Radiolabeling and Biological Evaluation of Novel 99mTc-Nitrido and 99mTc-Oxo Complexes with 4-Methoxy-L-Phenylalanine Dithiocarbamate for Tumor Imaging

To develop novel radiolabeled amino acid tumor imaging agents, 4-methoxy-L-phenylalanine dithiocarbamate (MOPADTC) was synthesized successfully, and two kinds of 99mTc-labeled complexes ([99mTc]TcN-MOPADTC and [99mTc]TcO-MOPADTC) with high radiochemical purities (RCP > 95%) were obtained. The in vitro stability and partition coefficient were determined, and the results show that both of these complexes have good in vitro stability; [99mTc]TcO-MOPADTC is hydrophilic, while [99mTc]TcN-MOPADTC is slightly lipophilic. The biodistribution of [99mTc]TcN-MOPADTC and [99mTc]TcO-MOPADTC in mice bearing S180 tumors shows that the tumor uptake and tumor/muscle ratio of [99mTc]TcO-MOPADTC were higher than the tumor uptake and tumor/muscle ratio of [99mTc]TcN-MOPADTC. In addition, the tumor retention of [99mTc]TcO-MOPADTC is better than the tumor retention of [99mTc]TcN-MOPADTC. A competitive inhibition assay was performed, and the results indicate that [99mTc]TcO-MOPADTC may enter cells primarily via the L-alanine/L-serine/L-cysteine (ASC) system. Single-photon emission computed tomography (SPECT) imaging of [99mTc]TcO-MOPADTC shows obvious accumulation in tumor sites, suggesting that [99mTc]TcO-MOPADTC is a novel potential tumor-imaging agent.


Introduction
Glucose-based [ 18 F]FDG ([ 18 F]fluorodeoxayglucose) is a positron emission tomography (PET) imaging agent, and is the most commonly used clinically [1,2]. Although [ 18 F]FDG has a high uptake of tumors, some limitations still exist, such as producing falsepositive or false-negative results and low image contrast in some cancer diagnoses [3,4]. The high uptake of [ 18 F]FDG is related to increased glycolysis in tumor cells [5]. Amino acid and protein metabolism are enhanced in certain cancer cells [6], and amino acids are regarded as the key components of proteins and have many other functions that are essential for life, such as cellular metabolic survival and division [7,8].
Amino acid transport mechanisms can be classified according to many factors, such as transport saturation, kinetic behavior, substrate specificity, dependence on Na + and sensitivity to pH [9,10]. The mechanisms can be divided into system A, system N, system ASC and system L [11][12][13]. System A is responsible mainly for the transport of small aliphatic amino acids, and system N is related mainly to the transport of amino acids with N atoms on the sidechain, such as histidine. System ASC transports small neutral amino acid molecules, such as alanine, cysteine and serine, while system L prefers to transport neutral large amino acids [14].
Recently, aromatic amino acids have played a significant role in the clinical diagnostics of cancer. The chemical structures of some mentioned radiolabeled amino acids were

Reagents and Chemicals
All chemicals were purchased from commercial sources and used without further purification. Mass spectrum was recorded on a Triple TOF 5600 spectrometer (Concord, Canada). The NMR results were acquired on a 400 MHz JNM-ECS spectrophotometer (JEOL, Tokyo, Japan). Technetium-99m solution ([ 99m Tc]TcO4 − ) was eluted freshly from a 99 Mo/ 99m Tc generator, which was purchased from ZHIBO Bio-Medical Tech, Beijing, China. Succinic dihydrazide (SDH) kits and glucoheptonate (GH) kits were obtained from Beijing Shihong Pharmaceutical Co., LTD (Bejing, China), Beijing Normal University, China. SPECT/CT imaging studies were performed on a SPECT/CT scanner, which was purchased from TriFoil imaging (Chatsworth, CA, USA).

