Zn Complex of Diaminedithiol Tetradentate Ligand as a Stable Precursor for 99mTc-Labeled Compounds.

The diaminedithiol (N2S2) tetradentate ligand constitutes a useful chelating molecule for preparing 99mTc-labeled compounds of high in vivo stability in high radiochemical yields. However, since the thiol groups in the N2S2 ligand are easy to be oxidized to disulfide bonds, they need to be protected with an appropriate protecting group, which hinders the broad applications of the N2S2 ligand for radiopharmaceuticals. In this study, a Zn chelate of N2S2 was evaluated as a precursor for purification-free 99mTc-labeled N2S2 under the mild and simple procedure. Zn-N2S2 was prepared by reacting Zn acetate with N2S2, and the Zn-N2S2 remained stable under aerobic conditions at room temperature. 99mTc-N2S2 was obtained over 90% radiochemical yields at room temperature by a one-pot reaction, consisting of Zn-N2S2 (10-5 M), 99mTcO4-, ethylenediaminetetraacetic acid (EDTA), and a reducing agent (Sn2+) at pH = 5.5 to 7.5. 99mTc-N2S2 was also obtained over 90% radiochemical yields when the reaction was conducted in the presence of an equimolar amount of IgG antibody. These findings indicate the Zn complex of N2S2 ligand constitutes a stable and useful precursor to prepare 99mTc-labeled N2S2 compounds in high yields under the mild and simple procedure.

1. Introduction

The growth and broad applications of diagnostic nuclear medicine have been mainly driven by the artificial radionuclide, technetium-99m (99mTc), due to its availability from a portable 99Mo-99mTc generator system and its almost ideal physical properties for external imaging. Although recent efforts are being made to develop radiopharmaceuticals derived from positron emitters such as 11C and 18F, over 70% of diagnostic practices are still conducted with 99mTc. The cost-effectiveness of 99mTc-based radiopharmaceuticals contributes to the medical economy in both developed and developing countries. Thus, 99mTc will continue to be used as one of the essential radionuclides for diagnostic nuclear medicine. Radioactive rhenium (186Re and 188Re), the congener of Tc, emits beta rays appropriate to targeted radiotherapy to form radiotheranostic pairs with their 99mTc counterparts due to the chemical analogy between Tc and Re. Thus, further development of 99mTc-radiopharmaceuticals constitutes a crucial issue for cost-effective and successful patient management.

A tetradentate chelating agent containing two amines and two thiol groups, referred to as a diaminedithiol (DADT) or a bis(amino ethanethiol: BAT), forms a neutral, lipophilic, and stable complex with the pentavalent oxo-Tc and oxo-Re (TcO3+ and ReO3+) [1,2]. Such characteristics render DADT attractive as a coordination molecule for 99mTc- and 186/188Re-labeled compounds [3,4,5,6]. A presentative structure of DADT ligand is shown in Scheme 1, compound 6 (N2S2). Indeed, 99mTc-labeled N,N′-ethylene bis (l-cysteine ethyl ester) has been used as a radiopharmaceutical for measuring cerebral blood flow [7]. However, further applications of DADT ligands are hindered due to the inherent easily oxidized property of the thiol groups during the storage.

Scheme 1

Synthetic procedure for N2S2 and its Zn, Re, and 99mTc complexes.

Some acylating agents have been used to protect the thiol groups in N2S2- or N3S1-type ligands such as mercaptoacetyl glycyl-glycyl-glycine. These include benzoyl [8], acetyl [9], and m-phthalic acid [10]. The deprotection of the benzoyl group is usually conducted at an elevated temperature (e.g., in boiling water) under alkaline conditions (pH > 10) [8,11]. The acetyl protecting group can be removed under milder conditions, whereas a long reaction time (1 h) is needed at room temperature [9]. The m-phthalic acid protecting group can be removed at room temperature in the presence of 1 M of NH2OH [10], which may necessitate post-labeling removal of NH2OH before injection to subjects. Thus, a protecting group that provides a precursor of N2S2 stable against oxidation and generates 99mTc-N2S2 in high radiochemical yields under mild conditions without post- or pre-labeling purification is highly useful for developing new 99mTc-radiopharmaceuticals.

Meanwhile, a transmetallation reaction between Zn chelate of dithiocarbamates (DTCs) and rhenium tricarbonyl has been reported. In this reaction, the thiol groups in the DTCs were stabilized upon complexation with Zn ion, and the direct reaction of the Zn complex with Re(CO)3Br3 provided Re(CO)3-DTCs in high radiochemical yields [12]. These results suggested that Zn ion would also be applicable as a protecting agent for the thiol groups in N2S2 ligands removable during the complexation reaction with 99mTc and Re. Indeed, a prior study showed the formation of Zn-N2S2 chelate [13]. Zn is classified as the least toxic group of all metals by the ICH Harmonized Guideline [14], which is advantageous to clinical applications.

