Synthesis and Biological Evaluation of Novel 99mTc(CO)3-Labeled Thymidine Analogs as Potential Probes for Tumor Proliferation Imaging

Achieving a 99mTc labeled thymidine radiotracer for single photon emission tomography (SPECT) is considered to be of interest. In this study, four novel thymidine analogs, 6a, 6b, 6c and 6d, were successfully synthesized via “click reaction” route and then radiolabeled using a [99mTc(CO)3]+ core to prepare the corresponding 99mTc(CO)3 complexes in high yields. These complexes were hydrophilic and had good in vitro stability. Biodistribution of these complexes in mice bearing S180 tumors showed that all of them exhibited accumulation in the tumors, suggesting that they would be potential tumor imaging agents.


Introduction
The sugar-based PET (Positron Emission Tomography) radiotracer 2-[ 18 F]fluoro-2-deoxy-D-glucose ( 18 F-FDG), the gold standard tracer for tumor detection and staging clinically, still has some limitations, like producing false-positive or negative results, low image contrast in brain tumor diagnosis and poor differentiation of tumor from inflammation [1,2].Continued proliferation is one of the hallmarks of cancer [3], and DNA synthesis is the most direct metabolic process relating to the cell proliferation [4].So many investigators have developed various radiolabeled DNA precursors, especially labeled thymidine and thymidine analogs.These radiolabeled tracers with positron emitters, such as 11 C-labeled nucleoside thymidine [5], 3 1 -deoxy-3 1 -[ 18 F]fluorothymidine ( 18 F-FLT) [6][7][8][9][10], 2 1 -fluoro-5-[ 11 C]-methyl-1-beta-D-arabinofuranosyluracil ( 11 C-FMAU) [11] and 2 1 -[ 18 F]fluoro-5-methyl-1-beta-D-arabinofuranosyluracil ( 18 F-FMAU) [12][13][14][15], are all substrates of the human thymidine kinase 1 (TK1). 18F-FLT, in particular, is an effective tracer of tumor proliferation, and is a specific and clinically relevant prognostic predictor in the treatment of cancer [16].However, an expensive cyclotron is essential in the production of these radionuclides, restricting their wide use in clinical practice.By comparison, 99m Tc can be obtained at a reasonable cost, which makes it readily available and affordable.The availability of a generator and kit chemistry to prepare 99m Tc based radiotracers may have a significant impact on nuclear medicine.Thus, using 99m Tc to label thymidine analogs is the focus of the ongoing research.To date, several 99m Tc labeled thymidine analogs have been reported [17][18][19][20][21][22][23].However, none of them showed the ideal properties.The preparation of novel 99m Tc labeled thymidine analogs is still considered to be of great necessity and a considerable challenge.
Recently, the [ 99m Tc(CO) 3 ] + complex has attracted significant attention due to its ease of preparation, readily substituted water molecules of the precursor fac-[ 99m Tc(CO) 3
In this study, four novel thymidine analogs, 6a, 6b, 6c and 6d, were successfully synthesized via "click chemistry" route and then radiolabeled using [ 99m Tc(CO) 3 ] + core to prepare the corresponding 99m Tc(CO) 3 complexes.The partition coefficient, stability in vitro, cell uptake, and biodistribution in mice were also evaluated.

Chemistry and Radiolabeling
Compounds 6a, 6b, 6c and 6d were synthesized by multi-step reactions from the starting materials thymidine via "click" reaction.The reaction equations were shown in Scheme 1.They were identified by 1 H-NMR, 13 C-NMR, ESI-MS (Electrospray Ionization Mass Spectrometry), HRMS (High Resolution Mass Spectrometry) and the results agreed well with the expected chemical structures.The final products were suitable tridentate precursors for the [ 99m Tc(CO) 3 ] + core (Scheme 2).Compounds 7a, 7b, 7c and 7d were obtained with high radiochemical purity (Figure 1).with technetium and rhenium tri-carbonyl as the substrates of TK1 [25,26].Among different tridentate ligands, a macrocyclic triamine compound, such as 1,5,9-triazacyclododecane (12N3), is a suitable ligand for radiolabeling with the [ 99m Tc(CO)3] + core [27].The copper-(I)-catalyzed "click chemistry" by reacting azide with terminal alkyne to form 1,2,3-triazoles (for the trizole's stability in biocircumstances) is used in the area of bioconjugation reactions.It has played a vital role in the synthesis of compounds, as well as in the field of radiopharmaceuticals and molecular imaging [28][29][30].
In this study, four novel thymidine analogs, 6a, 6b, 6c and 6d, were successfully synthesized via "click chemistry" route and then radiolabeled using [ 99m Tc(CO)3] + core to prepare the corresponding 99m Tc(CO)3 complexes.The partition coefficient, stability in vitro, cell uptake, and biodistribution in mice were also evaluated.