Synthesis of MOPADTC
MOPADTC was prepared according to a previously reported method [23]. NaOH (0.413 g, 10 mmol) was added to water (20 mL) and stirred at room temperature. Then, 4methoxy-L-phenylalanine (1.0 g, 5 mmol) was added to the above solution and stirred until it was completely dissolved. Carbon disulfide (3 mL, 50 mmol) was added to the above In the development of 99m Tc radiopharmaceuticals, such as [ 99m Tc]Tc-MIBI (MIBI: 2-methoxy-2-methylpropylisonitrile) as a myocardial perfusion imaging agent and a tumor imaging agent, an interesting structural feature was that it had a methoxy group. The methoxy group may affect the biodistribution properties of the complex. In order to develop novel 99m Tc-labeled aromatic amino acids, 4-methoxy-L -phenylalanine (MOPA) was selected as the raw material to synthesize the corresponding dithiocarbamate ligand, and it was radiolabeled with

Reagents and Chemicals
All chemicals were purchased from commercial sources and used without further purification. Mass spectrum was recorded on a Triple TOF 5600 spectrometer (Concord, Canada). The NMR results were acquired on a 400 MHz JNM-ECS spectrophotometer (JEOL, Tokyo, Japan). Technetium-99m solution ([ 99m Tc]TcO 4 − ) was eluted freshly from a 99 Mo/ 99m Tc generator, which was purchased from ZHIBO Bio-Medical Tech, Beijing, China. Succinic dihydrazide (SDH) kits and glucoheptonate (GH) kits were obtained from Beijing Shihong Pharmaceutical Co., LTD (Bejing, China), Beijing Normal University, China. SPECT/CT imaging studies were performed on a SPECT/CT scanner, which was purchased from TriFoil imaging (Chatsworth, CA, USA).

Synthesis of MOPADTC
MOPADTC was prepared according to a previously reported method [23]. NaOH (0.413 g, 10 mmol) was added to water (20 mL) and stirred at room temperature. Then, 4-methoxy-L -phenylalanine (1.0 g, 5 mmol) was added to the above solution and stirred until it was completely dissolved. Carbon disulfide (3 mL, 50 mmol) was added to the above solution, and the reaction was performed at 0 • C for 4 h. The reaction progress was monitored through the pH value of the reaction solution. Upon the disappearance of NaOH, the pH of the reaction solution was approximately 7-8. Most of solvent and remaining carbon disulfide were removed under reduced pressure. Then, the residue was recrystallized by methanol, and was obtained as a white powder after drying in vacuo.
Regarding MOPADTC (0.90 g, yield, 57%), the 1  − (37-370 MBq) was added into a SDH kit, which consisted of 0.05 mg of stannous chloride, 5.0 mg of SDH and 5.0 mg of propylenediamine tetraacetic acid (PDTA). The mixture was reacted at room temperature for 20 min. Then, 1.0 mg of MOPADTC dissolved in 1.0 mL of saline was added to the mixture, and the reaction was performed for 20 min at 100 • C. For preparing [ 99m Tc]TcO-MOPADTC, 1 mL of saline containing [ 99m Tc]TcO 4 − (37-370 MBq) was added to a GH kit, which consisted of 0.1 mg of stannous chloride and 20.0 mg of GH. The mixture was reacted at room temperature for 20 min. Then, 1.0 mg of MOPADTC dissolved in 1.0 mL of saline was added to the mixture, and the reaction was performed for 20 min at 100 • C.
The radiochemical purities (RCP) of both radiotracers were determined by thin-layer chromatography (TLC). TLC was performed using a polyamide strip as the solid phase and saline and acetonitrile as the eluents.

In Vitro Stability Study
The in vitro stability study of [ 99m Tc]TcN-MOPADTC and [ 99m Tc]TcO-MOPADTC was conducted according to the reported methods [23]. Briefly, the labeled complexes were kept at room temperature for 6 h. Then, the RCPs of the complexes were determined by TLC. In addition, 0.1 mL of radiolabeled complex was put into 0.1 mL of fresh mouse serum in a centrifuge tube, and the mixtures were incubated at 37 • C for 4 h. The in vitro serum stabilities were assessed by TLC.

Partition Coefficient (Log P)
The rate of dispersion of the radiolabeled complexes between the aqueous and organic phase, which is called partition coefficient, was measured. A total of 0.1 mL of the 99m Tc-complex was mixed with 1.0 mL of 1-octanol and 0.9 mL of phosphate buffer (PBS, 0.025 mol/L, pH 7.4). The mixtures were stirred vigorously, then centrifuged (3 min, 9000 rpm) so that the aqueous and organic phases were separated completely. For each phase, 0.1 mL of solution was removed, and the amount of radioactivity was measured in a γ-counter. The above experiments were repeated three times. The partition coefficient (P) was calculated as the radioactivity values of counts of the organic phases divided by those of the PBS. The final partition coefficient value was expressed as log P ± standard deviation (SD).