In the present study, the Zn ion was evaluated as a protecting agent for the thiol groups in N2S2 ligands using compound 6. After preparing the Zn complex of 6, the reaction parameters that affected the radiochemical yields of the 99mTc-N2S2 were investigated. The formation of 99mTc-N2S2 from Zn-N2S2 was also evaluated in the presence of an equimolar amount of IgG antibody to estimate the applicability to Zn-N2S2 as a chelating moiety of a bifunctional chelating agent for 99mTc-labeled polypeptides.

2. Results

2.1. Synthesis

The N2S2 ligand 6 was synthesized according to the procedure of Ohmomo et al. as shown in Scheme 1. 4-(Methoxyphyenyl)methanethiol 1 was reacted with ethyl 2-bromo-2-methyl propanoate 2 to prepare 3, followed by the condensation with ethylenediamine to provide 4. After reducing the amide bonds in 4 with BH3, the thiol protecting group in the resulting compound 5 was removed with TFA/anisole/methanesulfonic acid to obtain 6.

The Zn complex of the N2S2 was prepared by mixing Zn acetate with compound 6 in an aqueous solution at neutral pH. The Zn-N2S2 was obtained by extracting the reaction solution with dichloromethane, followed by recrystallization from the mixture of chloroform and hexane in 89% yield. The Zn-N2S2 remained stable at room temperature under aerobic conditions over several days.

The non-radioactive 185/187Re-N2S2 complex was synthesized by reacting 6 with ReO4− in aqueous ethanol using Sn2+ as a reducing agent. After silica-gel column chromatography, the oxorhenium complex of N2S2 was obtained in ca. 20% yield. The structure of this complex was confirmed by mass spectrometry (MS) and infrared spectroscopy (IR).

2.2. 99mTc Complexation Reaction

The reaction of Zn-N2S2 with 99mTc was evaluated at room temperature for future applications as a bifunctional chelating agent for labeling heat-sensitive polypeptides. The reaction parameters for preparing 99mTc-N2S2 from Zn-N2S2, which included the reaction pH, Zn-N2S2 concentration, presence or absence of ethylenediaminetetraacetic acid (EDTA) or glucoheptonate (GH), and the reaction time, were considered. Figure 1 and Figure 2 show typical reversed-phase high-performance liquid chromatography (RP-HPLC) and thin-layer radiochromatography (TLC) of 99mTc-N2S2 and Re-N2S2. The RP-HPLC retention time of 99mTc-N2S2 (8.8 min) was similar to that of 185/187Re-N2S2 (8.6 min) verified by MS, IR, elemental analysis, and proton nuclear magnetic resonance (1H-NMR). These results, along with previous studies [15,16], supported that the 99mTc-N2S2 would possess the chemical structure shown in Scheme 1.

Figure 1

Reversed-phase high-performance liquid chromatography (HPLC) radio-ultraviolet (UV) chromatograms: (a) 99mTc-N2S2; (b) Re-N2S2.

Figure 2

Thin-layer radiochromatography (TLC) of 99mTc-N2S2.

Figure 3 shows the results from a preliminary experiment as a function of reaction parameters. 99mTc-N2S2 was obtained in high radiochemical yields only when the reaction was conducted in the presence of EDTA. The reaction did not proceed without EDTA, while the presence of GH in place of EDTA resulted in low radiochemical yields (Figure 3a). The increase in EDTA concentration in the reaction mixture increased the radiochemical yields of 99mTc-N2S2 (Figure 3b). The radiochemical yields of 99mTc-N2S2 decreased as the reaction pH increased from pH = 5.5 to 7.5. However, the radiochemical yields reached similar to one another at 30 min (Figure 3c).

Figure 3

The radiochemical yields of 99mTc-N2S2 as a function of (a) ethylenediaminetetraacetic acid (EDTA) or glucoheptonate (GH) (pH = 7.5, Zn-N2S2: 1.0 × 10−5 M, EDTA: 1.5 × 10−4 M, GH: 1.5 × 10−4 M); (b) EDTA concentration (pH = 7.5, Zn-N2S2: 1.0 × 10−5 M); (c) reaction pH (Zn-N2S2: 1.0 × 10−5 M, EDTA: 1.5 × 10−4 M).

Figure 4 shows the radiochemical yields of 99mTc-N2S2 under the optimal conditions in the presence or absence of an IgG equimolar amount to that of Zn-N2S2. No significant differences were observed in the radiochemical yields of 99mTc-N2S2 between the two experimental conditions.

Figure 4

The radiochemical yields of 99mTc-N2S2 in the presence or absence of an equimolar amount of IgG (EDTA: 1.5 × 10−4 M, pH = 5.5, Zn-N2S2: 1.0 × 10−5 M, IgG: 0, or 1.0 × 10−5 M).

