Introduction

The sugar-based PET radiotracer [18F]fluorodeoxyglucose (18F-FDG), the golden 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 inflammatory [1–4]. Currently, great researches are being performed, with the aim to develop the alternative tracer of 18F-FDG. So far, there has been an increasing interest on a wide range of radiolabeled amino acids, because they are the substrates of various amino acids transport systems, which can be upregulated in certain tumors. Recently, since the success of 6-[F-18]fluoro-3,4-dihydroxy-l-phenylalanine (18F-DOPA) for tumor imaging, many studies have been focused on 18F labeled l-phenylalanine derivates [5–13]. However, the limited availability (requiring cyclotron) and high costs of 18F restrict its wide application in clinical practice, especially in many developing countries. Compared to radionuclide 18F, 99mTc is more widely accessible because it can be produced from a commercial 99Mo–99mTc generator and 99mTc labeled imaging agents can be easily prepared by using available kits, thus making 99mTc labeled tracers the mainstay of nuclear medicine imaging worldwide. Recently, we have reported the synthesis and evaluation of 99mTcN–PRODTC (PRODTC: proline dithiocarbamate) as a potential tumor imaging agent [14]. As reported, 99mTcN–PRODTC can be synthesized through easy procedures and exhibited relatively high tumor-to-muscle and tumor-to-blood ratio at 2 h post-injection. However, the uptake and retention in tumor of 99mTcN–PRODTC was not satisfactory. Thus, it may be of great interest to probe other better 99mTc labeled amino acids derivatives for tumor imaging. Among all the radiolabeled amino acids, radiolabeled aromatic amino acids have the tendency to be more suitable for tumor imaging [15]. Bearing in mind l-phenylalanine is a natural aromatic amino acid and its molecular structure has an active amine group, thus making it possible to react with carbon disulfide in NaOH solutions to produce the phenylalanine dithiocarbamate (PHEDTC). Based on our previous reported work [16, 17], it can be assumed that PHEDTC can also be used to form stable 99mTcO and 99mTcN complexes on the basis of efficient binding of the group to four sulfur atoms. This background encouraged us to synthesize 99mTcO–PHEDTC and 99mTcN–PHEDTC by ligand-exchange reaction to find good tumor imaging agents. In this study, the synthesis and biological evaluation of novel 99mTc-oxo and 99mTc-nitrido complexes with PHEDTC for tumor imaging are reported for the first time.

Experimental

Materials and methods

l–phenylalanine was purchased from J&K CHEMICA, China. Succinic dihydrazide (SDH) kit and glucoheptonate (GH) kit were obtained from Beijing Shihong Pharmaceutical Center, Beijing Normal University, China. All other chemicals were of reagent grade and were used without further purification. 99Mo/99mTc generator was obtained from the China Institute of Atomic Energy (CIAE). IR spectrum was obtained with an AVATAR 360 FT-IR spectrometer using KBr pellets. NMR spectrum was recorded on a 500 MHz Bruker Avance spectrophotometer. Elemental analyses were performed on a Vario EL elemental analyzer model.

Synthesis of phenylalanine dithiocarbamate (PHEDTC)

The synthesis of PHEDTC was carried out according to the literature [18]. Carbon disulfide (0.35 mL, 5.70 mmol) was dissolved in 5 mL anhydrous diethyl ether and cooled to 0 °C in an ice bath. l-phenylalanine (936 mg, 5.70 mmol) and NaOH (456 mg, 11.4 mmol) were dissolved in 10 mL anhydrous methanol and added dropwise to the carbon disulfide solution. The mixture was stirred for 3 h in an ice bath. The solvent was removed and the resulting residue was triturated with diethyl ether. The white solid was filtered and washed with Et2O and dried in vacuo to yield PHEDTC as a pale yellow solid (0.631 g, 39 %).

Radiolabeling of 99mTcO–PHEDTC and 99mTcN–PHEDTC and quality control techniques

99mTcO–PHEDTC and 99mTcN–PHEDTC were prepared by using GH kit and SDH kit, separately.

For preparing 99mTcO–PHEDTC, 1 mL of saline containing 99mTcO4 − (370 MBq) was added to a GH kit containing 0.1 mg of stannous chloride dihydrate, 20.0 mg of GH. The reaction vial was incubated at room temperature for 15 min. Successively, 1.0 mg of PHEDTC dissolved in 1.0 mL water was added and the resulting solution was heated at 100 °C for 30 min.

As for preparing 99mTcN–PHEDTC, 1 mL of saline containing [99mTcO4]− (370 MBq) was added to a SDH kit, which contained 0.05 mg of stannous chloride dihydrate, 5.0 mg of SDH and 5.0 mg of propylenediamine tetraacetic acid (PDTA). The mixture was kept at room temperature for 15 min. Then, 1.0 mg of PHEDTC dissolved in 1.0 mL water was added to the mixture and the reaction vial was incubated for 20 min on a boiling water bath.

