Next Article in Journal
Properties of the Sodium Naproxen-Lactose-Tetrahydrate Co-Crystal upon Processing and Storage
Previous Article in Journal
MP-V1 from the Venom of Social Wasp Vespula vulgaris Is a de Novo Type of Mastoparan that Displays Superior Antimicrobial Activities
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China
*
Author to whom correspondence should be addressed.
Molecules 2016, 21(4), 510; https://doi.org/10.3390/molecules21040510
Submission received: 31 December 2015 / Revised: 7 April 2016 / Accepted: 12 April 2016 / Published: 19 April 2016
(This article belongs to the Section Medicinal Chemistry)

Abstract

:
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.

1. Introduction

The sugar-based PET (Positron Emission Tomography) radiotracer 2-[18F]fluoro-2-deoxy-d-glucose (18F-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 11C-labeled nucleoside thymidine [5], 3′-deoxy-3′-[18F]fluorothymidine (18F-FLT) [6,7,8,9,10], 2′-fluoro-5-[11C]-methyl-1-beta-d-arabinofuranosyluracil (11C-FMAU) [11] and 2′-[18F]fluoro-5-methyl-1-beta-d-arabinofuranosyluracil (18F-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, 99mTc can be obtained at a reasonable cost, which makes it readily available and affordable. The availability of a generator and kit chemistry to prepare 99mTc based radiotracers may have a significant impact on nuclear medicine. Thus, using 99mTc to label thymidine analogs is the focus of the ongoing research. To date, several 99mTc 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 99mTc labeled thymidine analogs is still considered to be of great necessity and a considerable challenge.
Recently, the [99mTc(CO)3]+ complex has attracted significant attention due to its ease of preparation, readily substituted water molecules of the precursor fac-[99mTc(CO)3(H2O)3]+ by a variety of tridentate ligands, small size, and inertness [24]. Struthers et al. had synthesized many thymidine analogs labeled 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 [99mTc(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 [99mTc(CO)3]+ core to prepare the corresponding 99mTc(CO)3 complexes. The partition coefficient, stability in vitro, cell uptake, and biodistribution in mice were also evaluated.

2. Results and Discussion

2.1. 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 1H-NMR, 13C-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 [99mTc(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%.
Suzuki et al. [27] reported that 99mTc(CO)3-12N3 was not stable at an elevated temperature. Recently, we have discovered that the 99mTc(CO)3(H2O)3+ could be reconverted to 99mTcO4 at 100 °C without the protection of nitrogen. Moreover, 6a, 6b, 6c and 6d ligands should react with 99mTc(CO)3(H2O)3+ in order to obtain the desired products under nitrogen.

2.2. 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.

2.3. 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.

2.4. Biodistribution Study

The results of biodistributions of 7a, 7b, 7c and 7d in tumor-bearing mice were shown in Table 1, Table 2, Table 3 and Table 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 99mTc-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 99mTc-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.

3. Experimental Section

3.1. General

All chemical reagents were purchased from commercial sources and used without any further purification. 99Mo/99mTc 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 × 4.6 mm) (AkzoNobel, Bohus, Sweden). Murine sarcoma S180 cell line was obtained from Peking University Health Science Center (Beijing, China).