3. Discussion

99mTc radiopharmaceuticals are usually prepared under sterile conditions by mixing a solution of 99mTcO4−with a kit formulation consisting of a ligand and a reducing agent. The in situ deprotection of N2S2 ligand and subsequent 99mTc complexation reaction is preferable for clinical applications. In this study, the one-pot synthesis of 99mTc-N2S2 from Zn-N2S2 was investigated at room temperature for the applications to heat-sensitive molecules. The Zn-N2S2 concentration of 1.0 × 10−5 M was selected for future applications to imaging the saturable systems of the body [16].

Since the direct reaction of 99mTcO4− and Zn-N2S2 in the presence of a reducing agent, Sn2+, failed to produce 99mTc-N2S2 (Figure 3a), GH was added to the reaction mixture to stabilize the pentavalent 99mTcO3+ against hydrolysis [16]. Under the conditions, small amounts of 99mTc-N2S2 (ca. 10%) were obtained with unchanged radiochemical yields with the reaction time (Figure 3a). These results suggested that GH might rather act as a weak demetallation agent to produce N2S2 from Zn-N2S2. The concentration of GH was 15-times higher than that of Zn-N2S2. The stability constant for Zn-GH was assumed to be close to the stability constant for Zn-gluconic acid (1.70) [17], considering the similar chemical structures and acid deprotonation constants between GH and gluconic acid [18]. Thus, a deprotection agent of higher stability constant with Zn ion was then investigated to assess the working hypothesis.

To facilitate the removal of Zn ion from Zn-N2S2, EDTA was selected due to its high stability constant of 16.44 with Zn ion [19]. As shown in Figure 3a, over 90% radiochemical yields were achieved under the Zn-N2S2 concentration of 10−5 M at room temperature for 30 min. The radiochemical yields of 99mTc-N2S2 increased with an increase in the EDTA concentration (Figure 3b), indicating that the demetallation of Zn from Zn-N2S2 constituted the rate-determining step for the synthesis of 99mTc-N2S2 from Zn-N2S2. It should be noted that EDTA also forms a complex with the trivalent Tc [20,21]. However, since much higher EDTA concentrations (ca. >10−3 M) are needed to prepare 99mTc-EDTA [20,21], EDTA acted as a demetallation agent to generate N2S2 from Zn-N2S2 under the present reaction conditions. These results also indicated that N2S2 preferentially provided its 99mTc complex in high radiochemical yields under the presence of higher concentrations of labile chelating molecules. Indeed, 99mTc-N2S2 was obtained in high radiochemical yields in the presence of an equimolar amount of IgG, as shown in Figure 4. The formation of 99mTc-N2S2 under a wide range of reaction pH rendered Zn-N2S2 applicable to a variety of biomolecules of interest (Figure 3c). The gathered findings indicate that the Zn-N2S2 constitutes a useful precursor to prepare a variety of 99mTc-N2S2-based radiopharmaceuticals at low Zn-N2S2 concentrations under mild reaction conditions by a simple procedure.

4. Materials and Methods

4.1. Materials

All chemicals and an antibody were reagent grade and used without further purification. The pertechnetate-99m solution was obtained from a commercial 99Mo-99mTc Generator (Ultra-Techne Kow, FUJIFILM Toyama Chemical Co., Ltd., Tokyo, Japan).

4.2. Equipment

Radiochemical purities were determined with a Radio-Thin Layer Chromatography (TLC) Analyzer (GITA-STAR, Elysia-RAYTEST, Straubenhardt, Germany). High-performance liquid chromatography (HPLC) analyses were performed using a SHIMADZU model LC-20AD (Kyoto, Japan).

4.3. Syntheses

The N2S2 ligand, 1,1′-(ethane-1,2-diylbis(azanediyl))bis(2-methylpropane-2-thiol) dihydro-chloride, was synthesized according to the procedure of Ohmomo et al. [22].

Zn complex of N2S2 ligand: Under an argon atmosphere, 1,1′-(ethane-1,2-diylbis-(azanediyl))bis(2-methylpropane-2-thiol) dihydrochloride 50.0 mg (0.162 mmol) and anhydrous sodium acetate 19.1 mg (0.233 mmol) were dissolved in H2O (1.3 mL) at room temperature. A mixture of Zn(II) aceteate dehydrate 59.1 mg (0.269 mmol) and anhydrous sodium acetate 19.0 mg (0.232 mmol) in 1.3 mL of H2O was added dropwise to the solution. The reaction mixture was stirred at room temperature for 1.5 h. The solution was extracted with 10 mL of dichloromethane three times. The organic solution was dried over Na2SO4. After filtration, the filtrate was evaporated, and the residue was recrystallized from chloroform and hexane to afford Zn-N2S2 as a white powder. Yield 43.4 mg (89.4%). ESI-MS, C10H22N2S2Zn [M + H]+ m/z 299.06, found: 299.11. Anal. C10H22N2S2Zn·0.6Na2SO4: C, 31.19; H, 5.76; N, 7.28%, found: C, 31.59; H, 5.75; N, 7.09%.

