Introduction

Alzheimer’s disease (AD) is a form of dementia with progressive memory loss, irreversible cognitive decline, language impairment, and behavioral changes. The accumulation of β-amyloid (Aβ) aggregates in the brain is a defining pathologic feature for this disease [1, 2]. Nowadays, clinical diagnosis of AD is primarily done by assessment of clinical symptoms, which is often difficult and unreliable, and the only definitive diagnosis is established by pathological examination of the postmortem staining of affected brain tissue. Single photoemission tomography (SPECT) or positron emission tomography (PET) imaging of β-amyloid plaques in the living human brain may serve as a helpful tool for the early diagnosis of AD and monitoring of therapeutic effects.

Technetium-99m (t1/2 = 6 h, 140 keV) is the most commonly used radionuclide in routine clinical nuclear medicine. Because 99mTc can be easily prepared with a 99Mo/99mTc generator, emits the medium γ-ray energy suitable for γ-camera detection and has the ideal physical half-life compatible with biological localization and residence time required for imaging [3]. New 99mTc-labeled Aβ imaging agents will have more widespread clinical applicability for detecting and eventually quantifying β-amyloid plaques in living brain tissue.

The successful development of [Tc-99m]TRODAT-1 already showed the probability of 99mTc imaging agent that can penetrate the blood–brain barrier(BBB) via a simple diffusion mechanism and localize at sites in the central nervous system [4–6]. Based on this success, many efforts have been made so far to create a suitable Aβ plaques ligand based on 99mTc, but no clinical study of them has been reported [7–16].

(E)-2-(2-(2-(2-[18F]Fluoroethoxy)ethoxy)ethoxy)-5-(4-methylaminostyryl)pyridine ([18F]AV-45) with styrylpyridine core, and high affinity for Aβ plaques (Ki = 2.87 nM), which binds specifically to fibrillar Aβ and has favorable pharmacokinetic properties [17]. Preliminary clinical studies in patients with diagnosed mild AD showed notable retention of [18F]AV-45 in the cortex, known to contain large amounts of amyloid deposits in AD [18, 19]. To develop more useful 99mTc-labeled probes for clinical diagnosis, we synthesized AV-45 derivatives with bis-amino-bis-thiol (BAT).We selected BAT as chelation ligands taking into consideration the permeability of BBB with forming an electrically neutral complex with 99mTc [19].

Here, we report the synthesis of the precursor for labeling by coupling styrylpyridine derivatives(AV-45) with N1,N2-[bis(4-methoxybenzyl) thioethyl] ethanediamine (MBz- BAT),the final free thiol ligands (BAT-AV-45) were obtained by deblocking the 4-methooxybenzyl protecting group with Hg(OAc)2 (Scheme 1). Radiolabeling of BAT-AV-45 was carried outby using stannous chloride method. The quality control was determined by using thin layer chromatography and radio high performance liquid chromatography. The stability, partition coefficient and electrophoresis of 99mTc-BAT-AV-45 were investigated.

Scheme 1
scheme 1

Synthesis of AV-45 derivatives

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Experimental

Materials

All reagents were obtained commercially and used without further purification unless otherwise indicated.(E)-2-(2-(2-(5-(4-(tert-butoxycarbonyl(methyl)amino)styryl)pyridine-2-yloxy)-ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate(Boc-AV-45-OTs) [20] and N1,N2-[bis(4-methoxybenzyl) thioethyl]ethanediamine(MBz-BAT) were self-synthesized. Na99mTcO4 was supplied by Jiangsu Institute of Nuclear Medicine. Electron spray ion(ESI) mass spectra was measured using a Waters Platform ZMD4000LC/MS. NMR spectra was obtained on a Bruker DRX-500 spectrometer, and the chemical shift value was given relative to the internaltetramethylsilane (TMS). Wizard 1470 automatic gamma counter equipped with a multi-channel analyzer (U.S. Perkin Elmer Company). RHPLC was performed on a Waters 600-type high-performance liquid chromatography (the United States Waters Corporation) equipped with a dural λ absorbance detector (Waters 2487), binary HPLC pump (Waters 1525) and Cd (Te) detector equipped with a flow scintillation analyzer (Perkin Elmer).