3.2. Synthesis

The syntheses of 12N3-2-TdR (6a), 12N3-3-TdR (6b), 12N3-4-TdR (6c) and 12N3-5-TdR (6d) are depicted in Scheme 1. Compound 1a was synthesized according to the literature [31]. Compound 4 was synthesized according to the literature [32].
3-(2-Bromoethyl)-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (2a). Compound 2a was prepared according to the literature [25]. Namely, compound 1a (1 g, 2.13 mmol) was added into DMF (N,N-Dimethylformamide) (10 mL). Cs2CO3 (2.08 g, 6.39 mmol) was added and the mixture was stirred for 5 min at room temperature. After an addition of 1, 2-dibromoethane (11.94 g, 63.8 mmol), the reaction was stirred for another 2 h at room temperature, and was followed by TLC (Thin Layer Chromatography). Solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography with CH2Cl2 to give 2a as white solid (900 mg, yield 73%). 1H-NMR (400 MHz, CDCl3): δ 7.47 (d, J = 1.3 Hz, 1H), 6.34 (dd, J = 8.0, 5.8 Hz, 1H), 4.44–4.25 (m, 3H), 3.93 (q, J = 2.5 Hz, 1H), 3.80 (ddd, J = 42.1, 11.4, 2.6 Hz, 2H), 3.52 (t, J = 7.3 Hz, 2H), 2.25 (ddd, J = 13.1, 5.8, 2.6 Hz, 1H), 2.03–1.94 (m, 1H), 1.91 (s, 3H), 0.89 (d, J = 12.7 Hz, 18H), 0.10 (s, 6H), 0.07 (s, 3H), 0.06 (s, 3H). 13C-NMR (100 MHz, CDCl3): δ 163.0, 150.6, 133.8, 110.0, 87.9, 85.6, 72.3, 63.0, 42.1, 41.4, 27.2, 26.0, 25.8, 18.4, 18.0, 13.2, −4.6, −4.8, −5.4, −5.4. ESI-MS (m/s): 578.9 (calc. 579.2 [M + 3H+]). HRMS(m/s): 577.2122 [C24H45N2O5Si2Br]H+ (calc. 577.2123).
3-(3-Bromopropyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5 methylpyrimidine-2,4(1H,3H)-dione (2b). Compound 1b (1.9 g, 8 mmol) was added in DMF (30 mL). K2CO3 (3.3 g, 24 mmol) was added and the mixture was stirred for 5 min at room temperature. After an addition of 1,3-dibromopropane (1.62 g, 8 mmol), the reaction was stirred for another 18 h at room temperature and was followed by TLC. Solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography with EtOAc to give 2b as white solid (1.9 g, yield 67%). 1H-NMR (400 MHz, CDCl3): δ 7.46 (d, J = 1.3 Hz, 1H), 6.21 (t, J = 6.7 Hz, 1H), 4.54 (td, J = 5.1, 3.6 Hz, 1H), 4.09–4.02 (m, 2H), 3.99 (q, J = 3.3 Hz, 1H), 3.85 (ddd, J = 42.2, 11.4, 2.6 Hz, 2H), 3.40 (t, J = 6.9 Hz, 2H), 3.14 (s, 2H), 2.37–2.30 (m, 2H), 2.24–2.11 (m, 2H), 1.90 (d, J = 1.0 Hz, 3H). 13C-NMR (100 MHz, MeOD): δ 165.4, 152.4, 136.5, 110.7, 88.9, 87.2, 72.0, 62.8, 41.4, 41.3, 32.1, 31.1, 13.1. ESI-MS (m/s): 385.0 (calc. 385.0 [M + Na+]). HRMS (m/s): 363.0548 [C13H19N2O5Br]H+ (calc. 363.0550).
3-(4-Bromobutyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (2c). Compound 1b (300 mg, 1.23 mmol) was added in DMF (10 mL). K2CO3 (512 mg, 3.75 mmol) was added and the mixture was stirred for 5 min at room temperature. After an addition of 1,4-dibromobutane (267 mg, 1.23 mmol), the reaction was stirred for another 7 h at room temperature, and was followed by TLC. Solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography with EtOAc to give 2c as white solid (310 mg, yield 67%). 1H-NMR (400 MHz, CDCl3): δ 7.42 (d, J = 1.3 Hz, 1H), 6.21 (t, J = 6.8 Hz, 1H), 4.55 (dt, J = 6.2, 3.8 Hz, 1H), 4.00 (q, J = 3.2 Hz, 1H), 3.98–3.77 (m, 4H), 3.42 (t, J = 6.6 Hz, 2H), 3.04 (s, 2H), 2.42–2.27 (m, 2H), 1.96–1.82 (m, 5H), 1.76 (td, J = 8.1, 6.7 Hz, 2H). 13C-NMR (100 MHz, CDCl3): δ 163.5, 150.8, 134.8, 110.0, 87.0, 86.2, 71.1, 62.1, 40.3, 40.2, 33.3, 29.9, 26.2, 13.2. ESI-MS (m/s): 379.0 (calc. 379.1 [M + 3H+]). HRMS (m/s): 377.0710 [C14H21N2O5Br]H+ (calc. 377.0706).
3-(5-Bromopentyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (2d). Compound 1b (300 mg, 1.23 mmol) was added in DMF (10 mL). K2CO3 (512 mg, 3.75 mmol) was added and the mixture was stirred for 5 min at room temperature. After an addition of 1,5-dibromopentane (285 mg, 1.23 mmol), the reaction was stirred for another 7 h at room temperature, and was followed by TLC. Solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography with EtOAc to give 2d as white solid (300 mg, yield 62%). 1H-NMR (400 MHz, CDCl3): δ 7.40 (d, J = 1.4 Hz, 1H), 6.20 (t, J = 6.8 Hz, 1H), 4.55 (dt, J = 6.2, 3.8 Hz, 1H), 4.00 (q, J = 3.3 Hz, 1H), 3.95–3.76 (m, 4H), 3.39 (t, J = 6.7 Hz, 2H), 3.02 (s, 2H), 2.42–2.26 (m, 2H), 1.94–1.81 (m, 5H), 1.69–1.56 (m, 2H), 1.47 (tt, J = 10.2, 4.1 Hz, 2H). 13C-NMR (100 MHz, CDCl3): δ 163.5, 150.9, 134.7, 110.0, 87.1, 86.4, 71.2, 62.2, 41.0, 40.3, 33.7, 32.2, 26.6, 25.34, 13.3. ESI-MS (m/s): 392.9 (calc. 393.0 [M + 3H+]). HRMS (m/s): 391.0865 [C15H23N2O5Br]H+ (calc. 391.0863).
