumulated in the kidney, bladder and liver, which means that the clearances of 99m Tc-TMHEA and 99m Tc-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 99m Tc-TMHEA and 99m Tc-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 99m Tc-TMHEA and 99m Tc-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 N 2 S 2 ligand (i.e., from 99m Tc-ANMdU to 99m Tc-NHT). However, in the present work the ratio of 99m Tc-TMHHA was samller than that of 99m Tc-TMHEA (see Table 3), and the both ratios of 99m Tc-TMHHA and 99m Tc-TMHEA were smaller than that of 99m Tc-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 99m Tc-TMHHA was decreased with time. The uptake ratio of tumor to blood was increased with time for 99m Tc-TMHEA and 99m Tc-TMHHA, respectively.
In summary, 99m Tc-TMHEA and 99m Tc-TMHHA had similar biological behavior, however, the uptake ratios of tumor to muscle, tumor to bone and tumor to blood of 99m Tc-TMHEA and 99m Tc-TMHHA were smaller than those of 99m Tc-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. Table 3. Biodistribution of 99m Tc-TMHEA and 99m Tc-TMHHA in mice (mean ± SD, n = 5, %ID/g).

Tissue
Time (

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 99m Tc-TMHEA and 99m Tc-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.

General
All analytical chemical reagents employed were purchased from commercial sources and used without further purification. Na 99m TcO 4 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. Tc-TMHEA and 99m Tc-TMHHA   99m Tc-TMHEA and 99m Tc-TMHHA were synthesized according to the synthetic route summarized in Scheme 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-BOC 2 O, 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 CH 2 Cl 2 (60 mL) for three times. The organic layer was dried with anhydrous Na 2 SO 4 and the solvent was evaporated to give compound 1.

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).

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 H 2 O (50 mL) and dried with anhydrous Na 2 SO 4 . The compound 6 was purified by silica gel column chromatography using chloroform /methanol/triethylamine (5/2/0.1, v/v).

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). H 2 S 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. 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 SnCl 2 .2H 2 O 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.

TLC
About 3 μL 99m Tc-TMHEA and 99m Tc-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 99m Tc-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.

HPLC
The RCP of 99m Tc-TMHEA and 99m Tc-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 (SunFire TM 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.

In Vitro Stability of 99m Tc-TMHEA and 99m Tc-TMHHA
The in vitro stabilities of 99m Tc-TMHEA and 99m Tc-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.

Octanol-Water Partition Coefficients of 99m Tc-TMHEA and 99m Tc-TMHHA
The partition coefficients (logP) of 99m Tc-TMHEA and 99m Tc-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 99m Tc-TMHEA and 99m Tc-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)]．

Tumor Models
The mouse hepatoma HepA ascites tumor cells were maintained in ICR mice by weekly intraperitoneal 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 × 10 6 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.

Biodistribution in Tumor-Bearing Mice of 99m Tc-TMHEA and 99m Tc-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 ( 99m Tc-TMHEA and 99m Tc-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).

Conclusions
TMHEA and TMHHA, two kinds of novel thymidine derivative, have been prepared and successfully labeled with 99m Tc in a high labeling yield and good in vitro stability. 99m Tc-TMHEA and 99m Tc-TMHHA had similar biological behavior, however, the uptake ratios of tumor to muscle, tumor to bone and tumor to blood of 99m Tc-TMHEA and 99m Tc-TMHHA were smaller than those of 99m Tc-NHT, which means limitless extension of the carbon chain is not always beneficial to improve the uptake ratios.