Synthesis of BAT-AV-45 ligand

(E)-2-(2-(2-(2-(N-(4-methoxybenzylthio)-(N-(N-(4-methoxybenzylthioethyl)aminoethyl))aminoe-thoxy)ethoxy)ethoxy)-5-(4-tert-butoxycarbonylmethylaminostyryl)pyridin(MBz-BAT-AV-45, 1).

Boc-AV-45-OTs (0.21 g, 0.4 mmol) and N1,N2-[bis(4-methoxybenzyl) thioethyl] ethanediamine(MBz-BAT) (0.19 g, 0.45 mmol) were dissolved in anhydrous DMF(10 mL), Potassium carbonate(0.12 g, 0.81 mmol) and Potassium iodide(0.07 g, 0.09 mmol) were added. The reaction mixture was stirred at 60 °C for 24 h. After the evaporation of the solvent, water was added. The mixture was extracted with ethyl acetate. The organic layers were combined and dried with Na2SO4. Evaporation of the solvent gave a residue, which was purified by silica gel chromatography(methanol/methylene chloride/ammonia water;1:20:0.05;V/V/V) to give MBz-BAT-AV-45(0.18 g, 62%). 1H-NMR (500 MHz, CDCl3)δ:8.16(1H, s), 7.76(1H, d, d), 7.42(2H, d), 7.22(6H, m), 6.95(2H, s), 6.81(4H, m), 6.79(1H, d), 4.46(2H, m), 3.81(2H, m), 3.77(1H, m), 3.75–3.60(13H, m), 3.49(2H, t), 2.99(3H, s), 2.95(1H, br), 2.79(6H, br), 2.69(2H, t), 2.63(2H, t), 2.57(2H, t), 1.47(9H, s); MS(ESI):positive mode m/z = 862([M + 1]+).

(E)-2-(2-(2-(2-(N-mercaptoethyl)-(N-(N-mercaptoethylaminoethyl))aminoethoxy)ethoxy)ethoxy)-5-(4-methylaminostyryl)pyridin(BAT-AV-45, 2).

MBz-BAT-AV-45(0.18 g, 0.2 mmol) was dissolved in TFA (10 mL) and anisole (0.05 mL, 0.46 mmol) at 0 °C, and Hg(OAc)2 (0.39 g, 1.22 mmol) was added. The resulting mixture was stirred for 30 min and concentrated in vacuo to obtain a viscous oil that was dried in vacuo for 30 min. Dry ether (10 mL) was then added to the above oil, and the resulting suspension was sonicated for 5 min. The yellow solid that formed was collected by suction filtration, dried in vacuo for 20 min, and dissolved in absolute EtOH (10 mL). H2S gas was passed through the solution for 20 min, and the reaction mixture was filtered through a pad of Celite. The filtrate was concentrated in vacuo to obtain the trifluoroacetate salts of BAT-AV-45, which were used for further reactions without additional purification. MS(ESI):positive mode m/z = 521([M + 1]+).

Radiolabeling procedure

In the labeling of 99mTc-BAT-AV-45, BAT-AV-45(50 μg) was dissolved in 100 μL ethanol in an evacuated nitrogen filled vial. To this solution, sodium glucoheptonate (10 mg) dissolved in 0.2 mL water, stannous chloride (50 μg) dissolved in HCl(0.1 N) were added respectively, pH value(5–6) of the solution was adjusted by using HCl or NaOH. After addition of all reagents, 0.1 mL of [Tc-99 m]pertechnetate solution(~0.5 mCi) and additional some water were added into the vial. Reaction mixture volume used was about 0.9 mL. The vial was incubated at 90 °C in a water bath for 30 min. After cooling down to room temperature, 0.1 mL of injected sodium phosphate solution (0.1 M, pH 7.4) was added. A series of studies were performed to optimize labeling efficiency step by step, such as the amount of BAT-AV-45 ligand, reductant SnCl2, sodium glucoheptonate and the pH value of the reaction mixture.