3-(2-Azidoethyl)-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (3a). Compound 3a was prepared according to the literature [33]. Namely, compound 2a (1.1 g, 1.91 mmol) and NaN3 (1.24 g, 19.1 mmol) were added into CH3CN (20 mL). The mixture was refluxed for 16 h and was followed by TLC. The solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography with CH2Cl2 to give 3a as white solid (800 mg, yield 78%). 1H-NMR (400 MHz,CDCl3): δ 7.48 (d, J = 1.3 Hz, 1H), 6.35 (dd, J = 7.9, 5.8 Hz, 1H), 4.39 (dt, J = 5.6, 2.6 Hz, 1H), 4.19 (td, J = 6.3, 2.1 Hz, 2H), 3.94 (q, J = 2.6 Hz, 1H), 3.81 (ddd, J = 42.4, 11.4, 2.6 Hz, 2H), 3.53 (t, J = 6.3 Hz, 3H), 2.26 (ddd, J = 13.2, 5.8, 2.6 Hz, 1H), 1.99 (ddd, J = 13.5, 7.9, 6.0 Hz, 1H), 1.93 (d, J = 1.1 Hz, 3H), 0.90 (d, J = 13.0 Hz, 18H), 0.11 (s, 3H), 0.10 (s, 3H), 0.08 (s, 3H), 0.07 (s, 3H). 13C-NMR (100 MHz, CDCl3): δ 163.0, 150.6, 133.7, 110.0, 87.7, 85.4, 72.1, 62.8, 48.2, 41.3, 39.6, 25.8, 25.6, 18.2, 17.8, 13.0, −4.8, −5.0, −5.6, −5.6. ESI-MS (m/s): 540.1 (calc. 540.3 [M + H+]). HRMS (m/s): 540.3033 [C24H45N5O5Si2]H+ (calc. 540.3032).
3-(3-Azidopropyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (3b). Compound 2b (1 g, 2.75 mmol) and NaN3 (1.79 g, 27.5 mmol) were added into CH3CN (30 mL). The mixture was refluxed for 8 h and was followed by TLC. The solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography with EtOAc to give 3b as colorless oil (570 mg, yield 63%). 1H-NMR (400 MHz, CDCl3): δ 7.42 (d, J = 1.3 Hz, 1H), 6.20 (t, J = 6.8 Hz, 1H), 4.56 (dt, J = 6.2, 3.6 Hz, 1H), 4.05–3.97 (m, 3H), 3.85 (ddd, J = 42.3, 11.4, 2.6 Hz, 2H), 3.34 (t, J = 6.8 Hz, 2H), 2.94 (s, 2H), 2.43–2.26 (m, 2H), 1.96–1.83 (m, 5H). 13C-NMR (100 MHz, CDCl3): δ 163.7, 151.0, 135.0, 110.3, 87.3, 86.8, 71.5, 62.4, 49.3, 40.2, 39.1, 27.1, 13.4. ESI-MS (m/s): 326.0 (calc. 326.3 [M + H+]). HRMS (m/s): 326.1461 [C13H19N5O5]H+ (calc. 326.1458).
3-(4-Azidobutyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (3c). Compound 2c (310 mg, 0.825 mmol) and NaN3 (536 mg, 8.24 mmol) were added into CH3CN (10 mL). The mixture was refluxed for 5 h and was followed by TLC. The solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography with EtOAc to give 3c as colorless oil (180 mg, yield 64%). 1H-NMR (400 MHz, CDCl3): δ 7.33 (d, J = 1.2 Hz, 1H), 6.18 (t, J = 6.9 Hz, 1H), 4.61 (dt, J = 6.8, 3.6 Hz, 1H), 4.02 (q, J = 3.1 Hz, 1H), 3.99–3.79 (m, 4H), 3.31 (t, J = 6.7 Hz, 2H), 2.45 (dt, J = 13.8, 6.9 Hz, 2H), 2.31 (ddd, J = 13.7, 6.4, 3.6 Hz, 1H), 2.13 (s, 1H), 1.93 (d, J = 1.2 Hz, 3H), 1.76–1.57 (m,4H). 13C-NMR (100 MHz, CDCl3) δ 163.6, 150.9, 134.8, 110.1, 87.0, 86.5, 71.3, 62.2, 51.0, 40.7, 40.2, 26.2, 24.8, 13.2. ESI-MS (m/s): 339.9 (calc. 340.2 [M + H+]). HRMS (m/s): 340.1616 [C14H21N5O5]H+ (calc. 340.1615).
3-(5-Azidopentyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (3d). Compound 2d (300 mg, 0.769 mmol) and NaN3 (500 mg, 7.69 mmol) were added into CH3CN (10 mL). The mixture was refluxed for 5 h and was followed by TLC. The solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography with EtOAc to give 3d as colorless oil (130 mg, yield 48%). 1H-NMR (400 MHz,CDCl3): δ 7.34 (d, J = 1.3 Hz, 1H), 6.18 (t, J = 6.9 Hz, 1H), 4.59 (dt, J = 6.8, 3.5 Hz, 1H), 4.01 (q, J = 3.2 Hz, 1H), 3.97–3.80 (m, 4H), 3.27 (t, J = 6.9 Hz, 2H), 2.76–2.21 (m, 4H), 1.92 (d, J = 1.2 Hz, 3H), 1.72–1.56 (m, 4H), 1.51–1.35 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 163.4, 150.6, 134.6, 110.0, 86.9, 86.0, 71.0, 62.0, 50.9, 40.9, 40.0, 28.1, 26.7, 23.7, 13.0. ESI-MS (m/s): 353.9 (calc. 353.4 [M + H+]). HRMS (m/s): 354.1774 [C15H23N5O5]H+ (calc. 354.1771).
