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Synthesis and biodistribution of a novel 99mTc nitrido dithiocarbamate complex containing aromatic group for cerebral imaging
ARTICLE IN PRESS Applied Radiation and Isotopes 68 (2010) 101–104
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Synthesis and biodistribution of a novel 99mTc nitrido dithiocarbamate complex containing aromatic group for cerebral imaging JunBo Zhang , Xiao Lin, Jialei Ren, Jing Liu, XueBin Wang Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China
a r t i c l e in fo
abstract
Article history: Received 7 April 2009 Received in revised form 19 June 2009 Accepted 30 August 2009
In the present study, the N-benzyl dithiocarbamate (BZDTC) was synthesized and radiolabeled with [99mTcN]2 + intermediate to form the bis(N-benzyl dithiocarbamato) nitrido technetium-99m complex [99mTcN(BZDTC)2]. The radiochemical purity of the complex was over 90% by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). It was stable over 6 h at room temperature. The partition coefficient and electrophoresis results indicated that this complex was lipophilic and neutral. Biodistribution in mice showed that the complex accumulated in the brain with high uptake. The brain uptake (ID%/g) was 1.87, 1.21 and 0.85 and the brain/blood ratio was 0.75, 1.55 and 1.12 at 5, 30 and 60 min post-injection, respectively. These results suggest that this complex could be a potential brain perfusion imaging agent. & 2009 Elsevier Ltd. All rights reserved.
Keywords: [99mTcN]2 + core Aromatic group Dithiocarbamate Cerebral imaging
1. Introduction The search of 99mTc complex for brain imaging agents which accurately reflects regional cerebral blood flow in single-photon emission computed tomography (SPECT) remains the subject of great interest in the radiopharmaceutical field. Now, 99mTc-d, l-HMPAO (hexamethyl-propylene amine oxime) (Nowotnik et al., 1985) and 99mTc-l,l-ECD(ethyl cysteinate dimer) (Vallabhajosula et al., 1989) are accepted as valuable tracer agents for the determination of regional cerebral blood flow, but they are complexes containing the [99mTcQO]3 + core. The [TcRN]2 + core, which is isoelectronic with [TcQO]3 + and [OQTcQO] + , exhibits a very high chemical stability towards oxidation– reduction reactions and pH variations and the presence of the [99mTcN]2 + core in the molecular structure of a radiopharmaceutical may dramatically alter its biological behavior (Baldas and Bonnyman, 1985). Therefore, it may be of great interest to probe the 99mTc nitrido chemistry with some ligands to develop novel diagnostic agents. In the development of 99mTc radiopharmaceuticals for brain imaging, recently, several 99mTcN dithiocarbamate complexes containing aliphatic group on the nitrogen atom on the dithiocarbamate ligand have been reported (Zhang et al., 2005; Mallia et al., 2006; Zhang et al., 2007a, b). Bearing in mind that containing the aromatic group on the nitrogen atom on the dithiocarbamate ligand possibly alters the lipophilicity and biodistribution of
Corresponding author. Tel.: + 86 10 62208126; fax: + 86 10 62205562.
E-mail address: [email protected] (J. Zhang). 0969-8043/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2009.08.019
the complex (Pasqualini et al., 1994), our attention was drawn to study the 99mTcN(BZDTC)2 complex for finding a good imaging tracer. In this study, we report the synthesis and biodistribution in mice of a novel 99mTc nitrido complex with dithiocarbamate ligand containing the aromatic group in the ligating framework.
2. Materials and methods Succinic dihydrazide (SDH), propylenediamine tetraacetic acid (PDTA) and stannous chloride dihydrate were purchased from Aldrich Chemical Company, USA. 99Mo/99mTc generator was obtained from the China Institute of Atomic Energy (CIAE). All other chemicals were of reagent grade and were used without further purification. 2.1. Synthesis of sodium salt of N-benzyl dithiocarbamate (BZDTC) Benzylamine (0.1 mol) was dissolved in dried diethyl ether and the resulting solution was cooled in an ice–salt bath. Sodium hydroxide (0.1 mol) was added to this solution under stirring, followed by the addition of carbon disulfide (0.1 mol). The mixture was stirred for 2 h in an ice–salt bath. Most of the solvent were removed under reduced pressure, and the precipitate was collected by filtration. The crude product was recrystallized from isopropanol/diethyl ether to give white crystals of sodium N-benzyl dithiocarbamate (yield, 40%). HPLC analysis was carried out with a reversed-phase column (Kromasil 100-5C, 250 4.6 mm), Shimadzu SCL-10AVP series, by using methanol/ water (50:50, V/V) as a mobile phase, working at a flow rate of
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1.0 ml/min. HPLC chromatogram showed the impurities (about 5%) are small peaks right after the main peak (95.04%), indicating the purity of the product was over 95%. IR (KBr)/cm 1: 3314(nN–H); 3084, 3054, 3024 (nAr–H); 2970 (nC–H); 1505, 1454 (nCQC); 1047 (nCQS). 1 H NMR(D2O, 400 MHz) d (ppm): 7.24–7.13 (m, 5H, Ar–H); 4.59(s, 2H, C–H). 13 C NMR(D2O, 400 MHz) d (ppm): 211.60, 137.70, 128.62, 128.62, 127.13, 126.86, 126.86, 50.83. ESI-MS: 182 ([M–Na] ).
