2.1.1 N-[2″-(p-methoxybenzylmercapto)ethyl]-2-[(2′-(p-methoxybenzylmercapto) ethyl)(N’-hydroxypropyl)amino] acetamide (2)

N-[2″-(p-methoxybenzylmercapto)ethyl]-2-[(2′-(p-methoxybenzylmercapto)ethyl) amino] acetamide (1) (1.0 g, 2.3 mmol) was dissolved in anhydrous acetonitrile (25 mL), and then 3-bromopropanol (624 μL, 6.9 mmol) and triethylamine (1.6 mL, 11.5 mmol) were added. The mixture was stirred and heated at 90 °C for 24 h under nitrogen. The solvent was removed under reduced pressure. The residue was partitioned between ethyl acetate (50 mL) and water (25 mL). The layers were separated, and the organic extract was concentrated in vacuum. The crude product was purified by column chromatography silica gel (MeOH/CH₂Cl₂ 2/100, v/v) to afford 540 mg of compound 2 as pale yellow oil in 47.7 % yield. MS, m/z: 494 [M + H]⁺; 516 [M + Na]⁺. IR (ν, cm⁻¹): 3332; 2933; 2834; 1657; 1610; 1512; 1463. ¹H NMR (CDCl₃, 400 MHz), δ: 7.23～7.18 (m, 4H), 6.85～6.82 (m, 4H), 3.78 (s, 6H), 3.69～3.65 (q, 6H), 3.44～3.39 (q, 2H), 3.04 (s, 2H), 2.65～2.51 (m, 8H), 1.68～1.61 (m, 2H).

2.1.2 N-[2″-(p-methoxybenzylmercapto)ethyl]-2-[(2′-(p-methoxybenzylmercapto) ethyl)(N’-tosylatepropyl)amino] acetamide (3)

To compound 2 (480 mg, 0.98 mmol) in dry dichloromethane (25 mL), DMAP (239 mg, 1.95 mmol) and triethylamine (272 μL, 1.95 mmol) were added under nitrogen. The mixture was cooled to 0 °C, then p-tosyl chloride (224 mg, 1.17 mmol) was added and the mixture was stirred overnight at room temperature. Next, distilled water (25 mL) was added and the layers were separated. The aqueous phase was extracted with dichloromethane (3 × 20 mL). The combined organic extracts were dried over Na₂SO₄, evaporated and purified by column chromatography silica gel (MeOH/CH₂Cl₂ 1/100, v/v) to give 410 mg of compound 3 in 64.7 % yield. MS, m/z: 647 [M + H]⁺; 669 [M + Na]⁺. IR (ν, cm⁻¹): 3338; 2933; 2835; 1672; 1610; 1512; 1463. ¹H NMR (CDCl₃, 400 MHz), δ: 7.77 (d, J = 8.28 Hz, 2H), 7.65～7.62 (m, 1H), 7.32 (d, J = 8.06 Hz, 2H), 7.23～7.18 (m, 4H), 6.85～6.83 (m, 4H), 4.08 (t, J = 6.04 Hz, 2H), 3.79 (s, 3H), 3.78 (s, 3H), 3.65 (s, 2H), 3.63 (s, 2H), 3.42～3.37 (m, 2H), 2.99 (s, 2H), 2.59 (t, J = 6.71 Hz, 2H), 2.53 (t, J = 6.62 Hz, 4H), 2.50～2.47 (m, 2H), 2.44 (s, 3H), 1.76～1.71 (m, 2H).

2.1.3 9-Desmethyl-dihydrotetrabenazine (5)

N-methylaniline (438 μL, 4 mmol) was added dropwise at 95 °C to a stirred suspension of 80 % sodium hydride (400 mg, 16 mmol) and hexamethylphosphoramide (700 μL, 4 mmol) in dry xylene (20 mL) under nitrogen. After 15 min, dihydrotetrabenazine (4) (638 mg, 2 mmol) was added to the above mixture. The suspension was stirred for 48 h at 95 °C. The reaction mixture was cooled to room temperature, and then 20 % sodium hydroxide aqueous solution (50 mL) was added slowly with stirring. The aqueous phase was separated and washed several times with ether. Next, the aqueous phase was neutralized by 6 M of HCl until the pH reached 7 – 9 and extracted with ether (3 × 50 mL). The combined organic extracts were dried over Na₂SO₄, evaporated and purified by column chromatography silica gel (MeOH/CH₂Cl₂ 4/100, v/v). Yield of 9-desmethyl-dihydrotetrabenazine was 460 mg (75.4 %). MS, m/z: 306.2 [M + H]⁺; 288.2 [M−OH]⁺; 260.1 [M−C2H5O]⁺. IR (ν, cm⁻¹): 3099; 2949; 1609; 1529; 1463. ¹H NMR (CDCl₃, 400 MHz), δ: 6.74～6.55 (m, 2H), 5.47 (s, 1H), 3.84 (s, 3H), 3.41～3.36 (m, 1H), 3.14～2.97 (m, 4H), 2.64 (d, J = 9.27 Hz, 2H), 2.59～2.50 (m, 2H), 2.48～2.42 (m, 1H), 1.98 (t, J = 11.0 Hz, 1H), 1.73～1.61 (m, 3H), 1.09～1.02 (m, 1H), 0.94 (d, J = 6.52 Hz, 3H), 0.91 (d, J = 6.59 Hz, 3H).