Di-tert-butyl-9-((1-(2-(3-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-1,5,9-triazacyclododecane-1,5-dicarboxylate (5a). Compound 3a (400 mg, 0.74 mmol) was dissolved in THF (10 mL) and compound 4 (364 mg, 0.89 mmol) was added. After an addition of vitamin C (40 mg, 0.23 mmol), CuSO4·5H2O (15 mg, 0.06 mmol) was added under nitrogen. The reaction was stirred overnight at room temperature. The solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography with CH2Cl2/EtOAc (v/v = 20:1) to give 5a as white solid (500 mg, yield 71%). 1H-NMR (400 MHz, CDCl3): δ 7.49–7.41 (m, 2H), 6.25 (dd, J = 7.8, 5.8 Hz, 1H), 4.61 (t, J = 6.3 Hz, 2H), 4.45–4.31 (m, 3H), 3.91 (q, J = 2.6 Hz, 1H), 3.87–3.70 (m, 4H), 3.44–3.22 (m, 8H), 2.48–2.32 (m, 4H), 2.21 (ddd, J = 13.1, 5.8, 2.7 Hz, 1H), 1.96 (ddd, J = 13.5, 7.9, 6.0 Hz, 1H), 1.91–1.78 (m, 9H), 1.43 (s, 18H), 0.89 (d, J = 12.6 Hz, 18H), 0.09–0.06 (m, 12H). 13C-NMR (100 MHz, CDCl3): δ 162.9, 156.2, 150.4, 133.9, 122.7, 109.6, 87.7, 85.4, 80.0, 72.0, 62.8, 49.5, 47.2, 46.0, 45.4, 43.6, 41.3, 40.4, 28.4, 26.8, 26.1, 25.9, 25.7, 18.3, 17.9, 13.1, −4.7, −4.9, −5.4, −5.5. ESI-MS (m/s): 949.4 (calc. 949.6 [M + H+]). HRMS (m/s): 949.5972 [C46H84N8O9Si2]H+ (calc. 949.5972).
Di-tert-butyl-9-((1-(3-(3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)-1,5,9 triazacyclododecane-1,5-dicarboxylate (5b). Compound 3b (200 mg, 0.62 mmol) was dissolved in THF (10 mL) and compound 4 (251 mg, 0.62 mmol) was added. After an addition of vitamin C (40 mg, 0.23 mmol), CuSO4·5H2O (20 mg, 0.08 mmol) was added under nitrogen. The reaction was stirred overnight at room temperature. The solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography with EtOAc/MeOH (v/v = 20:1) to give 5b as white solid (270 mg, yield 60%). 1H-NMR (400 MHz, DMSO-d6): δ 8.02 (s, 1H), 7.77 (d, J = 1.4 Hz, 1H), 6.19 (t, J = 6.7 Hz, 1H), 5.23 (d, J = 4.2 Hz, 1H), 5.03 (t, J= 5.1 Hz, 1H), 4.36 (t, J = 7.1 Hz, 2H), 4.24 (p, J = 4.3 Hz, 1H), 3.83 (t, J = 7.2 Hz, 2H), 3.77 (q, J = 3.6 Hz, 1H), 3.69 (s, 2H), 3.65–3.51 (m, 2H), 3.24 (dt, J = 6.8, 3.6 Hz, 8H), 2.30 (t, J = 6.1 Hz, 4H), 2.15–2.01 (m, 4H), 1.87–1.69 (m, 9H), 1.38 (s, 18H). 13C-NMR (100 MHz, CDCl3): δ 163.6, 156., 150.9, 135.2, 123.1, 109.8, 87.4, 86.4, 79.6, 77.4, 70.9, 62.0, 49.6, 48.2, 46.8, 45.4, 43.8, 40.6, 38.5, 28.5, 28.3, 27.0, 26.1, 13.3. ESI-MS (m/s): 736.1 (calc. 736.4 [M + 2H+]). HRMS (m/s): 735.4396 [C35H58N8O9]H+ (calc. 735.4399).
Di-tert-butyl-9-((1-(4-(3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)butyl)-1H-1,2,3-triazol-4-yl)methyl)-1,5,9-triazacyclododecane-1,5-dicarboxylate (5c). Compound 3c (400 mg, 1.18 mmol) was dissolved in THF (10 mL) and compound 4 (578 mg, 1.41 mmol) was added. After an addition of vitamin C (60 mg, 0.34 mmol), CuSO4·5H2O (20 mg, 0.06 mmol) was added under nitrogen. The reaction was stirred overnight at room temperature. The solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography EtOAc/MeOH (v/v = 20:1) to give 5c as white solid (500 mg, yield 57%). 1H-NMR (400 MHz, CDCl3): δ 7.61–7.53 (m, 2H), 6.27 (t, J = 6.7 Hz, 1H), 4.54 (dt, J = 6.6, 3.4 Hz, 1H), 4.40 (q, J = 7.0, 5.3 Hz, 2H), 4.04–3.91 (m, 3H), 3.91–3.72 (m, 4H), 3.39–3.23 (m, 8H), 2.50–2.20 (m, 6H), 2.02 (d, J = 1.8 Hz, 1H), 1.96–1.78 (m, 12H), 1.66–1.55 (m, 2H), 1.42 (s, 18H). 13C-NMR (100 MHz, CDCl3): δ 163.5, 156.4, 150.8, 142.7, 134.9, 123.1, 109.8, 87.3, 86.1, 79.5, 71.00, 62.0, 49.4, 46.2, 45.5, 43.8, 40.5, 30.0, 28.4, 27.5, 27.0, 25.8, 24.2, 13.2. ESI-MS (m/s): 749.3 (calc. 749.4 [M + H+]). HRMS (m/s): 749.4557 [C36H60N8O9]H+ (calc. 749.4556).
Di-tert-butyl-9-((1-(5-(3-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)pentyl)-1H-1,2,3-triazol-4-yl)methyl)-1,5,9-triazacyclododecane-1,5-dicarboxylate (5d). Compound 3d (300 mg, 0.849 mmol) was dissolved in THF (10 mL) and compound 4 (578 mg, 1.02 mmol)was added. After an addition of vitamin C (45 mg, 0.26 mmol), CuSO4·5H2O (15 mg, 0.08 mmol) was added under nitrogen. The reaction was stirred overnight at room temperature. The solvent was evaporated under a reduced pressure and the crude product was purified by column chromatography EtOAc/MeOH (v/v = 20:1) to give 5d as white solid (552 mg, yield 62%). 1H-NMR (400 MHz, CDCl3): δ 7.58 (d, J = 1.4 Hz, 1H), 7.42 (s, 1H), 6.22 (t, J = 6.5 Hz, 1H), 4.55 (dt, J = 6.2, 4.1 Hz, 1H), 4.32 (t, J = 7.0 Hz, 2H), 4.06–3.98 (m, 2H), 3.93–3.73 (m, 6H), 3.31 (t, J = 6.7 Hz, 8H), 2.50–2.36 (m, 4H), 2.30 (m, 2H), 1.96–1.76 (m, 12H), 1.71–1.54 (m, 2H), 1.42 (s, 18H), 1.26–1.36 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 163.6, 156.5, 1501.0, 143.1, 135.0, 122.7, 109.9, 87.3, 86.5, 79.1, 70.9, 62.0, 60.5, 50.1, 49.5, 46.5, 45.5, 43.9, 40.7, 40.6, 29.7, 28.5, 26.7, 26.0, 23.6, 13.3. ESI-MS (m/s): 763.3 (calc. 763.5 [M + H+]). HRMS (m/s): 763.4716 [C37H62N8O9]H+ (calc. 763.4712).
