Bioorganic & Medicinal Chemistry Letters 24 (2014) 3964–3967

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Preparation and biodistribution of a 99mTc tricarbonyl complex with deoxyglucose dithiocarbamate as a tumor imaging agent for SPECT Xiao Lin a, Xiaoyu Chao a, Junbo Zhang a,⇑, Zhonghui Jin b,⇑, Yanyan Zhang b a b

Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China Nuclear Medicine Department, Peking University 3rd Hospital, Beijing 100191, PR China

a r t i c l e

i n f o

Article history: Received 2 December 2013 Revised 9 June 2014 Accepted 12 June 2014 Available online 21 June 2014 Keywords: 99m Tc tricarbonyl complex Tumor imaging agent Deoxyglucose Dithiocarbamate

a b s t r a c t The deoxyglucose dithiocarbamate (DGDTC) was successfully labeled with the 99mTc(CO)3 core to provide the corresponding 99mTc(CO)3–DGDTC complex in good yields. The radiochemical purity of the 99m Tc(CO)3–DGDTC complex was over 90%, as measured by high performance liquid chromatography (HPLC). The complex possessed good stability in saline at room temperature and in mouse plasma at 37 °C. Its partition coefficient result indicated that it was a hydrophilic complex. The electrophoresis results showed the complex was neutral. The biodistribution of 99mTc(CO)3–DGDTC in mice bearing S 180 tumor showed that the complex clearly accumulated in tumor, exhibiting high tumor/blood and tumor/muscle ratios and good tumor retention. Single photon emission computed tomography (SPECT) image studies showed there was a visible uptake in tumor sites, suggesting 99mTc(CO)3–DGDTC could be considered as a potential tumor imaging agent. Ó 2014 Elsevier Ltd. All rights reserved.

As well known, an enhanced glucose uptake and consumption is observed in tumor cells than in normal cells. Most notably, [18F]fluorodeoxyglucose (18F-FDG) is the most widely used agent for the diagnosis of tumor.1–3 However, the necessity of the presence of a nearby cyclotron and high cost of the [18F] isotope restrict its wide use in clinical practice. By comparison, generator produced isotope, such as 99mTc, has low cost and is readily available and affordable. The availability of a generator and kit chemistry to prepare 99mTc based radiopharmaceuticals may have a significant impact on nuclear medicine. Thus, using 99mTc to label glucose analogues is the major focus of ongoing research. To date, several 99m Tc labeled glucose derivatives have been reported by many groups.4–11 However, none of them exhibited the ideal properties. Thus, further investigation in this area is still necessary. Recently, we have reported the synthesis of deoxyglucose dithiocarbamate (DGDTC) and its 99mTc labeling using the 99mTcN core and 99mTcO core as targeted agents for tumor imaging.12,13 However, the tumor uptake of the 99mTcN-DGDTC was not high enough and 99mTcO-DGDTC had a relative lower tumor/blood ratio. Up to now, 99mTc(CO)3+ core has been an attractive core due to its small size, high stability and convenient preparation method. Bearing in mind the presence of the [99mTc(CO)3]+ core in the molecular structure of a radiopharmaceutical may possibly improve its biological behavior. As part of our ongoing research in the application ⇑ Corresponding authors. Tel.: +86 10 62208126; fax: +86 10 62205562. E-mail addresses: [email protected] (J. Zhang), [email protected] (Z. Jin). http://dx.doi.org/10.1016/j.bmcl.2014.06.037 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

of different 99mTc cores, the synthesis and biological evaluations of the 99mTc(CO)3–DGDTC complex as a potential agent to target tumor are reported for the first time. The sodium salt of deoxyglucose dithiocarbamate (DGDTC) was prepared by reacting 2-amino-2-deoxy-D-glucose hydrochloride with an equivalent amount of carbon disulfide in NaOH solutions.12 The preparation of 99mTc(CO)3–DGDTC was carried out using the following procedure in Scheme 1.14 The DGDTC ligand is a bidentate chelator which has two sulfur atoms that can easily replace the two H2O molecules in the fac[99mTc(CO)3(H2O)3]+ precursor. With the displacement of the H2O molecules, 99mTc(CO)3–DGDTC was formed. The reaction of [M(CO)3]+ (M = Re, Tc) with dithiocarbamate ligands leads to complexes of the structure shown in Scheme 1. The radiochemical purity of the complex was routinely checked by HPLC.15 The retention time of [99mTc(CO)3(H2O)3]+ was 17.21 min, while that of 99m Tc(CO)3–DGDTC was found to be 3.25 min (Fig. 1). A single product with high radiochemical purity (>95%) was obtained. To verify the proposed structure of 99mTc(CO)3–DGDTC, corresponding Re(CO)3 analogue was also synthesized according to the literature method.16–18 After co-injection 99mTc(CO)3–DGDTC and its Re(CO)3 analogue, the HPLC elution profile of Re(CO)3-analogue nearly matched with the corresponding 99mTc(CO)3–DGDTC complex (Fig. 2), suggesting 99mTc(CO)3–DGDTC possesses the proposed structure. The stability of the complex was assayed by measuring the radiochemical purity by HPLC at different times after preparation. The complex was stable over 6 h in the reaction mixture at