2.1.4 p-Methoxybenzyl-monoaminomonoamidedithiol-propyl-dihydrotetrabenazine (6)

To a yellow solution of compound 5 (198 mg, 0.65 mmol) in absolute DMF (20 mL), cesium carbonate (635 mg, 1.95 mmol) was added. The mixture was stirred for 30 min at room temperature. A DMF (5 mL) solution of compound 3 (420 mg, 0.65 mmol) was then added to the resultant orange solution. The mixture was stirred at 120 °C for 24 h under nitrogen, during which the solution turned dark red. The solvent was removed under reduced pressure. The residue was partitioned between ethyl acetate (50 mL) and water (25 mL). The layers were separated. The organic phase was dried over Na₂SO₄ and concentrated in vacuum. The crude product was purified by column chromatography silica gel (MeOH/EtOAc 2/100, v/v) to obtain 180 mg of compound 6 as yellowish oil in 35.5 % yield. MS, m/z: 781 [M + H]⁺; 803 [M + Na]⁺. IR (ν, cm⁻¹): 3335; 2952; 2920; 2868; 2835; 1664; 1609; 1512; 1464. ¹H NMR (CDCl₃, 400 MHz), δ: 7.86～7.77 (m, 1H), 7.20～7.15 (dd, 4H), 6.83～6.81 (d, 4H), 6.67 (s, 1H), 6.59 (s, 1H), 4.03～4.00 (m, 1H), 3.92～3.90 (d, 1H), 3.79～3.77 (d, 9H), 3.61～3.59 (d, 4H), 3.41～3.33 (m, 3H), 3.12～2.96 (m, 6H), 2.69～2.63 (m, 5H), 2.54～2.41 (m, 6H), 1.99～1.90 (m, 6H), 1.62～1.55 (m, 1H), 1.52～1.43 (m, 1H), 1.09～1.01 (m, 1H), 0.94 (d, J = 6.50 Hz, 3H), 0.92 (d, J = 6.47 Hz, 3H).

2.1.5 p-Methoxybenzyl-bisaminodithiol-propyl-dihydrotetrabenazine (7)

Compound 6 (300 mg, 0.38 mmol) was added to 1 M borane-tetrahydrofuran solution (7.6 mL, 7.6 mmol) under nitrogen, and the resulting mixture was heated at reflux overnight. The reaction mixture was cooled to 0 °C, and distilled water was added dropwise until no more gas evolution was observed, and then the mixture was concentrated in vacuo. The residue was added to a mixture of ethanol (20 mL) and 1 M hydrochloric acid (20 mL) and hydrolyzed at 60 °C for 3 h. The solution was then basified with concentrated NH₄OH. The product was extracted into CH₂Cl₂ and purified by column chromatography silica gel (MeOH/EtOAc 1/9, v/v) to produce compound 7 (70 mg, 24 %). MS, m/z: 767.3 [M + H]⁺; 788.7 [M + Na]⁺. IR (ν, cm⁻¹): 3356; 2951; 2834; 1610; 1511; 1464. ¹H NMR (CDCl₃, 400 MHz), δ: 7.20～7.18 (d, 4H), 6.83～6.80 (dd, 4H), 6.71～6.57 (m, 2H), 4.05～3.99 (m, 2H), 3.80 (s, 3H), 3.78～3.76 (d, 6H), 3.63～3.60 (d, 4H), 3.41～3.35 (m, 1H), 3.12～3.10 (br. d, 1H), 3.05～2.96 (m, 3H), 2.76～2.72 (m, 2H), 2.65～2.64 (br. d, 2H), 2.62～2.58 (m, 8H), 2.56～2.52 (m, 4H), 2.46～2.39 (m, 2H), 1.99～1.96 (d, 2H), 1.94～1.87 (m, 2H), 1.75～1.65 (m, 2H), 1.61～1.55 (m, 1H), 1.52～1.43 (m, 1H), 1.10～1.02 (m, 1H), 0.94 (d, J = 6.49 Hz, 3H), 0.92 (d, J = 6.46 Hz, 3H).