Chemistry and Radiolabeling
Compounds 6a, 6b, 6c and 6d were synthesized by multi-step reactions from the starting materials thymidine via "click" reaction.The reaction equations were shown in Scheme 1.They were identified by 1 H-NMR, 13 C-NMR, ESI-MS (Electrospray Ionization Mass Spectrometry), HRMS (High Resolution Mass Spectrometry) and the results agreed well with the expected chemical structures.The final products were suitable tridentate precursors for the [ 99m Tc(CO)3] + core (Scheme 2).Compounds 7a, 7b, 7c and 7d were obtained with high radiochemical purity (Figure 1).with technetium and rhenium tri-carbonyl as the substrates of TK1 [25,26].Among different tridentate ligands, a macrocyclic triamine compound, such as 1,5,9-triazacyclododecane (12N3), is a suitable ligand for radiolabeling with the [ 99m Tc(CO)3] + core [27].The copper-(I)-catalyzed "click chemistry" by reacting azide with terminal alkyne to form 1,2,3-triazoles (for the trizole's stability in biocircumstances) is used in the area of bioconjugation reactions.It has played a vital role in the synthesis of compounds, as well as in the field of radiopharmaceuticals and molecular imaging [28][29][30].
In this study, four novel thymidine analogs, 6a, 6b, 6c and 6d, were successfully synthesized via "click chemistry" route and then radiolabeled using [ 99m Tc(CO)3] + core to prepare the corresponding 99m Tc(CO)3 complexes.The partition coefficient, stability in vitro, cell uptake, and biodistribution in mice were also evaluated.

Chemistry and Radiolabeling
Compounds 6a, 6b, 6c and 6d were synthesized by multi-step reactions from the starting materials thymidine via "click" reaction.The reaction equations were shown in Scheme 1.They were identified by 1 H-NMR, 13 C-NMR, ESI-MS (Electrospray Ionization Mass Spectrometry), HRMS (High Resolution Mass Spectrometry) and the results agreed well with the expected chemical structures.The final products were suitable tridentate precursors for the [ 99m Tc(CO)3] + core (Scheme 2).Compounds 7a, 7b, 7c and 7d were obtained with high radiochemical purity (Figure 1).According to the results of HPLC analysis, the radiochemical purities of the complexes amount to more than 90%.

Stability and Partition Coefficients
The stability of the complexes was assayed by measuring the radiochemical purity by HPLC (High Performance Liquid Chromatography).The complex was stable over 6 h in the reaction mixture at room temperature (HPLC chromatograms can be found in the Supplementary Materials Figure S1).Only a little decomposition of the complexes was observed in the mouse serum at 37 °C for 6 h (HPLC chromatograms can be found in the Supplementary Materials Figure S2), suggesting that they had good in vitro stability.
The log p values of 7a, 7b, 7c and 7d were −1.16 ± 0.01, −0.97 ± 0.01, −1.09 ± 0.01 and −1.13 ± 0.01, respectively.The results suggested that all of them were hydrophilic.According to the results of HPLC analysis, the radiochemical purities of the complexes amount to more than 90%.
Suzuki et al. [27] reported that 99m Tc(CO) 3 -12N3 was not stable at an elevated temperature.Recently, we have discovered that the 99m Tc(CO) 3 (H 2 O) 3 + could be reconverted to 99m TcO 4 ´at 100 ˝C without the protection of nitrogen.Moreover, 6a, 6b, 6c and 6d ligands should react with 99m Tc(CO) 3 (H 2 O) 3 + in order to obtain the desired products under nitrogen.

Stability and Partition Coefficients
The stability of the complexes was assayed by measuring the radiochemical purity by HPLC (High Performance Liquid Chromatography).The complex was stable over 6 h in the reaction mixture at room temperature (HPLC chromatograms can be found in the Supplementary Materials Figure S1).Only a little decomposition of the complexes was observed in the mouse serum at 37 ˝C for 6 h (HPLC chromatograms can be found in the Supplementary Materials Figure S2), suggesting that they had good in vitro stability.