Quality control of 99mTc-BAT-AV-45

The labeling yield and radiochemical purity were determined by thin layer chromatographic method using polyamide strip. The reaction product was spotted on polyamide strips and developed in methanol/ethyl acetate(1:1; V:V) solution. After developing, the strips were dried at room temperature. Then, they were cut into 0.5 cm pieces and counted by using an automatic γ counter. Retention factor (Rf) and labeling yield were determined from TLC chromatogram data. For comparison, samples of Na99mTcO4, 99mTc-GH and reduced/hydrolyzed 99mTc(RH-99mTc) were also run under identical conditions.

99mTc-BAT-AV-45 was further confirmed by radioHPLC system. The flow rate was maintained at 0.8 mL/min after an injection 10 μL from the reaction mixture into analytical reversed-phase column (Amethyst C18, 4.6 × 150 mm 5 μm, Sepax Technologies, Inc.). The eluting solvent consisted of H2O/acetonitrile (1:4;V:V),and the elution was monitored by observing the radioactivity profile. For comparison, samples of Na99mTcO4, 99mTc-GH were also run under identical condition. The chromatograms for Na99mTcO4, 99mTc-GH, 99mTc-BAT-AV-45 are showing in Fig. 2.

Stability of 99mTc-BAT-AV-45

The stability of freshly prepared 99mTc-BAT-AV-45 at physiological pH was measured at different times at room temperature (~25 °C).

Partition coefficient

A 25 μL aliquot of 99mTc-BAT-AV-45 was added to a test tube containing 3 mL of 1-octanol and 3 mL of 0.02 M phosphate buffer pH 7.4. The test tube was vortexed at room temperature for 5 min and then centrifuged at 3,500 rpm for 10 min. A 100 μL aliquot was taken from the 1-octanol phase and a 100 μL aliquot from the aqueous phase, taking care to avoid cross contamination between the phases and weighed. The radioactivity of the aliquots was counted using an automatic γ counter and the partition coefficient P was calculated using the following equation:

$$ p = \frac{\text{cpm/ml\,octanol}}{\text{cpm/ml\,bufffer}} $$

with cpm = counts per minute.

Experiments were performed at least in triplicate.

Electrophoresis

A 5 μL was spotted on a paper strip (3.5 × 13 cm, Whatman 1 chromatography paper) wetted with a mixture of 0.02 M phosphate buffer pH 7.4 and methanol (50:50, V/V). Electrophoresis was performed during 60 min using the described mixture as the electrolyte solution and an applied voltage of 300 V. After drying, the paper was cut into 0.5 cm strips and the activity on each strip was counted using an automatic γ counter.

Results and discussion

Synthesis and radiolabeling procedure

As shown in Scheme 1, the labeling precursor was synthesized by coupling styrylpyridine derivatives(AV-45) with N1,N2-[bis(4-methoxybenzyl) thioethyl] ethanediamine (MBz- BAT), and then deblocking the protecting group. The key compound MBz-BAT-AV-45 was identified by 1H NMR and MS. The labeling precursor BAT-AV-45 was not additionally purified because of its easy oxidation and just identified by MS.

99mTc-BAT-AV-45 was prepared by a ligand exchange reaction employing the precursor 99mTc-GH. Several reaction parameters were optimized by varying relative reaction parameter step by step, such as the amount of BAT-AV-45 ligand, reductant SnCl2, sodium glucoheptonate and the pH value of the reaction mixture.

The effects of the amount of BAT-AV-45 ligand were summarized in Fig. 1. The data showed that the radiolabeling yield of 99mTc-BAT-AV-45 reached to 95.7 ± 1% at the optimum amount of ligand (50 μg). Below the amount of 50 μg, the labeling yield was low due to the substrate concentrations being insufficient to form complex with all of the reduced technetium.

Fig. 1
figure 1

Effects of reaction parameters on the labeling yield of 99mTc-BAT-AV-45: a the amount of ligand; b the amount of SnCl2; c the amount of sodium glucoheptonate; d the pH value

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The effects of the amount of stannous chloride were shown in Fig. 1. The data showed that the radiolabeling yield was dependent on the amount of stannous chloride, and the highest labeling efficiency was 95.4 ± 1% by using 50 μg of SnCl2. Below the amount of 50 μg, stannous chloride was not sufficient for complete reduction of pertechnetate to form 99mTc-complex because of easy oxidation. Above the amount of 50 μg, the radiochemical yield was decreased. This may be due to the fact that the excess amount of stannous chloride leaded to the formation of stannous hydroxide colloid in basic medium or by oxidation and stannous hydroxide colloid adsorbed the free technetium-99 m or 99mTc-complex.