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X. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3964–3967

CO 99m

TcO4-

0.9%NaCl/Na2CO 3

+ OH2

OC Tc

1atm CO/NaBH4

OC

DGDTC

OH2 OH2 OH HO

OC

OH 2

S

Tc OC

S CO

O

N H

OH OH

Scheme 1. Preparation procedure and proposed structure of

99m

Tc(CO)3–DGDTC.

room temperature. The stability in mouse plasma was determined by incubating 99mTc(CO)3–DGDTC in the solution of murine plasma at 37 °C for 4 h. Plasma proteins were precipitated by adding acetonitrile and removed by centrifugation. The supernatant was injected in HPLC to determine the stability of the complex. No decomposition of 99mTc(CO)3–DGDTC occurred over 4 h at 37 °C in mouse plasma. The partition coefficient was determined by mixing the complex with an equal volume of 1-octanol and phosphate buffer (0.025 mol/L, pH 7.4) in a centrifuge tube. The measurements were repeated three times and reported as an average of three measurements plus the standard deviation. The partition coefficient (log P) value of 99mTc(CO)3–DGDTC was calculated as 0.96 ± 0.03, suggesting it was hydrophilic. As compared to 99mTcN-DGDTC and 99m TcO-DGDTC, the log P of the 99mTc(CO)3–DGDTC complex was more than that of 99mTcN-DGDTC (log P = 1.30), but was less than that of 99mTcO-DGDTC (log P = 0.73). Paper electrophoresis was performed by using an already established procedure.12 The electrophoresis results showed that 99mTc(CO)3–DGDTC remained at the point of spotting (percentage of radioactivity >90%), suggesting it was a neutral complex, which was in accordance with the proposed structure. Murine sarcoma S180 cell lines were extracted from tumor-bearing mice. In vitro cell uptake assay was assessed and a blocking experiment using D-glucose was conducted according to the literature method.13 Cellular uptake of the 99mTc(CO)3–DGDTC complex was increasing through time with the highest value of 7.60% at 4 h post-incubation (Fig. 3). Adding D-glucose at a concentration of 1–2 mg per well led to a decreased uptake of 99mTc(CO)3–DGDTC

Figure 1. HPLC pattern of

99m

Figure 2. HPLC pattern of

99m

Tc(CO)3–DGDTC and Re(CO)3-DGDTC.

in murine sarcoma S180 cells (Fig. 4). The results suggest 99m Tc(CO)3–DGDTC is transported via the glucose transporters. Biodistribution studies were carried out on Kunming male mice (weighing 18–20 g) bearing S 180 tumor. The tumors were allowed to grow till they reached 1.0–1.5 cm in diameter. Animal studies were carried out in compliance with the national laws related to the ethics during experimentation. 0.1 ml of 99mTc(CO)3–DGDTC (0.74 MBq) was injected via a tail vein and the injected radioactivity was measured with a well-type NaI(Tl) detector. The mice were killed in group of five at 0.5 h, 2 h and 4 h post-injection. The tumor, other organs of interest and blood were collected, weighed and measured for radioactivity. The results were expressed as the percent uptake of injected dose per gram of tissue (%ID/g). The final results are expressed as mean ± standard deviation. The results of biodistribution of 99mTc(CO)3–DGDTC are shown in Table 1. As noted from Table 1, 99mTc (CO)3-DGDTC has a significant uptake and good retention in tumor. The blood and muscle clearance is faster than that of the tumor, therefore the tumor/blood and tumor/muscle ratios increase with time. The kidney uptake is appreciable and is little higher than that of the liver, suggesting the main routes of excretion are via the urinary tract and through the hepatobiliary. As seen from Table 2, among the three complexes, 99mTcNDGDTC is more hydrophilic than the others, thus possibly making

Tc(CO)3–DGDTC (A), fac-[99mTc(CO)3(H2O)3]+ precursor (B).

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X. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3964–3967

Figure 3. In vitro cell uptake of

99m

Tc(CO)3–DGDTC.

Table 2 Comparison of

Figure 4. In vitro cell uptake of D-Glucose was administered.