3-(2-(4-((1,5,9-Triazacyclododecan-1-yl)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (6a, 12N3-2-TdR). Compound 5a (500 mg, 0.53 mmol) was dissolved in ethanol (30 mL). After an addition of concentrate hydrochloric acid (1 mL), the reaction mixture was refluxed for 1 h. Solvent was removed under a reduced pressure. The mixture was recrystallized from ethanol (100 mL) to give 6a as white solid (200 mg, yield 72.7%). 1H-NMR (400 MHz, D2O): δ 8.12 (s, 1H), 7.69 (d, J = 1.4 Hz, 1H), 6.25 (t, J = 6.7 Hz, 1H), 4.82–4.80 (m, 2H), 4.50 (q, J = 4.6 Hz, 1H), 4.45 (dd, J = 7.0, 4.9 Hz, 2H), 4.07 (dt, J = 5.2, 3.8 Hz, 1H), 4.03 (s, 2H), 3.73 (ddd, J = 42.3, 11.4, 2.6 Hz, 2H), 3.39 (dt, J = 12.3, 5.8 Hz, 8H), 2.91 (s, 4H), 2.43–2.31 (m, 4H), 2.18–2.08 (m, 4H), 1.90 (s, 3H). 13C-NMR (100 MHz, D2O): δ(ppm) 164.7, 151.2, 135.7, 128.1, 110.2, 86.6, 85.7, 70.3, 61.2, 57.4, 48.3, 47.3, 42.1, 41.3, 41.1, 38.7, 20.0, 17.9, 16.9, 12.3. ESI-MS (m/s): 521.1 (calc. 521.3 [M + H+]). HRMS (m/s): 521.3196 [C24H40N8O5]H+ (calc. 521.3194).
3-(3-(4-((1,5,9-Triazacyclododecan-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (6b, 12N3-3-TdR). Compound 5b (270 mg, 0.37 mmol) was dissolved in ethanol (20 mL). After an addition of concentrate hydrochloric acid (1 mL), the reaction mixture was refluxed for 1 h. Solvent was removed under a reduced pressure. The mixture was recrystallized from ethanol (50 mL) to give 6b as white solid (120 mg, yield 60.7%). 1H-NMR (400 MHz, D2O): δ 8.26 (s, 1H), 7.55 (d, J = 1.4 Hz, 1H), 6.17 (t, J = 6.7 Hz, 1H), 4.53–4.44 (m, 4H), 4.43–4.33 (m, 1H), 3.95 (dt, J = 5.2, 3.7 Hz, 1H), 3.87 (t, J = 6.9 Hz, 2H), 3.73 (ddd, J = 42.1, 11.4, 2.6 Hz, 2H), 3.46–3.28 (m, 12H), 2.34–2.17 (m, 10H), 1.83 (s, 3H). 13C-NMR(100 MHz, D2O): δ(ppm) 165.1, 151.3, 135.5, 127.4, 110.4, 86.5, 85.8, 70.4, 61.2, 57.4, 48.6, 48.5, 47.5, 42.1, 41.1, 38.7, 26.7, 20.0, 18.1, 16.9, 12.3. ESI-MS (m/s): 535.0 (calc. 535.3 [M + H+]). HRMS (m/s): 535.3349 [C25H42N8O5]H+ (calc. 535.3350).
3-(4-(4-((1,5,9-Triazacyclododecan-1-yl)methyl)-1H-1,2,3-triazol-1-yl)butyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (6c, 12N3-4-TdR). Compound 5c (500 mg, 0.67 mmol) was dissolved in ethanol (20 mL). After an addition of concentrate hydrochloric acid (1 mL), the reaction mixture was refluxed for 1 h. Solvent was removed under a reduced pressure. The mixture was recrystallized from ethanol (50 mL) to give 6c as white solid (216 mg, yield 58.9%). 1H-NMR (400 MHz, D2O): δ 8.22 (s, 1H), 7.57 (d, J = 1.3 Hz, 1H), 6.19 (t, J = 6.7 Hz, 1H), 4.51–4.31 (m, 5H), 3.94 (q, J = 4.1 Hz, 1H), 3.82 (t, J = 7.2 Hz, 2H), 3.79–3.64 (m, 2H), 3.45–3.25 (m, 12H), 2.34–2.14 (m, 8H), 1.94–1.73 (m, 5H), 1.49 (p, J = 7.6 Hz, 2H). 13C-NMR (100 MHz, D2O): δ (ppm) 165.3, 151.5, 135.5, 127.4, 110.5, 86.5, 85.8, 70.4, 61.2, 57.4, 50.1, 47.7, 42.3, 41.2, 40.7, 38.7, 26.7, 23.6, 20.0, 18.1, 16.9, 12.3. ESI-MS (m/s): 549.0 (calc. 549.0 [M + H+]). HRMS (m/s): 549.3505 [C26H44N8O5]H+ (calc. 549.3507).
3-(5-(4-((1,5,9-Triazacyclododecan-1-yl)methyl)-1H-1,2,3-triazol-1-yl)pentyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (6d, 12N3-5-TdR). Compound 5d (552 mg, 0.73 mmol) was dissolved in ethanol (20 mL). After an addition of concentrate hydrochloric acid (1 mL), the reaction mixture was refluxed for 1 h. Solvent was removed under a reduced pressure. The mixture was recrystallized from ethanol (50 mL) to give 6d as white solid (220 mg, yield 54%). 1H-NMR (400 MHz, D2O): δ 8.19 (s, 1H), 7.58 (s, 1H), 6.21 (t, J = 6.6 Hz, 1H), 4.49–4.32 (m, 5H), 3.95 (q, J = 4.3 Hz, 1H), 3.85–3.64 (m, 4H),3.45–3.19 (m, 12H), 2.42–2.08 (m, 8H), 1.95–1.76 (m, 5H), 1.52 (p, J = 7.6 Hz, 2H), 1.20 (p, J = 7.8 Hz, 2H). 13C-NMR (100 MHz, D2O): δ (ppm) 165.3, 151.5, 135.5, 127.1, 110.5, 86.5, 85.8, 70.4, 61.2, 57.4, 50.4, 48.1, 42.5, 41.3, 41.2, 38.7, 28.8, 25.6, 22.9, 19.9, 18.4, 16.8, 12.3. ESI-MS (m/s): 563.4 (calc. 563.4 [M + H+]). HRMS (m/s): 563.3662 [C27H46N8O5]H+ (calc. 563.3663).