In Vitro Cell Experiments
In vitro cell uptake of 7d using S180 cells showed that there was no significant difference (p > 0.05) between control and blocking groups (Figure 2).The results indicated the tumor uptake of 7d was related to a nonspecific diffusion.The complex possibly exhibits the overall positive charge, thus making it pass the tumor cell membrane.

In Vitro Cell Experiments
In vitro cell uptake of 7d using S180 cells showed that there was no significant difference (p > 0.05) between control and blocking groups (Figure 2).The results indicated the tumor uptake of 7d was related to a nonspecific diffusion.The complex possibly exhibits the overall positive charge, thus making it pass the tumor cell membrane.

Biodistribution Study
The results of biodistributions of 7a, 7b, 7c and 7d in tumor-bearing mice were shown in Tables 1-4, respectively.At 30 min post-injection, the tumor uptakes of 7a, 7b, 7c and 7d were 2.16% ± 0.59%, 1.28% ± 0.46%, 1.57% ± 0.31% and 1.90% ± 0.58%ID/g.At 360 min post-injection, the tumor uptakes of the complexes were 0.55% ± 0.14%, 0.46% ± 0.08%, 0.40% ± 0.06% and 0.36% ± 0.16%ID/g.These results indicated that all of them exhibited an accumulation in the tumor.At 30 min post-injection, 7d exhibited a higher tumor/muscle ratio (3.56) and 7a had the highest tumor uptake (2.16% ± 0.59%ID/g).It was found that the tumor/muscle ratio at 30 min post-injection increased with the increase of the carbon chain length between thymidine and the 12N3.Desbouis et al. [25] had discovered that a long spacer between the thymidine and organometallic core would improve the ability of the complexes to be accommodated in the binding site.The discovery might be the reason for the increasing uptake ratio of tumor to muscle.

Biodistribution Study
The results of biodistributions of 7a, 7b, 7c and 7d in tumor-bearing mice were shown in Tables 1-4 respectively.At 30 min post-injection, the tumor uptakes of 7a, 7b, 7c and 7d were 2.16% ˘0.59%, 1.28% ˘0.46%, 1.57% ˘0.31% and 1.90% ˘0.58%ID/g.At 360 min post-injection, the tumor uptakes of the complexes were 0.55% ˘0.14%, 0.46% ˘0.08%, 0.40% ˘0.06% and 0.36% ˘0.16%ID/g.These results indicated that all of them exhibited an accumulation in the tumor.At 30 min post-injection, 7d exhibited a higher tumor/muscle ratio (3.56) and 7a had the highest tumor uptake (2.16% ˘0.59%ID/g).It was found that the tumor/muscle ratio at 30 min post-injection increased with the increase of the carbon chain length between thymidine and the 12N3.Desbouis et al. [25] had discovered that a long spacer between the thymidine and organometallic core would improve the ability of the complexes to be accommodated in the binding site.The discovery might be the reason for the increasing uptake ratio of tumor to muscle.Compared with 99m Tc-12N3 [27], the complexes 7a, 7b, 7c and 7d were also mainly accumulated in the excretory organs such as liver, kidneys and intestines, suggesting that the major route of excretion was renal and hepatobiliary.The bone uptakes of the four complexes were more than that of 99m Tc-12N3, which demonstrates a selective uptake in the marrow, a tissue with a large number of proliferative cells [6].Low uptake in the stomach and thyroid was indicative of in vivo stability of these complexes.

General
All chemical reagents were purchased from commercial sources and used without any further purification. 99Mo/ 99m Tc generator was obtained from the China Institute of Atomic Energy (CIAE).NMR spectra were obtained on a 400 MHz Bruker Avance 500 spectrometer (Bruker, Billerica, MA, USA).ESI-MS spectra were obtained on a LC-MS Shimadzu 2010 series (Shimadzu, Kyoto, Japan).HRMS spectra were obtained on a AB SCIEX TripleTOF™ 5600(AB Sciex, Concord, ON, Canada).HPLC analysis was performed on a Waters 600 binary HPLC pump (Waters, Milford, MA, USA) and a Waters 2487 UV absorbance dual λ detector (Waters, Milford, MA, USA) with a reversed-phase column (Kromaisl C18, 250 mm ˆ4.6 mm) (AkzoNobel, Bohus, Sweden).Murine sarcoma S180 cell line was obtained from Peking University Health Science Center (Beijing, China).