Table 1 Biodistribution of

99m

99m

99m

TcN-DGDTC,

99m

Tc(CO)3–DGDTC and

99m

TcO-DGDTC

Complex

99m

99m

Time p.i.(h) Log P Tumor uptake (%ID/g) Liver uptake (%ID/g) Kidney uptake (%ID/g) T/N ratio T/B ratio

4

4

4

1.30 1.16 ± 0.57

0.96 2.77 ± 0.11

0.73 3.53 ± 0.85

2.00 ± 0.34

4.08 ± 0.38

15.29 ± 5.22

7.71 ± 1.40

5.21 ± 0.51

15.12 ± 3.56

1.68 2.32

7.29 1.67

4.21 0.76

TcN–DGDTC

Tc(CO)3–DGDTC

99m

TcO–DGDTC

Tc(CO)3–DGDTC when different amount

a

Tc(CO)3–DGDTC in mice bearing S 180 tumor (%ID/g)

Tissue

0.5 h

2h

4h

Heart Liver Lung Kidney Brain Spleen Bone Muscle Blood Tumor T/N ratio T/B ratio

1.45 ± 0.17 5.03 ± 0.58 3.59 ± 0.51 7.74 ± 0.45 0.17 ± 0.03 1.50 ± 0.23 1.17 ± 0.07 0.66 ± 0.06 3.42 ± 0.19 2.22 ± 0.32 3.36 0.65

1.44 ± 0.47 6.43 ± 0.57 3.41 ± 0.94 6.88 ± 0.43 0.19 ± 0.06 1.40 ± 0.51 1.15 ± 0.27 0.67 ± 0.20 2.08 ± 0.12 2.70 ± 0.21 4.03 1.30

0.89 ± 0.40 4.08 ± 0.38 2.23 ± 0.66 5.21 ± 0.51 0.12 ± 0.04 0.85 ± 0.30 0.71 ± 0.50 0.38 ± 0.11 1.66 ± 0.04 2.77 ± 0.11 7.29 1.67

T/N = tumor-to-muscle, T/B = tumor-to-blood. a All data are the mean percentage (n = 5) of the injected dose of DGDTC per gram of tissue, ±the standard deviation of the mean.

99m

Tc(CO)3– Figure 5. SPECT image of tumor.

TcN-DGDTC lower tumor and liver uptakes than 99mTcO-DGDTC and 99mTc(CO)3–DGDTC. By comparison, 99mTc(CO)3–DGDTC shows much higher tumor uptake and the tumor-to-muscle (T/N) ratio than 99mTcN-DGDTC, but the tumor-to-blood (T/B) ratio of 99m Tc(CO)3–DGDTC is less than that of 99mTcN-DGDTC. As compared to 99mTcO-DGDTC, 99mTc(CO)3–DGDTC has a higher T/B ratio and T/N ratio, whereas 99mTcO-DGDTC has a little higher tumor uptake than 99mTc(CO)3–DGDTC.

99m

Tc(CO)3–DGDTC in a rabbit bearing hepatic VX2

99m

0.1 ml of 99mTc(CO)3–DGDTC (185 MBq) was injected intravenously through ear vein in a rabbit (weighing 2–3 kg) bearing hepatic VX2 tumor. A dual-head SPECT (Skylight; Philips, Milpitas, CA, USA), using a low-energy parallel-hole collimator, was used for SPECT imaging studies. Static images were acquired at 4 h after injection. The SPECT imaging results showed the tumor uptake

X. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3964–3967

was clearly observable (Fig. 5), however, the liver and kidney uptakes of 99mTc(CO)3–DGDTC are appreciable, thus making it unsuitable to localize tumor in liver and kidney. The imaging data were in accordance with the biodistribution results in mice. In summary, DGDTC was successfully radiolabeled with [99mTc(CO)3(H2O)3]+ precursor in high yield. The biodistribution studies in mice bearing tumor showed that the 99mTc(CO)3–DGDTC complex exhibited the most promising properties for tumor imaging as compared to 99mTcN-DGDTC and 99mTcO-DGDTC, suggesting the incorporation of the [99mTc(CO)3]+ core into the DGDTC ligand can improve the biological features for tumor imaging. In the present case, it may be of great interest to further probe the 99m Tc(CO)3–DGDTC complex as a novel potential tumor imaging agent. Acknowledgments The work was financially supported, in part, by National Natural Science Foundation of China (21171024, 81101069), Beijing Natural Science Foundation (7112035), Fundamental Research Funds for the Central Universities (105566), and Beijing Nova Program (xxhz201306). References and notes 1. 2. 3. 4.

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