3.3. 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), Na2CO3 (5 mg), and NaBH4 (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 [99mTcO4]. The vial was heated at 80 °C for 30 min and then the [99mTc(CO)3(H2O)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.

3.4. 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.

3.5. 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.

3.6. 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 × 104 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.

3.7. 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 7ad was evaluated in Kunming male mice (18–22 g) bearing S180 tumors. The complex (0.1 mL, 3.7 × 105 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).

4. Conclusions

In the present study, four novel thymidine analogs were synthesized via “click reaction” route, and their 99mTc(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 99mTc(CO)3 complexes as radiotracers for tumor proliferation imaging.

Supplementary Materials

Supplementary materials can be accessed at: https://www.mdpi.com/1420-3049/21/4/510/s1.

Acknowledgments

The work was financially supported, in part, by the National Natural Science Foundation of China (21541001, 21171024), the Foundation of Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education.

Author Contributions

Xiaojiang Duan, Teli Liu and Yichun Zhang performed the chemical synthesis, radiolabeling and biodistribution studies. Xiaojiang Duan prepared the manuscript. Junbo Zhang designed the whole research and corrected the final manuscript. All authors read and approved the final version.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Rosenbaum, S.J.; Lind, T.; Antoch, G.; Bockisch, A. False-positive FDG PET uptake—The role of PET/CT. Eur. Radiol. 2006, 16, 1054–1065. [Google Scholar] [CrossRef] [PubMed]
  2. Kubota, R.; Yamada, S.; Kubota, K.; Ishiwata, K.; Tamahashi, N.; Ido, T. Intratumoral distribution of fluorine-18-fluorodeoxyglucose in vivo: High accumulation in macrophages and granulation tissues studied by microautoradiography. J. Nucl. Med. 1992, 33, 1972–1980. [Google Scholar] [PubMed]
  3. Hanahan, D.; Weinberg, R.A. Hallmarks of cancer: The next generation. Cell 2011, 144, 646–674. [Google Scholar] [CrossRef] [PubMed]
  4. Alauddin, M.M. Nucleoside-based probes for imaging tumor proliferation using positron emission tomography. J. Label. Compd. Radiopharm. 2013, 56, 237–243. [Google Scholar] [CrossRef] [PubMed]
  5. Shields, A.F.; Mankoff, D.A.; Link, J.M.; Graham, M.M.; Eary, J.F.; Kozawa, S.M.; Zheng, M.; Lewellen, B.; Lewellen, T.K.; Grierson, J.R. Carbon-11-thymidine and FDG to measure therapy response. J. Nucl. Med. 1998, 39, 1757–1762. [Google Scholar] [PubMed]
  6. Shields, A.F.; Grierson, J.R.; Dohmen, B.M.; Machulla, H.-J.; Stayanoff, J.C.; Lawhorn-Crews, J.M.; Obradovich, J.E.; Muzik, O.; Mangner, T.J. Imaging proliferation in vivo with [F-18]FLT and positron emission tomography. Nat. Med. 1998, 4, 1334–1336. [Google Scholar] [CrossRef] [PubMed]
  7. Grierson, J.R.; Shields, A.F. Radiosynthesis of 3′-deoxy-3′-[18F] fluorothymidine:[18F]FLT for imaging of cellular proliferation in vivo. Nucl. Med. Biol. 2000, 27, 143–156. [Google Scholar] [CrossRef]
  8. Vesselle, H.; Grierson, J.; Muzi, M.; Pugsley, J.M.; Schmidt, R.A.; Rabinowitz, P.; Peterson, L.M.; Vallières, E.; Wood, D.E. In vivo validation of 3′ deoxy-3′-[18F]fluorothymidine ([18F]FLT) as a proliferation imaging tracer in humans correlation of [18F]FLT uptake by positron emission tomography with Ki-67 immunohistochemistry and flow cytometry in human lung tumors. Clin. Cancer Res. 2002, 8, 3315–3323. [Google Scholar] [PubMed]
  9. Buck, A.K.; Halter, G.; Schirrmeister, H.; Kotzerke, J.; Wurziger, I.; Glatting, G.; Mattfeldt, T.; Neumaier, B.; Reske, S.N.; Hetzel, M. Imaging proliferation in lung tumors with PET: 18F-FLT versus 18F-FDG. J. Nucl. Med. 2003, 44, 1426–1431. [Google Scholar] [PubMed]
  10. McKinley, E.T.; Watchmaker, J.M.; Chakravarthy, A.B.; Meyerhardt, J.A.; Engelman, J.A.; Walker, R.C.; Washington, M.K.; Coffey, R.J.; Manning, H.C. [18F]-FLT PET to predict early response to neoadjuvant therapy in KRAS wild-type rectal cancer: A pilot study. Ann. Nucl. Med. 2015, 29, 535–542. [Google Scholar] [CrossRef] [PubMed]
  11. Conti, P.S.; Alauddin, M.M.; Fissekis, J.R.; Schmall, B.; Watanabe, K.A. Synthesis of 2′-fluoro-5-[11C]-methyl-1-β-d-arabinofuranosyluracil ([11C]-FMAU): A potential nucleoside analog for in vivo study of cellular proliferation with PET. Nucl. Med. Biol. 1995, 22, 783–789. [Google Scholar] [CrossRef]
  12. Alauddin, M.M.; Conti, P.S.; Fissekis, J.D. Synthesis of [18F]-labeled 2′-deoxy-2′-fluoro-5-methyl-1-β-d-arabinofuranosyluracil ([18F]-FMAU). J. Label. Compd. Rad. 2002, 45, 583–590. [Google Scholar] [CrossRef]
  13. Lu, L.; Samuelsson, L.; Bergström, M.; Sato, K.; Fasth, K.-J.; Långström, B. Rat studies comparing 11C-FMAU, 18F-FLT, and 76Br-BFU as proliferation markers. J. Nucl. Med. 2002, 43, 1688–1698. [Google Scholar] [PubMed]
  14. Sun, H.; Mangner, T.J.; Collins, J.M.; Muzik, O.; Douglas, K.; Shields, A.F. Imaging DNA synthesis in vivo with 18F-FMAU and PET. J. Nucl. Med. 2005, 46, 292–296. [Google Scholar] [PubMed]