Biodistribution Studies
All biodistribution studies were performed in compliance with the national laws and regulations related to the conduct of animal experimentations. The S180 cell line was cultured in Kunming mice ascites. The S180 tumor models were established by injecting S180 cells (about 1 × 10 6 in 0.1 mL of saline) into the left upper limb armpit of Kunming mice (female, 18-22 g) by subcutaneous injection. The tumors were allowed to grow for about one week, with diameters ranging from 5 to 8 mm.
Then, 0.1 mL of 99m Tc complex (740 kBq/mL) was injected into Kunming female mice bearing S180 tumors via the tail vein. Mice were sacrificed in groups of five at different times post-injection. The tumors, blood, muscles and other organs of interest were collected, weighed and measured for radioactivity. The counting tubes, including a standard equivalent to 1% of the injected dose, were measured with a γ-counter. The results were calculated as the percent uptake of injected dose per gram of organ (%ID/g). The final results were expressed as the mean ± SD.

Cellular Inhibition Studies of [ 99m Tc]TcO-MOPADTC
The murine sarcoma S180 cell line was used to characterize the transport mechanism of the radiotracers. The S180 tumor cells were obtained from the abdominal cavity of a sacrificed ascites mouse. The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The lower-layer cells were washed three times with saline. Then, the S180 cells were suspended in fresh Dulbecco's modified eagle medium (DMEM) at a cell concentration of 5 × 10 6 per mL for later use.
To characterize the transport mechanism of [ 99m Tc]TcO-MOPADTC, competitive inhibition studies were conducted with the S180 cell line according to a previously reported method [24][25][26]. A series of inhibitors was added to the cells in DMEM at a concentration of 20 mM. These inhibitors included selected amino acid transport inhibitors, such as 2-(methylamino)isobutyric acid hydrate (MeAIB, 40 mM, the specific inhibitor of system A) and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH, 40 mM, the specific inhibitor of system L). Additionally, natural amino acids were used as inhibitors, such as L -histidine (His, 40 mM, the inhibitor of system N) and a mixture of L -alanine/ L -serine/ L -cysteine (ASC, 13.3 mM of each amino acid, the inhibitor of system ASC). For each group (n = 5), 0.2 mL of cell solution, 0.5 mL (20 mM) of inhibitor, 0.1 mL of radiotracer and 0.2 mL of DMEM were added to the centrifuge tube successively. A total of 0.2 mL of cell solution, 0.7 mL of DMEM and 0.1 mL of radiotracer were added to the centrifuge tube as the control group. Then, they were incubated at 37 • C for 20 min. At the end of the incubation period, the samples were centrifuged at 9000 rpm for 5 min. Then, the supernatant was removed, and the residual sample with cells was washed with 0.5 mL of PBS twice. Finally, the residual sample with cells was counted with γ-counter. The results are expressed as the percentage of the cellular uptake in the control group.

SPECT/CT Imaging Studies
For the SPECT/CT imaging studies, Kunming female mice (18-22 g) bearing S180 tumors were injected with 0.1 mL of [ 99m Tc]TcO-MOPADTC (18 MBq) via the tail vein. SPECT/CT images were obtained from anesthetized mice using 1.5% isoflurane at 4 h post-injection. SPECT/CT images were analyzed using HISPECT software and VivoQuant 2.5 software.

Synthesis of MOPADTC
The synthesis route is shown in Scheme 1. MOPADTC was successfully prepared by reacting MOPA with carbon disulfide (CS 2 ) under basic conditions in 57% yield. It was characterized by 1 H NMR, 13

Radiolabeling and Quality Control
The

Stability Experiments and Partition Coefficient
The in vitro stabilities of [ 99m Tc]TcN-MOPADTC and [ 99m Tc]TcO-MOPADTC were assayed by measuring their radiochemical purities (RCP). As shown in Figure 2, the RCP of both complexes was more than 90% after incubation either in saline at room temperature for 6 h or in mouse serum at 37 °C for 4 h, indicating good in vitro stabilities of the 99m Tc-labeled complexes. According to the reported literature [27][28][29], it can be presumed that [ 99m Tc]TcN-MOPADTC or [ 99m Tc]TcO-MOPADTC would form a structure with a Tc≡N bond or Tc=O bond linked to MOPADTC ligands through the four sulfur atoms. One dithiocarbamate ligand contains two sulfur atoms, whose coordination ability with technetium is better than that of nitrogen and oxygen atoms.
The radiochemical purities of the complexes were checked by TLC.