The effects of the amount of sodium glucoheptonate were summarized in Fig. 1. The data interestingly showed that large amount of sodium glucoheptonate (10 mg) was required for optimal preparation of 99mTc-BAT-AV-45 (RCP .95%), contrast to a minimal amount of ligand. A small amount of sodium glucoheptonate played a transitional ligand role as the form of 99mTc-GH in the radiolabeling procedure. While excess of glucoheptonate with a multi- hydroxyl moiety may facilitate the solubility of BAT-AV-45 ligand by dispersing the compound into reaction solution. Thus 99mTc-GH complex may trans-chelate more effectively with BAT-AV-45 ligand to form 99mTc-BAT-AV-45. Similar results were previously reported in the preparation of [Tc-99m]TRODAT-1 [21].

The effects of pH value were shown in Fig. 1. The data showed that the radiolabeling yield was dependent on the pH value, and the highest labeling efficiency was 95.5 ± 1% with the pH value (5–6). Above the optimum pH value, the radiochemical yield was drastically decreased because of forming RH-99mTc which is the main radiochemical impurities at alkaline medium.

Quality control of 99mTc-BAT-AV-45

In thin layer chromatographic method using methanol/ethyl acetate(1:1;V:V) as solvent, The Rf value of 99mTc-BAT-AV-45 was 0.7–1, and the Rf value of 99mTc-GH was 0–0.2, while RH-99mTc and free Technetium-99m remained at the point of spotting (Rf = 0).The average radiolabeling yield was 95 ± 1%.

HPLC radiochromatograms of Na99mTcO4, 99mTc-GH, 99mTc-BAT-AV-45 are presented in Fig. 2. The radio peak at 2.7 and 4.7 min represent the free Na99mTcO4 and 99mTc-GH respectively. 99mTc-BAT-AV-45 gives one radio peak at 7 min of retention.

Fig. 2
figure 2

RHPLC chromatogram: a Na99mTcO4; b 99mTc-GH; c 99mTc-BAT-AV-45

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Stability of 99mTc-BAT-AV-45

To avoid the radio decomposed side products which may accumulate in non-target organs, the stability of 99mTc-BAT-AV-45 at physiological pH was studied in order to determine the suitable time for injection. The results of the stability of 99mTc-BAT-AV-45 are presented in Fig. 3 . The data clearly shows that 99mTc-BAT-AV-45 is stable (RCP. > 90%) for up to 6 h which is suitable for nuclear medicine applications.

Fig. 3
figure 3

Stability of 99mTc-BAT-AV-45 at physiological pH at different intervals

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Partition coefficient

Previous studies suggested that the optimal lipophilicity for entry into the brain is obtained with log P values of between 1 and 3 [22]. 99mTc-BAT-AV-45 displayed suitable lipophilicity with the value of log P(2.07 ± 0.087) and the results are listed in Table 1.

Table 1 The liquid–water partition coefficient of 99mTc-BAT-AV-45
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Electrophoresis

In electrophoresis experiments the applied radioactivity of 99mTc-BAT-AV-45 remained at the spotted point, which indicates that the compounds is uncharged at physiological pH.

Conclusions

In conclusion, we successfully designed and synthesized novel styrylpyridine derivative conjugated with 99mTc. Satisfactory results were obtained by optimizing the radiolabeling conditions. 99mTc-BAT-AV-45 was prepared at an average yield of 95 ± 1% by mixing 50 μg of BAT-AV-45, 10 mg of sodium glucoheptonate, and stannous chloride (50 μg) dissolved in HCl (0.1 N) at pH value (5–6) at 90 °C for 30 min. 99mTc-BAT-AV-45 with suitable lipophilicity (log P = 2.07 ± 0.087) was stable and uncharged at physiological pH. Further studies are needed to apply 99mTc-BAT-AV-45 for Aβ plaques imaging.