  15. Jadvar, H.; Yap, L.P.; Park, R.; Li, Z.B.; Chen, K.; Hughes, L.; Kouhi, A.; Conti, P. [18F] -2’-Fluoro-5-methyl-1-β-d-arabinofuranosyluracil (18F-FMAU) in prostate cancer: Initial preclinical observations. Mol. Imaging 2012, 11, 426–432. [Google Scholar] [PubMed]
  16. Peck, M.; Pollack, H.A.; Friesen, A.; Muzi, M.; Shoner, S.C.; Shankland, E.G.; Fink, J.R.; Armstrong, J.O.; Link, J.M.; Krohn, K.A. Applications of PET imaging with the proliferation marker 18F-FLT. Q. J. Nucl. Med. Mol. Imaging 2015, 59, 95–104. [Google Scholar] [PubMed]
  17. Kim, J.Y.; Oh, S.J.; Ryu, J.S.; Choi, S.J.; Ha, H.J.; Moon, D.H. Synthesis of 99mTc(CO)3-deoxyuridine derivatives as potential HSV1-tk gene expression imaging agents. Appl. Radiat. Isot. 2008, 66, 489–496. [Google Scholar]
  18. Zhang, Y.; Dai, X.; Kallmes, D.F.; Pan, D. Synthesis of a technetium-99m-labeled thymidine analog: a potential HSV1-TK substrate for non-invasive reporter gene expression imaging. Tetrahedron Lett. 2004, 45, 8673–8676. [Google Scholar] [CrossRef]
  19. Celen, S.; de Groot, T.; Balzarini, J.; Vunckx, K.; Terwinghe, C.; Vermaelen, P.; Van Berckelaer, L.; Vanbilloen, H.; Nuyts, J.; Mortelmans, L.; et al. Synthesis and evaluation of a 99mTc-MAMA-propyl-thymidine complex as a potential probe for in vivo visualization of tumor cell proliferation with SPECT. Nucl. Med. Biol. 2007, 34, 283–291. [Google Scholar] [CrossRef] [PubMed]
  20. Teng, B.; Bai, Y.P.; Chang, Y.; Chen, S.Z.; Li, Z.L. Technetium-99m-labeling and synthesis of thymidine analogs: Potential candidates for tumor imaging. Bioorg. Med. Chem. Lett. 2007, 17, 3440–3444. [Google Scholar] [CrossRef] [PubMed]
  21. Lu, C.X.; Jiang, Q.F.; Tan, C.; Tang, J.; Zhang, J.K. Preparation and preliminary biological evaluation of novel 99mTc-Labelled thymidine analogs as tumor imaging agents. Molecules 2012, 17, 8518–8532. [Google Scholar] [CrossRef] [PubMed]
  22. Schmid, M.; Neumaier, B.; Vogg, A.T.; Wczasek, K.; Friesen, C.; Mottaghy, F.M.; Buck, A.K.; Reske, S.N. Synthesis and evaluation of a radiometal-labeled macrocyclic chelator-derivatised thymidine analog. Nucl. Med. Biol. 2006, 33, 359–366. [Google Scholar] [CrossRef] [PubMed]
  23. Chun Xiong, L.; Zheng Wu, W.; Quan Fu, J.; Jie, T.; Cheng, T.; Jian Kang, Z. Synthesis and preliminary biological evaluation of a technetium-99m labeled thymidine analog. Chin. Chem. Lett. 2011, 22, 1309–1312. [Google Scholar]
  24. Alberto, R.; Schibli, R.; Egli, A.; Schubiger, A.P.; Abram, U.; Kaden, T.A. A novel organometallic aqua complex of technetium for the labeling of biomolecules: Synthesis of [99mTc(OH2)3(CO)3]+from [99mTcO4] in aqueous solution and its reaction with a bifunctional ligand. J. Am. Chem. Soc. 1998, 120, 7987–7988. [Google Scholar] [CrossRef]
  25. Desbouis, D.; Struthers, H.; Spiwok, V.; Kuster, T.; Schibli, R. Synthesis, in vitro, and in silico evaluation of organometallic technetium and rhenium thymidine complexes with retained substrate activity toward human thymidine kinase type 1. J. Med. Chem. 2008, 51, 6689–6698. [Google Scholar] [CrossRef] [PubMed]
  26. Struthers, H.; Hagenbach, A.; Abram, U.; Schibli, R. Organometallic [Re(CO)3]+ and [Re(CO)2(NO)]2+ labeled substrates for human thymidine kinase 1. Inorg.Chem. 2009, 48, 5154–5163. [Google Scholar] [CrossRef] [PubMed]
  27. Suzuki, K.; Shimmura, N.; Thipyapong, K.; Uehara, T.; Akizawa, H.; Arano, Y. Assessment of macrocyclic triamine ligands as synthons for organometallic 99mTc radiopharmaceuticals. Inorg. Chem. 2008, 47, 2593–2600. [Google Scholar] [CrossRef] [PubMed]
  28. Struthers, H.; Spingler, B.; Mindt, T.L.; Schibli, R. “Click-to-Chelate”: Design and incorporation of triazole-containing metal-chelating systems into biomolecules of diagnostic and therapeutic interest. Chem. Eur. J. 2008, 14, 6173–6183. [Google Scholar] [CrossRef] [PubMed]
  29. Kolb, H.C.; Finn, M.G.; Sharpless, K.B. Click chemistry: Diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 2001, 40, 2004–2021. [Google Scholar] [CrossRef]
  30. Kluba, C.A.; Mindt, T.L. Click-to-chelate: Development of technetium and rhenium-tricarbonyl labeled radiopharmaceuticals. Molecules 2013, 18, 3206–3226. [Google Scholar] [CrossRef] [PubMed]
  31. Kachare, D.; Song, X.P.; Herdewijn, P. Phospho-carboxylic anhydride of a homologated nucleoside leads to primer degradation in the presence of a polymerase. Bioorg. Med. Chem. Lett. 2014, 24, 2720–2723. [Google Scholar] [CrossRef] [PubMed]
  32. Guo, Z.F.; Yan, H.; Li, Z.F.; Lu, Z.L. Synthesis of mono- and di-[12]aneN3 ligands and study on the catalytic cleavage of RNA model 2-hydroxypropyl-p-nitrophenyl phosphate with their metal complexes. Org. Biomol. Chem. 2011, 9, 6788–6796. [Google Scholar] [CrossRef] [PubMed]
  33. Struthers, H.; Viertl, D.; Kosinski, M.; Spingler, B.; Buchegger, F.; Schibli, R. Charge dependent substrate activity of C3’ and N3 functionalized, organometallic technetium and rhenium-labeled thymidine derivatives toward human thymidine kinase 1. Bioconjug. Chem. 2010, 21, 622–634. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Alberto, R.; Schibli, R.; Schubiger, A.P.; Abram, U.; Pietzsch, H.J.; Johannsen, B. First application of fac-[99mTc(OH2)3(CO)3]+ in bioorganometallic chemistry: Design, structure, and in vitro affinity of a 5-HT1A receptor ligand labeled with 99mTc. J. Am. Chem. Soc. 1999, 121, 6076–6077. [Google Scholar] [CrossRef]