The radiochemical purities of the complexes were routinely checked by thin layer chromatography (TLC). TLC was performed on a polyamide strip and eluted with saline and acetonitrile, respectively.

In vitro stability study

99mTcO–PHEDTC and 99mTcN–PHEDTC were kept in the labeling milieu at room temperature (25 °C) and the radiochemical purities were assayed by TLC analysis for up to 6 h after labeling. To test the stabilities of the complexes in serum, 0.5 mL of each labeled complex was separately incubated in 1.0 mL human serum (1 mg/mL) at 37 °C for 4 h and then the radiochemical purity of each complex was analyzed by TLC.

Human serum albumin binding assay

10 µL of labeled complex (370 KBq) was added in 100 µL human serum albumin (100 mg/mL) in the centrifuge tube. After the mixture was incubated at 37 °C for 2 h, the serum protein was precipitated by adding 1 mL trichloroacetic acid (250 mg/mL) to the mixture. The supernatant and precipitate were separated by centrifugation at 2,000 g for 5 min. Then the radioactivity of each phase was measured separately. This experimental procedure was repeated three times and the percentage of human serum protein binding was determined as the following equation:

$$ {\text{Serum protein binding}}\;\% = {{{\text{(counts per minute in precipitate)}}} \mathord{\left/ {\vphantom {{{\text{(counts per minute in precipitate)}}} {{\text{(counts per minute in precipitate + counts per minute in supernatant)}}}}} \right. \kern-\nulldelimiterspace} {{\text{(counts per minute in precipitate + counts per minute in supernatant)}}}} \times 100 \% . $$

Determination of the partition coefficient

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, containing 2 mL of each phase, 0.1 mL of the labeled complex solution was added, and the mixture was shacked on a Vortex mixer for 1 min and then centrifuged at 5,000 g for 5 min. Three samples (0.1 mL each) from each layer were counted in a well gamma γ-counter. The partition coefficient, P, was calculated as the mean value of counts per minute in octanol divided by that of the buffer. Usually the final partition coefficient value was expressed as log P. The log P value was reported as an average of three measurements plus the standard deviation.

Biodistribution study

All biodistribution studies were carried out in compliance with the national laws related to the conduct of animal experimentation. 0.1 mL of 99mTcO–PHEDTC (7.4 × 105 Bq) was injected into the Kunming male mice (18–20 g) bearing S180 tumor via a tail vein. The mice were sacrificed in groups of five at 2 and 4 h post-injection. The tumor, other organs of interest and blood were collected, weighed and measured for radioactivity. The counting tubes, including a standard equivalent to 1 % of the injected dose, were assayed in a well-type NaI (Tl) detector and 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 biodistribution study of 99mTcN–PHEDTC was conducted in the same way.

Results and discussion

Synthesis and radiolabeling

PHEDTC was prepared by reacting l-phenylalanine with an equivalent amount of carbon disulfide in NaOH solutions. The reaction equation is shown in Scheme 1. It was characterized by IR, 1H NMR, 13C NMR and Elemental analysis. Specific rotation [α] 25D (H2O) = +53.9°. IR(KBr)/cm−1: 3418 (N–H), 1651 (C=O), 1614 (C=C), 1111 (C=S). 1H-NMR (D2O) δ: 7.11–7.26 (m, 5H), 4.77–4.80 (dd, 1H), 2.88–3.17 (m, 2H). 13C-NMR (D2O) δ: 210.54, 177.79, 137.55, 129.342, 129.37, 129.02, 128.57, 126.74, 63.61, 37.24. Elemental analysis calculated (%) for C10H9NNa2O2S2·0.5H2O: C, 40.82; N, 4.76; H, 3.40. Found: C, 41.21; N, 4.82; H, 3.98.

Scheme 1
scheme 1

Synthesis of PHEDTC The preparations of 99mTcO–PHEDTC and 99mTcN–PHEDTC were carried out using the following procedure in Scheme 2

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Scheme 2
scheme 2

Preparation procedures and speculative structures of 99mTcO–PHEDTC and 99mTcN–PHEDTC

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For preparing 99mTcO–PHEDTC, the method is based on the reaction of [99mTcO4]− with GH in the presence of stannous chloride as reducing agent to form 99mTc(V)–GH which contains the [99mTcO]3+ core and 99mTc(V)–GH is a suitable substrate for the substitution reaction with PHEDTC to produce the final complex 99mTcO–PHEDTC.

As for 99mTcN–PHEDTC, it was prepared by adding PHEDTC to the [99mTcN]2+ intermediate, which was produced by the reaction of [99mTcO4]− with SDH in the presence of stannous chloride as reducing agent. The [99mTcN]2+ core is an appropriate substrate for the substitution reaction with PHEDTC to obtain 99mTcN–PHEDTC with high yield.

Based on a previous characterization of the molecular structure of 99mTc(V)O-DMSA (DMSA: dimercaptosuccinic acid) and bis(N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium-99m complex [99mTcN(NOEt)2] [19–21], it can be presumed that 99mTcO–PHEDTC or 99mTcN–PHEDTC would have a square pyramidal geometry with an apical Tc=O bond or Tc≡N bond and two PHEDTC ligands spanning the four positions in the basal plane through the four sulfur atoms. Clearly, its detailed structure needs to be determined by further research work.