Stability Experiments and Partition Coefficient
The in vitro stabilities of [ 99m Tc]TcN-MOPADTC and [ 99m Tc]TcO-MOPADTC were assayed by measuring their radiochemical purities (RCP). As shown in Figure 2, the RCP of both complexes was more than 90% after incubation either in saline at room temperature for 6 h or in mouse serum at 37 • C for 4 h, indicating good in vitro stabilities of the 99m Tc-labeled complexes.

Biodistribution Studies
The As shown in Table 1, the tumor uptake of [ 99m Tc]TcO-MOPADTC increased with time in the first 2 h and was maintained at a constant value in the following 2 h, indicating that the tumor retention of [ 99m Tc]TcO-MOPADTC is excellent. At each time point, the accumulation in muscle was relatively low and diminished from 0.63 ± 0.14 %ID/g to 0.44 ± 0.11 %ID/g and 0.34 ± 0.12 %ID/g at 0.5 h, 2 h and 4 h, respectively. The initial uptake values of the heart, lung and stomach were not high, and most radioactivity was cleared after 4 h. The uptake doses of the liver and kidney remained high, indicating that the labeled complexes are metabolized and excreted mainly through the liver and kidney.

Biodistribution Studies
The accumulation in muscle was relatively low and diminished from 0.63 ± 0.14 %ID/g to 0.44 ± 0.11 %ID/g and 0.34 ± 0.12 %ID/g at 0.5 h, 2 h and 4 h, respectively. The initial uptake values of the heart, lung and stomach were not high, and most radioactivity was cleared after 4 h. The uptake doses of the liver and kidney remained high, indicating that the labeled complexes are metabolized and excreted mainly through the liver and kidney.

Cellular Inhibition Studies of [ 99m Tc]TcO-MOPADTC
Based on the results of the biodistribution studies, the tumor uptake and tumor/muscle ratios of [ 99m Tc]TcO-MOPADTC were higher than those of [ 99m Tc]TcN-MOPADTC. Thus, [ 99m Tc]TcO-MOPADTC was chosen for further cellular studies by S180 cells to characterize the mechanisms of amino acid transport.
To investigate the transport mechanisms of [ 99m Tc]TcO-MOPADTC, a variety of natural amino acids or amino acid transporter inhibitors were used to determine which amino acid transport systems were responsible for the uptake of [ 99m Tc]TcO-MOPADTC. As shown in Figure 3, the uptake of [ 99m Tc]TcO-MOPADTC was inhibited in S180 cells under ASC conditions (a decrease of 43%, p < 0.01) mainly and in MeAIB conditions (a decrease of 5%, p < 0.05) slightly. The results indicate that [ 99m Tc]TcO-MOPADTC may be transported mainly through system ASC and slightly through system A.

Cellular Inhibition Studies of [ 99m Tc]TcO-MOPADTC
Based on the results of the biodistribution studies, the tumor uptake and tumor/muscle ratios of [ 99m Tc]TcO-MOPADTC were higher than those of [ 99m Tc]TcN-MOPADTC. Thus, [ 99m Tc]TcO-MOPADTC was chosen for further cellular studies by S180 cells to characterize the mechanisms of amino acid transport.
To investigate the transport mechanisms of [ 99m Tc]TcO-MOPADTC, a variety of natural amino acids or amino acid transporter inhibitors were used to determine which amino acid transport systems were responsible for the uptake of [ 99m Tc]TcO-MOPADTC. As shown in Figure 3, the uptake of [ 99m Tc]TcO-MOPADTC was inhibited in S180 cells under ASC conditions (a decrease of 43%, p  0.01) mainly and in MeAIB conditions (a decrease of 5%, p  0.05) slightly. The results indicate that [ 99m Tc]TcO-MOPADTC may be transported mainly through system ASC and slightly through system A.   Figure 4, which were consistent with the biodistribution results. The tumor uptake was clearly observable. Additionally, the accumulations of the liver and kidney were obviously detectable from the imaging at 4 h post-injection.

SPECT/CT Imaging
As [ 99m Tc]TcO-MOPADTC had a higher tumor uptake and tumor/muscle ratio than [ 99m Tc]TcN-MOPADTC, [ 99m Tc]TcO-MOPADTC was selected as a promising agent for further SPECT/CT imaging studies. The SPECT/CT imaging results of [ 99m Tc]TcO-MOPADTC are shown in Figure 4, which were consistent with the biodistribution results. The tumor uptake was clearly observable. Additionally, the accumulations of the liver and kidney were obviously detectable from the imaging at 4 h post-injection.