2.1.6 Bisaminodithiol-propyl-dihydrotetrabenazine (8)

Compound 7 (70 mg, 0.09 mmol) was dissolved in trifluoroacetic acid (2.1 mL) and anisole (31 μL) at 0 °C, and methanesulfonic acid (310 μL) was added. The resulting mixture was stirred for 1 h under nitrogen and concentrated in vacuo to obtain a viscous oil. Distilled water (15 mL) was then added to the above oil at 0 °C, and the resulting suspension was washed several times with ether (3 × 15 mL). The aqueous phase was basified by sodium bicarbonate and extracted with dichloromethane (2 × 15 mL). The combined organic extracts were dried over Na₂SO₄. Next, dry HCl gas was passed through the above organic phase for 2 min and the organic phase was concentrated in vacuo to obtain the hydrochloride salt of compound 8 (40 mg, 70 %). MS, m/z: 526.3 [M + H]⁺; 492.3 [M−SH]⁺. HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₇H₄₈N₃O₃S₂⁺ 526.3137; found 526.3109. ¹H NMR (CD₃OD, 400 MHz), δ: 6.98～6.84 (m, 2H), 4.49～4.46 (br. d, 1H), 4.20 (br. s, 2H), 3.90～3.85 (m, 5H), 3.83～3.55 (m, 11H), 3.45～3.36 (m, 4H), 3.05～2.89 (m, 4H), 2.46～2.27 (m, 2H), 2.14～1.89 (m, 2H), 1.83～1.69 (m, 3H), 1.21～1.14 (m, 2H), 1.01 (d, J = 5.95 Hz, 3H), 0.98 (d, J = 5.93 Hz, 3H).

2.3.1 Radiochemical purity

The radiochemical purity (RCP) of ^99mTc-BAT-P-DTBZ was estimated by high performance liquid chromatography (HPLC) system. Chromatographic analysis was performed on a Waters liquid chromatography system equipped with a 1525 binary HPLC pump and a Radiomatic 610TR detector. A reversed-phase HPLC column (C18, 5 µm, 4.6 × 150 mm, Waters, Symmetry) was employed, and the column temperature was maintained at room temperature. The mobile phase consisted of acetonitrile, water and triethylamine (48: 52: 0.1, v/v/v) was filtered through a 0.45 µm membrane filter and degassed with ultrasound before use. The flow rate of the mobile phase was kept at 1.0 mL/min under isocratic elution. The sample solution (10 µL) was directly injected into the chromatographic system.

2.3.2 Stability test

The stability of freshly produced ^99mTc-BAT-P-DTBZ was performed in phosphate buffer solution (PBS, pH = 7.4) and fetal bovine serum (FBS). An aliquot of ^99mTc-BAT-P-DTBZ (37 MBq) was added to PBS (2.0 mL) and FBS (2.0 mL) respectively, and the in vitro incubation was kept at 37 °C. The RCP of ^99mTc-BAT-P-DTBZ was determined by the chromatographic method as mentioned above at 0, 1, 2, 4, and 6 h.

2.3.3 Partition coefficient

Partition coefficient was determined by mixing ^99mTc-BAT-P-DTBZ solution with n-octanol and phosphate buffer at room temperature. Two phosphate buffers (pH = 7.0 and 7.4) were adopted for the test respectively. A 10 µL aliquot of ^99mTc-BAT-P-DTBZ (37 MBq/mL) was added into two-phase mixture of 3 mL n-octanol and 3 mL phosphate buffer. The test tube was vortexed for 5 min and then centrifuged for 5 min. Two pipetted samples (1 mL each) from the n-octanol and buffer layers were measured in a gamma counter. Samples from the n-octanol layer were repartitioned and all measurements were tested in quadruplicate. The logP value was obtained by the formula: logP = log(CPM_n-octanol/CPM_PBS).