The radiochemical purities of the complexes were routinely checked by TLC. For 99mTcO–PHEDTC, in saline, 99mTcO4 − and 99mTcO–PHEDTC migrated at R f = 0.1 while 99mTc–GH migrated at R f = 0.8–1.0. In acetonitrile, 99mTcO4 − migrated at R f = 0.3–0.5, while 99mTcO–PHEDTC and 99mTc–GH migrated at R f = 0.1. For 99mTcN–PHEDTC, in saline, 99mTcO4 − and 99mTcN–PHEDTC remained at the origin while [99mTcN]2+ migrated with the front. In acetonitrile, 99mTcO4 − migrated at R f = 0.3–0.5, while 99mTcN–PHEDTC and [99mTcN]2+ remained at the origin.

Stability study

The radiochemical purities of 99mTcO–PHEDTC and 99mTcN–PHEDTC were both over 90 % at 6 h after preparation. On the other hand, in serum at 37 °C, the radiochemical purities of the complexes were higher than 90 % even up to 4 h after synthesis, suggesting they possessed a great stability in human serum.

Human serum albumin binding assay

As described in the experiment of human serum albumin binding assay [14], the percentage of human serum protein binding ability for both complexes was measured and calculated. The results showed that the percentage of human serum protein binding of 99mTcO–PHEDTC and 99mTcN–PHEDTC were high, 85.68 ± 0.61 and 86.32 ± 0.19 %, respectively. When compared with 99mTcN–PRODTC (87.42 ± 0.01 %), there was no great difference in the serum protein binding ability among these three complexes.

Partition coefficient (log P)

The log P values of 99mTcO–PHEDTC and 99mTcN–PHEDTC were found to be −1.33 ± 0.07 and −1.52 ± 0.03, respectively, indicating both tracers were hydrophilic. Moreover, 99mTcN–PHEDTC was more hydrophilic than 99mTcO–PHEDTC. As compared to 99mTcN–PRODTC (log P: −2.80 ± 0.11), 99mTcO–PHEDTC and 99mTcN–PHEDTC had a higher lipophilicity due to their structures containing aromatic groups.

Biodistribution study

The results of biodistribution of 99mTcO–PHEDTC and 99mTcN–PHEDTC in mice bearing S180 tumor are shown in Table 1. Results of biodistribution of recently reported 99mTc complexes as tumor imaging agents are shown in Table 2 for comparison.

Table 1 Biodistribution of 99mTcO–PHEDTC and 99mTcN–PHEDTC in mice bearing S 180 tumor (%ID/g) a
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Table 2 Comparison of biodistribution of some reported 99mTc complexes
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As shown in Table 1, both 99mTcO–PHEDTC and 99mTcN–PHEDTC have a high uptake and good retention in tumor. The muscle uptakes are low so the T/N ratios are better. Activity accumulation in the kidneys and liver shows that the major route of excretion is renal and hepatobiliary. Low uptake in the stomach is suggestive of in vivo stability of the above 99mTc labeled complexes. As compare to 99mTcN–PHEDTC, 99mTcO–PHEDTC has a higher tumor and liver uptake than 99mTcN–PHEDTC and this possibly be related to its higher lipophilicity. 99mTcO–PHEDTC has a lower muscle uptake and higher tumor uptake, so the T/N ratio of 99mTcO–PHEDTC is much higher than that of 99mTcN–PHEDTC. However, due to the higher blood uptake, the T/B ratio of 99mTcO–PHEDTC is lower than that of 99mTcN–PHEDTC.

From Table 2, it is observed that 99mTcO–PHEDTC and 99mTcN–PRODTC show a much higher tumor uptake when compared to the other three complexes. A decrease in the order is observed: 99mTcO–PHEDTC > 99mTcN–PHEDTC > [99mTc(CO)3(IDA–PEG3–CB)]− > 99mTcN–PRODTC > [99mTc(CO)3(PA–TZ–CHC)]+. In the limits of our study, among 99mTcO–PHEDTC, 99mTcO–PHEDTC and 99mTcN–PRODTC, the results demonstrated that the 99mTc labeled complexes containing aromatic amino acid moieties possibly increased the tumor uptake, confirming our hypothesis that radiolabeled aromatic amino acids are possibly more promising for tumor imaging. Moreover, different 99mTc core for preparing the complexes may exhibit significant impact on the tumor uptake, T/B and T/N ratios.

Conclusion

In present study, a novel ligand PHEDTC was synthesized and its 99mTc-oxo core and 99mTc-nitrido core complexes were successfully prepared in high yields through a ligand-exchange reaction. The preliminary in vivo studies showed both of them had a relative high tumor uptake and good tumor-to-muscle ratios. Especially for 99mTcO–PHEDTC, it would be a promising tumor imaging agent, justifying further investigations.