Oxorhenium(V) complex of N2S2 ligand: The N2S2 ligand 62.3 mg (2.01 mmol) in 7 mL of 50% (v/v) ethanol/water was added to a solution of potassium perrhenate 58.2 mg (0.201 mmol) in 30 mL of 50% (v/v) ethanol/water. Tin(II) chloride dehydrate 45.5 mg (0.202 mmol) in 7 mL of 50% (v/v) ethanol/water was then added to the mixture, and the reaction mixture was stirred at room temperature for 2 h. After removing ethanol by evaporation, an aqueous solution of 1 M potassium carbonate was added to bring the reaction pH neutral. The solution was extracted with 100 mL of ethyl acetate three times. The organic solution was dried over Na2SO4. After filtration, the filtrate was evaporated to dryness. The residue was purified by silica gel chromatography eluted with a mixture of dichloromethane and methanol (95:5) to provide a purple crystal of ReO-N2S2. Yield 16.8 mg (19.2%). ESI-MS, Mass C10H21N2OReS2 [M + H]+ m/z 437.07, found 437.19. Anal. C10H21N2OReS2: C, 27.57; H, 4.86; N, 6.43%, found: C, 27.34; H, 4.73; N, 6.22%. IR (KBr) 920 cm−1 (Re=O). 1H-NMR (400 MHz, CD3OD) δ1.46 (s, 6H), 1.60 (s, 3H), 1.80 (s, 3H), 2.99 (br, 2H), 3.37 (br, 2H), 3.76 (br, 4H).

4.4. Technetuim-99m Complexatoin Reaction

All the solutions were bubbled with a stream of N2 before use. A 1-mL solution of pertechnetate-99m (99mTcO4−, 581 ± 185 MBq) was added to 0.5 mL of 0.2 M of sodium phosphate buffer (pH = 5.5, 6.5 and 7.5) and 0.1 mL of Zn-N2S2 (6.39, 38.3 and 230 μg) ethanol solution. Then, 0.5 mL of SnCl2·2H2O (2.1 × 10−4 M) solution or SnCl2·2H2O (2.1 × 10−4 M) containing a transfer ligand (EDTA·2Na·2H2O 6.4 × 10−4 M or GH sodium salt 6.4 × 10−4 M) aqueous solution was added to the mixture. The mixture was stood at room temperature for 5, 15, and 30 min. The effect of EDTA concentrations (1.0, 6.1, and 37 × 10−5 M) on the radiochemical yields was also evaluated.

The radiochemical yields of 99mTc-N2S2 were also determined in the presence of an equimolar amount of IgG at pH = 5.5 in the presence of 1.5 × 10−4 M of EDTA. The concentration of Zn-N2S2 and the IgG were 1.0 × 10−5 M.

4.5. Measurement of Radiochemical Yields of 99mTc-N2S2

Radiochemical yields of 99mTc-N2S2 were determined by TLC method with the C18 reversed-phase TLC plate (NAGEL RP-18W/UV254) eluted with a mixture of acetone and 0.5 M of ammonium acetate (65:35). Under the conditions, 99mTc-N2S2 had a Rf value of 0.6–0.7, while the Rf values of 99mTcO4− and 99mTcO2.nH2O were 0.9 to 1.0 and 0, respectively.

4.6. Characterization of 99mTc-N2S2 and Re-N2S2

The HPLC retention time of 99mTc-N2S2 and Re-N2S2 was compared with SHISEIDO CAPCELL PAK UG120 (5 μm, 4.6 × 150 mm) eluted with 45% (v/v) aqueous methanol at 40 °C under a flow rate of 1 mL/min.

4.7. Statistical Analysis

Results were statistically analyzed using EXSUS Version 8.1. Differences were considered statistically significant when p was < 0.05. A Shapiro–Wilk test was used to determine normality. When data were normally distributed for two groups, a student’s t-test was used. If that was not the case, the nonparametric Wilcoxon’s rank sum test was used.

5. Conclusions

The Zn-N2S2 was easily synthesized, remained stable under aerobic conditions, and provided 99mTc-N2S2 in high radiochemical yields under a mild one-pot reaction at the Zn-N2S2 concentration of 10−5 M. The formation of 99mTc-N2S2 was not hindered by the presence of labile chelators, such as EDTA and an IgG antibody. The gathered findings would facilitate the development of cost-effective kit formulations for 99mTc-radiopharmaceuticals using the N2S2 ligand as the chelating moiety.