3.2.1 Effect of the reaction temperature and time

Reaction temperature and time represent the minimum temperature and time required to achieve high RCC. Fig. 4a indicates that the RCCs are all<90 % at 70 °C with increasing the reaction time from 5 to 30 min, indicating that the rate of formation of ^99mTc-BAT-P-DTBZ complex is strongly dependent on reaction temperature. Consequently, the reaction temperature was increased to 85 °C or 100 °C, and the experimental results at the two different temperatures were basically consistent (Fig. 4b and Fig. 4c). The labelling efficiency was approximately 83 % at 5 min. After 10 min, the efficiency remained almost constant with RCC > 90 % and it reached a relative maximum of 92 % at 30 min. Consequently, 30 min was selected as the optimal reaction time. The temperature of 100 °C was chosen as the best reaction temperature due to that this temperature could be easily achieved by boiling water bath.

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Fig. 4

Effect of reaction parameters (a. the reaction time at 70 °C; b. the reaction time at 85 °C; c. the reaction time at 100 °C; d. EDTA-2Na amount; e. GH amount; f. the pH value of the reaction solution; g. labelling precursor amount; h. SnCl₂·2H₂O amount) on the radiochemical conversion (RCC) of ^99mTc-BAT-P-DTBZ.

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3.2.2 Effect of EDTA-2Na amount

Disodium edetate (EDTA-2Na) is commonly used as a stabilizer in technetium-99m labelling research (Mock and Wellman 1984, Meegalla et al., 1997, Ahlgren et al., 2010). Fig. 4d clearly depicts the effect of EDTA-2Na amount on the RCC. The radiolabelling efficiency was almost constant over the entire experiment (0.2–2 mg) and the intermediate amount (1 mg EDTA-2Na) was selected.

3.2.3 Effect of GH amount

Sodium glucoheptonate played a transitional ligand role as the form of ^99mTc-GH in the preparation of ^99mTc-BAT-P-DTBZ. Excess of glucoheptonate with a multi-hydroxyl moiety might facilitate the solubility of labelling precursor by dispersing the compound, and which is conducive for the ligand exchange reaction (Choi et al., 1999). Therefore, the GH amount range of 1–25 mg (much more than the amount of precursor) was investigated in our study. The effect of the GH amount was summarized in Fig. 4e. The results indicated that 10 mg GH is best for maximum RCC (approximately 95 %). The labelling efficiency was not increased significantly in the case of that the amount of GH was more than 10 mg.

3.2.4 Effect of the pH value of the reaction solution

The RCCs are influenced by the variations in pH of reaction solution as shown in Fig. 4f. At pH 2.6, the labelling efficiency was approximately 87 %. The RCCs were increased with the increase of pH. The maximum conversion was near 95 %, while pH was equal to 7.2. This phenomenon may be due to the deprotonation of the labelling precursor at a neutral pH, which is beneficial for the formation of ^99mTc-BAT-P-DTBZ. With the continuous increase of pH, the labelling efficiency began to decrease. At alkaline pH value, the presence of hydroxyl ions in the reaction solution may lead to incomplete reduction of pertechnetate to the desired oxidation state and this could produce an unreliable conversion of ^99mTc-BAT-P-DTBZ. Therefore, pH = 7.2 is the optimum radiolabelling condition.

3.2.5 Effect of labelling precursor amount

DTBZ, a bioactive molecule, was labelled with technetium-99m using the indirect technique in which the reduced technetium-99m reacted with the BAT group in the DTBZ derivative structure to form a radioactive complex. The labelling precursor amount actually influenced the RCC as shown in Fig. 4g. The reaction was carried out at different labelling precursor amounts (10–200 µg). At the low amount of precursor (<25 µg), the RCC was relatively low due to that the precursor concentration was insufficient to form complex with all of the reduced technetium-99m. At the amount of 25–200 µg, the conversion varied in the range of 94–96 %. After comprehensive consideration, 50 µg was chosen as the optimum ligand amount required to obtain maximum RCC.

3.2.6 Effect of SnCl₂·2H₂O amount

Stannous chloride (SnCl₂·2H₂O) is usually used as reducing agent for the reduction of the pertechnetate, which favors the chelation of technetium-99m with labelling precursor. Fig. 4h demonstrates the variation of the labelling efficiency with altered amount of SnCl₂·2H₂O. In the whole experiment (10–100 µg), the labelling efficiency was more than 90 %. Our findings indicated that the most favorable labeling efficiency with a RCC of approximately 97 % was found at the range of 10–20 µg. At the amount of 20–100 µg, the labelling efficiency showed a slightly downward trend. This phenomenon might be explained by the formation of tin colloid (Eid Moustapha et al., 2016, Shahzadi et al., 2019). The raise of SnCl₂·2H₂O amount may lead to consume the reduced pertechnetate for the formation of ^99mTc-sn-colloid, which results in a decline in RCC of ^99mTc-BAT-P-DTBZ. In this test, 10–20 µg SnCl₂·2H₂O was the optimal amount that reduced the maximum amount of pertechnetate to offer the maximum labelling efficiency. Considering the vulnerability of stannous chloride to oxidation, 20 µg SnCl₂·2H₂O was selected as the optimum amount.