  • Sample Availability: Samples of the compounds are available from the authors.
Scheme 1. The syntheses of 6ad.
Scheme 1. The syntheses of 6ad.
Molecules 21 00510 sch001
Scheme 2. The radiochemical syntheses of 7ad and their proposed structures.
Scheme 2. The radiochemical syntheses of 7ad and their proposed structures.
Molecules 21 00510 sch002
Figure 1. HPLC chromatograms of 99mTcO4 (tR = 4.78 min), [99mTc(CO)3]+ (tR = 11.68 min), 7a (tR = 10.53 min), 7b (tR =10.46 min), 7c (tR = 10.63 min) and 7d (tR = 10.75 min).
Figure 1. HPLC chromatograms of 99mTcO4 (tR = 4.78 min), [99mTc(CO)3]+ (tR = 11.68 min), 7a (tR = 10.53 min), 7b (tR =10.46 min), 7c (tR = 10.63 min) and 7d (tR = 10.75 min).
Molecules 21 00510 g001
Figure 2. In vitro cell uptake of 7d when different amount thymidine was administered.
Figure 2. In vitro cell uptake of 7d when different amount thymidine was administered.
Molecules 21 00510 g002
Table 1. Biodistribution of 7a in mice bearing S180 tumor (mean ± SD, n = 4, %ID/g).
Table 1. Biodistribution of 7a in mice bearing S180 tumor (mean ± SD, n = 4, %ID/g).
Tissue30 min120 min240 min360 min
Heart2.48 ± 1.151.65 ± 1.121.26 ± 0.480.74 ± 0.29
Liver9.94 ± 2.339.12 ± 2.098.10 ± 0.547.98 ± 1.00
Lung2.92 ± 0.631.41 ± 0.330.92 ± 0.080.85 ± 0.15
Kidney14.12 ± 1.569.90 ± 1.247.71 ± 1.048.35 ± 0.91
Spleen1.77 ± 0.681.58 ± 0.900.86 ± 0.070.83 ± 0.09
Stomach2.06 ± 0.691.57 ± 1.071.35 ± 0.481.31 ± 0.20
Bone3.41 ± 0.852.73 ± 1.222.47 ± 0.302.14 ± 0.76
Muscle1.65 ± 0.800.67 ± 0.110.88 ± 0.320.59 ± 0.24
Intestine6.20 ± 1.074.06 ± 1.432.57 ± 0.802.62 ± 1.50
Tumor2.16 ± 0.590.96 ± 0.450.57 ± 0.150.55 ± 0.14
Blood2.50 ± 0.411.06 ± 0.480.54 ± 0.050.43 ± 0.09
Thyroid (ID%)0.14 ± 0.010.11 ± 0.050.14 ± 0.050.05 ± 0.03
Tumor/Blood0.860.902.021.29
Tumor/Muscle1.311.441.230.94
Table 2. Biodistribution of 7b in mice bearing S180 tumor (mean ± SD, n = 4, %ID/g).
Table 2. Biodistribution of 7b in mice bearing S180 tumor (mean ± SD, n = 4, %ID/g).
Tissue30 min120 min240 min360 min
Heart1.08 ± 0.240.77 ± 0.170.46 ± 0.060.51 ± 0.11
Liver17.49 ± 4.409.22 ± 1.198.52 ± 0.327.13 ± 0.90
Lung2.25 ± 0.351.15 ± 0.140.95 ± 0.110.79 ± 0.13
Kidney14.54 ± 2.246.52 ± 0.695.18 ± 0.634.58 ± 0.85
Spleen1.12 ± 0.171.58 ± 0.090.61 ± 0.050.45 ± 0.12
Stomach1.04 ± 0.271.00 ± 0.410.87 ± 0.270.94 ± 0.33
Bone1.77 ± 0.241.26 ± 0.930.84 ± 0.310.66 ± 0.32
Muscle0.72 ± 0.100.29 ± 0.060.29 ± 0.050.25 ± 0.13
Intestine5.21 ± 0.193.12 ± 1.012.50 ± 0.832.27 ± 0.36
Tumor1.28 ± 0.460.70 ± 0.160.63 ± 0.150.46 ± 0.08
Blood1.87 ± 0.280.92 ± 0.140.67 ± 0.060.65 ± 0.29
Thyroid (ID%)0.07 ± 0.030.05 ± 0.020.04 ± 0.010.02 ± 0.01
Tumor/Blood0.680.760.950.71
Tumor/Muscle1.792.392.221.83
Table 3. Biodistribution of 7c in mice bearing S180 tumor (mean ± SD, n = 4, %ID/g).
Table 3. Biodistribution of 7c in mice bearing S180 tumor (mean ± SD, n = 4, %ID/g).
Tissue30 min120 min240 min360 min
Heart0.72 ± 0.140.48 ± 0.200.43 ± 0.200.41 ± 0.11
Liver8.70 ± 1.707.54 ± 0.959.58 ± 0.577.08 ± 1.52
Lung2.64 ± 1.231.07 ± 0.141.03 ± 0.441.13 ± 0.42
Kidney15.79 ± 1.3012.23 ± 2.039.84 ± 1.778.48 ± 0.78
Spleen0.85 ± 0.071.58 ± 0.140.64 ± 0.160.61 ± 0.18
Stomach0.63 ± 0.340.32 ± 0.070.24 ± 0.050.36 ± 0.07
Bone1.09 ± 0.330.79 ± 0.290.57 ± 0.260.97 ± 0.46
Muscle0.47 ± 0.180.30 ± 0.070.19 ± 0.060.24 ± 0.11
Intestine2.38 ± 0.301.10 ± 0.320.95 ± 0.550.98 ± 0.35
Tumor1.57 ± 0.310.63 ± 0.180.47 ± 0.110.40 ± 0.06
Blood1.83 ± 0.320.60 ± 0.090.53 ± 0.100.44 ± 0.03
Thyroid (ID%)0.07 ± 0.030.05 ± 0.020.04 ± 0.010.02 ± 0.01
Tumor/Blood0.861.060.900.92
Tumor/Muscle3.362.112.491.70
Table 4. Biodistribution of 7d in mice bearing S180 tumor (mean ± SD, n = 4, %ID/g).
Table 4. Biodistribution of 7d in mice bearing S180 tumor (mean ± SD, n = 4, %ID/g).
Tissue30 min120 min240 min360 min
Heart1.12 ± 0.270.63 ± 0.220.53 ± 0.140.36 ± 0.04
Liver14.21 ± 2.4011.70 ± 4.838.27 ± 2.098.86 ± 2.60
Lung2.72 ± 0.561.39 ± 0.281.21 ± 0.290.96 ± 0.33
Kidney11.40 ± 2.636.79 ± 1.205.24 ± 1.114.38 ± 0.53
Spleen1.46 ± 0.471.58 ± 0.440.77 ± 0.130.65 ± 0.12
Stomach1.24 ± 0.620.31 ± 0.120.26 ± 0.080.28 ± 0.05
Bone2.40 ± 0.590.88 ± 0.310.69 ± 0.220.41 ± 0.18
Muscle0.53 ± 0.100.22 ± 0.050.20 ± 0.080.25 ± 0.10
Intestine4.04 ± 0.791.31 ± 0.420.80 ± 0.300.66 ± 0.11
Tumor1.90 ± 0.580.63 ± 0.140.33 ± 0.050.36 ± 0.16
Blood1.75 ± 0.230.67 ± 0.080.52 ± 0.080.47 ± 0.05
Thyroid (ID%)0.07 ± 0.030.05 ± 0.020.04 ± 0.010.09 ± 0.01
Tumor/Blood1.080.950.640.77
Tumor/Muscle3.562.881.661.48

Share and Cite

MDPI and ACS Style

Duan, X.; Liu, T.; Zhang, Y.; Zhang, J. Synthesis and Biological Evaluation of Novel 99mTc(CO)3-Labeled Thymidine Analogs as Potential Probes for Tumor Proliferation Imaging. Molecules 2016, 21, 510. https://doi.org/10.3390/molecules21040510

AMA Style

Duan X, Liu T, Zhang Y, Zhang J. Synthesis and Biological Evaluation of Novel 99mTc(CO)3-Labeled Thymidine Analogs as Potential Probes for Tumor Proliferation Imaging. Molecules. 2016; 21(4):510. https://doi.org/10.3390/molecules21040510

Chicago/Turabian Style

Duan, Xiaojiang, Teli Liu, Yichun Zhang, and Junbo Zhang. 2016. "Synthesis and Biological Evaluation of Novel 99mTc(CO)3-Labeled Thymidine Analogs as Potential Probes for Tumor Proliferation Imaging" Molecules 21, no. 4: 510. https://doi.org/10.3390/molecules21040510

Article Metrics

Back to TopTop