Discussion
Currently, aromatic amino-acid-based radiopharmaceuticals play an important rol in the field of oncologic imaging. In addition, 99m Tc is an ideal radionuclide with suitabl energy and a suitable half-life for SPECT/CT. The 99m Tc-labeled radiotracer can be pre pared in the kit form, which is convenient and simple for application. Moreover, SPECT scanners are much more numerous worldwide than PET scanners [30]. Furthermore, th diagnosis efficacy of cadmium zinc telluride (CTZ) SPECT will be nearly consistent with those of PET [31]. Consequently, it is necessary to develop novel 99m Tc-labeled aromati amino-acid-based radiopharmaceuticals as tumor imaging agents for SPECT/CT.
More importantly, chelators are required to conjugate the amino acid derivative with 99m Tc [6,32]. Due to the fact that the coordination ability with 99m Tc of sulfur atoms i better those that of nitrogen and oxygen atoms, MOPADTC containing two sulfur atom can make efficiently stable binding with To verify whether the addition of a methoxy group on the benzene ring has an effec on the uptake of tumors, biodistribution studies of [ 99m Tc]TcO-TYRDTC (tyrosine dithio carbamate was abbreviated to TYRDTC) at 4 h post-injection were also performed (n = 5 (the synthesis of TYRDTC (Scheme S1), radiolabeling (Scheme S2) and biodistribution re sults (Table S1) of [ 99m Tc]TcO-TYRDTC are provided in Supplementary data). As show in Figure 5, the tumor uptake of [ 99m Tc]TcO-MOPADTC (1.58 ± 0.35 %ID/g) was highe than the tumor uptake of [ 99m Tc]TcO-TYRDTC (0.78 ± 0.37 %ID/g). The main structura difference among the 99m Tc-oxo complexes was that the group at the para-position of th benzene ring was different: the para-position of MOPA was methoxy, and the groups o tyrosine were phenolic hydroxyl respectively. [ 99m Tc]TcO-MOPADTC should be more lip ophilic than [ 99m Tc]TcO-TYRDTC, thus making it easier for the former to penetrate th lipophilic membrane of tumor cells.