3.3.1 Radiochemical purity

Radiochemical purity of ^99mTc-BAT-P-DTBZ was estimated by an HPLC method. The retention time of ^99mTc-BAT-P-DTBZ was 14.9 min, while the undesirable radiochemical species such as free ^99mTcO₄⁻, ^99mTc-radiocolloid and ^99mTc-GH appeared at 1.2, 1.3 and 1.1 min, respectively. The details were depicted in the radiochromatograms (Fig. 5).

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Fig. 5

Radio-HPLC chromatograms of ^99mTc-BAT-P-DTBZ and its possible radiochemical impurities.

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For quality control of the RCP, in addition to the undesirable radiochemical species including free ^99mTcO₄⁻, ^99mTc-radiocolloid and ^99mTc-GH, we also investigated other possible impurity of ^99mTc-BAT-P-DTBZ. Ether bond could be hydrolyzed without any catalyst under high temperature (Yoon et al., 2018). The radiolabelling of compound 8 was performed through boiling water bath and the hydrolysis of phenol ether bond might occur. Probable degradation route of compound 8 was illustrated in Scheme 3A. The hydrolysis product bisaminoethanethiol-propanol (BAT-NPA) may form a possible impurity ^99mTc-BAT-NPA during radiolabelling procedure. Therefore, we prepared this possible impurity as a reference (Scheme 3B). In the radiochromatogram, the retention time of ^99mTc-BAT-NPA was 2.2 min as shown in Fig. 5. Fortunately, no trace of this possible impurity was observed during the preparation of ^99mTc-BAT-P-DTBZ. In other words, the phenol ether bond in compound 8 is stable at ∼ 100 °C.

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Scheme 3

(A) The hypothetical degradation route of compound 8. (B) Preparation of ^99mTc-BAT-NPA. (a) 1 M Borane-THF, 80 °C, overnight. (b) TFA, anisole, methanesulfonic acid, 0 °C to room temperature, 1 h. (c) Na^99mTcO₄, GH, SnCl₂·2H₂O, EDTA-2Na, citric acid-sodium citrate buffer solution, 100 °C, 30 min.

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3.3.2 Stability test

The stability of ^99mTc-BAT-P-DTBZ was carried out to detect any dissociation of the title complex and to find the suitable time for the use of ^99mTc-BAT-P-DTBZ to avoid the formation of undesired radiolysis product. For this purpose, ^99mTc-BAT-P-DTBZ was incubated in PBS (pH = 7.4) and FBS at 37 °C for 6 h respectively. At each time point, the RCP was the mean value of three repeated measurements. The results showed that ^99mTc-BAT-P-DTBZ is stable (RCP was approximately 96 %) over the period of 6 h (Fig. 6), indicating successful appositeness for the needs of late-stage biological evaluation experiments.

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Fig. 6

In vitro stability of ^99mTc-BAT-P-DTBZ in phosphate buffer solution (a) and fetal bovine serum (b).

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3.3.3 Partition coefficient

The ability of a compound to be freely across the blood–brain barrier (BBB) is related to its lipophilicity. To verify the potential of ^99mTc-BAT-P-DTBZ as a brain tracer, the lipophilicity test was performed by the “shake flask” method. The technetium-99m-labelled dihydrotetrabenazine was added to a two-phase mixture of n-octanol and phosphate buffer and then its distribution across the two phases was determined. The results were listed in Table S2. All experiments were performed with four extractions. The values of LogP_7.0 and LogP_7.4 were found to be 1.69 ± 0.04 and 1.69 ± 0.01, respectively (n = 3, the values of the first ones in Table S2 were discarded due to the largest error). According to the literature (Dischino et al., 1983), the optimum LogP value for penetrating BBB is in the range of 0.9–2.5. In other words, the tracer molecules falling within this range may be free to diffuse across the BBB. Therefore, ^99mTc-BAT-P-DTBZ might be suitable for permitting intercalation into the lipid bilayer of the endothelial cells and then crossing BBB.