Radiolabeling
The tricarbonyl technetium precursor was prepared according to the literature published by Alberto and his coworkers [24,34] with little modification.Namely, potassium sodium tartrate (15 mg), Na 2 CO 3 (5 mg), and NaBH 4 (10 mg) were added to a 10 mL glass vial.The vial was sealed and flushed with CO for 15 min, which is followed by the addition of 1 mL of saline containing [ 99m TcO 4 ] ´.The vial was heated at 80 ˝C for 30 min and then the [ 99m Tc(CO) 3 (H 2 O) 3 ] + precursor was prepared.After being cooled to the room temperature, 1.0 mol/L HCl was added to adjust the pH to approximately 7.Then, 0.5 mL of phosphate-buffer (0.2 mol/L, pH = 7.2) containing 1.0 mg of ligand 6 was added, and the reaction mixture was heated at 100 ˝C for 30 min under nitrogen (Scheme 2).After being cooled to the room temperature, the radiochemical purity (RCP) of the complexes was evaluated by HPLC.Water (containing 0.1% TFA) (A) and acetonitrile (containing 0.1% TFA) were used as the mobile phase.The gradient elution technique was adopted for the preparation: 0 min 10% B, 2 min 10% B, 10 min 90% B, 18 min 10% B.

Stability Studies
The complexes were incubated in saline at room temperature for 6 h, and then the stabilities of the complexes were measured by HPLC.To evaluate the serum stability of the complexes, 0.1 mL (3.7 MBq) of 7a, 7b, 7c and 7d were incubated in 0.5 mL of mouse serum at 37 ˝C for 6 h, and then the RCP of the complex was measured by HPLC after removing the proteins.

Octanol/Water Partition Coefficient
The partition coefficient was measured by mixing the complex with an equal volume of 1-octanol and phosphate buffer (0.025 mol/L, pH 7.4) in a 10 mL centrifugal tube.The mixture was vigorously vortexed for 5 min, and then centrifuged at 14000 rpm for another 5 min.Three samples (100 µL) in triplets from 1-octanol and phosphate buffer were pipetted and measured in a well γ-counter.The partition coefficient was calculated using the following equation: P = (counts per minute in octanol/counts per minute in buffer).Usually, Log P was expressed as the final partition coefficient value.

In Vitro Cell Experiments
Murine sarcoma S180 cell lines were extracted from tumor-bearing mice.The cells were washed three times by saline.S180 cells were grown in DMEM (Dulbecco Modified Eagle Medium) medium containing 10% (v/v) of fetal bovine serum at a cell concentration of 2 ˆ10 4 cells/mL.One culture flask containing 1.0 mL cell suspension was added to 0.074 MBq of 7d, and the others were added to 0.074 MBq of 7d and 0.1 mL of saline which contained different amount of thymidine.After incubation for 2 h, the cells were centrifuged at 10,000 rpm for 5 min for pellet formation.The cells after pelleting were washed three times with phosphate-buffer (0.2 mol/L, pH = 7.2).Each supernatant was removed for counting purposes.The percentage of cell uptake is calculated as residue counts/the total counts 100%.The studies were measured five times.The final results were expressed as an average of five measurements plus the standard deviation.

Biodistribution Study
Animal studies were carried out in compliance with the Regulations on Laboratory Animals of Beijing Municipality and the guidelines of the Ethics Committee of Beijing Normal University.The experiments were approved by the Ethics Committee of Beijing Normal University.The biodistribution of 7a-d was evaluated in Kunming male mice (18-22 g) bearing S180 tumors.The complex (0.1 mL, 3.7 ˆ10 5 Bq) was injected into the mice via a tail vein.At 0.5, 2, 4 and 6 h post-injection, the mice were sacrificed by neck dislocation.The tumors and other interesting organs including blood were collected, weighed and measured for radioactivity.The final results were expressed as the percent uptake of injected dose per gram of tissue (%ID/g).

Conclusions
In the present study, four novel thymidine analogs were synthesized via "click reaction" route, and their 99m Tc(CO) 3 complexes were successfully prepared in high yields through a ligand-exchange reaction.They were hydrophilic and stable in vitro.The preliminary in vivo studies showed that all of them had a relative high tumor uptake and tumor-to-muscle ratio.Further studies should be conducted to evaluate the possibilities of these 99m Tc(CO) 3 complexes as radiotracers for tumor proliferation imaging.

Figure 2 .
Figure 2. In vitro cell uptake of 7d when different amount thymidine was administered.

Figure 2 .
Figure 2. In vitro cell uptake of 7d when different amount thymidine was administered.