Discussion
Currently, aromatic amino-acid-based radiopharmaceuticals play an important role in the field of oncologic imaging. In addition, 99m Tc is an ideal radionuclide with suitable energy and a suitable half-life for SPECT/CT. The 99m Tc-labeled radiotracer can be prepared in the kit form, which is convenient and simple for application. Moreover, SPECT scanners are much more numerous worldwide than PET scanners [30]. Furthermore, the diagnosis efficacy of cadmium zinc telluride (CTZ) SPECT will be nearly consistent with those of PET [31]. Consequently, it is necessary to develop novel 99m Tc-labeled aromatic amino-acidbased radiopharmaceuticals as tumor imaging agents for SPECT/CT.
More importantly, chelators are required to conjugate the amino acid derivatives with 99m Tc [6,32]. Due to the fact that the coordination ability with 99m Tc of sulfur atoms is better those that of nitrogen and oxygen atoms, MOPADTC  To verify whether the addition of a methoxy group on the benzene ring has an effect on the uptake of tumors, biodistribution studies of [ 99m Tc]TcO-TYRDTC (tyrosine dithiocarbamate was abbreviated to TYRDTC) at 4 h post-injection were also performed (n = 5) (the synthesis of TYRDTC (Scheme S1), radiolabeling (Scheme S2) and biodistribution results (Table S1) of [ 99m Tc]TcO-TYRDTC are provided in Supplementary data). As shown in Figure 5, the tumor uptake of [ 99m Tc]TcO-MOPADTC (1.58 ± 0.35 %ID/g) was higher than the tumor uptake of [ 99m Tc]TcO-TYRDTC (0.78 ± 0.37 %ID/g). The main structural difference among the 99m Tc-oxo complexes was that the group at the para-position of the benzene ring was different: the para-position of MOPA was methoxy, and the groups of tyrosine were phenolic hydroxyl respectively. Currently, most amino-acid-based tracers are transported into cells via system A, ASC or L. Anti-1-amino-2-[ 18 F]-fluorocyclobutane-1-carboxylic acid (anti-3-[ 18 F]FACBC) is a radiolabeled probe used for its utility in the detection of recurrent prostate carcinoma and gliomas. The [ 18 F]FACBC seems to enter cells through system ASC and system L [19]. Moreover, p-[ 123 I]iodo-L-phenylalanine (IPA) and L-3-[ 123 I]iodo-α-methyltyrosine (IMT) showed high accumulation in glioblastomas, and entered tumor cells through systems ASC and L [33]. By comparison, the inhibition studies in this study show that [ 99m Tc]TcO-MOPADTC is transported via system ASC mainly and via system A slightly in S180 tumor cells. We speculated that the conversion amino acid to amino acid dithiocarbamate, the addition of methoxy group and the different 99m Tc cores labeling may have an effect on the transport mechanisms of the tracer. Radiotracers targeting exact amino acid transport systems need to be further studied.
Due to its better biodistribution performance, [ 99m Tc]TcO-MOPADTC was chosen for further SPECT/CT imaging study. As predicted, [ 99m Tc]TcO-MOPADTC imaging exhibits evident tumor uptake. The uptakes of the kidneys and liver are also obvious, which indicates that the urinary and intestinal tracts are the major routes of excretion. The imaging results suggest that [ 99m Tc]TcO-MOPADTC has the potential to be used in molecular imaging to diagnose tumors. By comparing the biodistribution data of the new tracers, it can be concluded that the radioactivity accumulation was similar in the majority of organs, except for the kidneys. The kidney uptake of [ 99m Tc]TcO-MOPADTC was remarkably higher than the kidney uptake of [ 99m Tc]TcN-MOPADTC, which may be related to the hydrophilicity of the 99m Tcoxo complex, while in the majority of other normal organs the uptake was similar. Currently, most amino-acid-based tracers are transported into cells via system A, ASC or L. Anti-1-amino-2-[ 18 F]-fluorocyclobutane-1-carboxylic acid (anti-3-[ 18 F]FACBC) is a radiolabeled probe used for its utility in the detection of recurrent prostate carcinoma and gliomas. The [ 18 F]FACBC seems to enter cells through system ASC and system L [19]. Moreover, p-[ 123 I]iodo-L -phenylalanine (IPA) and L -3-[ 123 I]iodo-α-methyltyrosine (IMT) showed high accumulation in glioblastomas, and entered tumor cells through systems ASC and L [33]. By comparison, the inhibition studies in this study show that [ 99m Tc]TcO-MOPADTC is transported via system ASC mainly and via system A slightly in S180 tumor cells. We speculated that the conversion amino acid to amino acid dithiocarbamate, the addition of methoxy group and the different 99m Tc cores labeling may have an effect on the transport mechanisms of the tracer. Radiotracers targeting exact amino acid transport systems need to be further studied.
Due to its better biodistribution performance, [ 99m Tc]TcO-MOPADTC was chosen for further SPECT/CT imaging study. As predicted, [ 99m Tc]TcO-MOPADTC imaging exhibits evident tumor uptake. The uptakes of the kidneys and liver are also obvious, which indicates that the urinary and intestinal tracts are the major routes of excretion. The imaging results suggest that [ 99m Tc]TcO-MOPADTC has the potential to be used in molecular imaging to diagnose tumors.

Conclusions
In summary, MOPADTC was synthesized, and the corresponding radiolabeled complexes ([ 99m Tc]TcN-MOPADTC and [ 99m Tc]TcO-MOPADTC) were prepared with high radiochemical purity. The biodistribution and imaging studies in mice bearing S180 tumors show that [ 99m Tc]TcO-MOPADTC has a higher uptake of tumors and a higher tumor/muscle ratio than [ 99m Tc]TcN-MOPADTC. Compared with our previously synthesized dithiocarbamate tracer [ 99m Tc]TcO-PHEDTC [23], which shows a better tumor uptake and tumor/muscle ratio, the new tracer [ 99m Tc]TcO-MOPADTC shows better tumor retention and a higher tumor/blood ratio, characteristics that qualify it for diagnostic imaging.
[ 99m Tc]TcO-MOPADTC is mainly transported via system ASC. It has the potential to be a novel 99m Tc-labeled tumor imaging agent.

Institutional Review Board Statement:
Animal studies were carried out in accordance with the principles of laboratory animal care and the guidelines of the Ethics Committee of Beijing Normal University (permit no. BNUCC-EAW-2022-001, 11 January 2022).

Conflicts of Interest:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.