2.2. Preparation of the
99m
TcN(BZDTC)2 complex
The preparation of the complex was carried out using the following procedure: 1 ml of saline containing [99mTcO4] (15 MBq) was added to a kit containing 0.05 mg of stannous chloride dihydrate, 5.0 mg of succinic dihydrazide (SDH) and 5.0 mg of propylenediamine tetraacetic acid (PDTA). The mixture was kept at room temperature for 15 min.
buffer, in an electrophoresis bath. Across 12 cm of the strip, 150 V was applied for 1.5 h. The strips were dried, and the distribution of radioactivity on the strip was determined. 2.6. Biodistribution study A solution of 99mTcN(BZDTC)2 (0.1 ml, 740 KBq) was administered via a tail vein to Kunming mice (18–20 g) and the injected radioactivity measured with a well-type NaI(Tl) detector. The mice were sacrificed at 5, 30 and 60 min after injection. The organs of interest and the blood were collected, weighed and measured for radioactivity. All biodistribution studies were carried out in compliance with the national laws related to the conduct of animal experimentation.
3. Results and discussion 3.1. Chemistry of the
99m
TcN(BZDTC)2 complex
The preparation of the 99mTcN(BZDTC)2 complex can be carried out by the following method:
SDH kit 99m
[
99m
-
[
TcO4]
r.t., 15 min
2+
CH2NHCS2Na
TcN]
99m
TcN(
CH2NHCS2)2
r.t., 10 min
Successively, 5.0 mg of the sodium salt of N-benzyl dithiocarbamate dissolved in 1.0 ml of ethanol was added and the reaction was allowed to proceed for 10 min at room temperature. The radiochemical purity of the products was evaluated by TLC and HPLC ranged from 90–99%. The TLC was performed on a polyamide strip and was eluted with saline and CH2Cl2: CH3OH= 9:1(V/V). HPLC analysis was carried out with a reversed-phase column (Kromasil 100-5C, 250 4.6 mm), Shimadzu SCL-10AVP series, by using methanol/water (70:30, V/V) as a mobile phase, working at a flow rate of 1.0 ml/min. 2.3. Determination of the partition coefficient for the complex The partition coefficient was determined by mixing the complexes with an equal volume of 1-octanol and phosphate buffer (0.025 M, pH 7.0 and 7.4) in a centrifuge tube. The mixture was vortexed at room temperature for 1 min and then centrifuged at 5000 rpm for 5 min. From each phase, 0.1 ml of the aliquot was pipetted and counted in a well g-counter. The measurement was repeated three times. Care was taken to avoid cross contamination between the phases. The partition coefficient, P, was calculated using the following equation: P ¼ ðcpm in octanol cpm in backgroundÞ=ðcpm in buffer cpm in backgroundÞ
Usually the final partition coefficient value was expressed as log P. 2.4. Stability study The stability of the complex was assayed by measuring the radiochemical purity at different times after preparation. 2.5. Paper electrophoresis One ml sample was spotted on a piece of Whatman 1 chromatography paper, saturated with 0.05 M pH 7.4 phosphate
The method is based on the reaction of [99mTcO4] with succinic dihydrazide (SDH) in the presence of stannous chloride as reducing agent to form a technetium-99m nitrido intermediate. The [99mTcRN]2 + is a suitable substrate for the substitution reaction with the sodium salt of N-benzyl dithiocarbamate at room temperature to give the final complex 99m TcN(BZDTC)2. The radiochemical purity of the complex was routinely checked by TLC. In saline, 99mTcO4 , 99mTcO2 nH2O and 99m TcN(BZDTC)2 remained at the origin while [99mTcRN]2int+ migrated with the front. In CH2Cl2: CH3OH= 9:1 (V/V), 99m TcN(BZDTC)2 migrated with the front while 99mTcO4 , 99m TcO2 nH2O and [99mTcRN]2int+ remained at the origin. HPLC retention times (Rt) for some selected complexes were reported as 99m TcO4 : 3.2 min, [99mTcRN]2int+ : 2.6 min, follows: 99m TcN(BZDTC)2: 13.8 min. The HPLC pattern of 99mTcN(BZDTC)2 is shown in Fig. 1. Single peak suggested only one product was formed. The mean radiochemical purity of the product was over 90% immediately after the preparation. Bis(diethyldithiocarbamato) nitrido technetium-99m [99mTcN(DDC)2] is neutral and has a square pyramidal geometry with an apical TcRN bond and two monoanionic dithiocarbamate ligands spanning the four positions in the basal plane through the four sulfur atoms (Baldas et al., 1981). It can be presumed that 99mTcN(BZDTC)2 would also have a similar structure. Clearly, further studies should be performed, using macroscopic levels of the long-lived 99Tc, to determine and characterize the structure of the novel complex.
3.2. Partition coefficient (log P) of the complex The partition coefficient (log P) of the complex was 1.33 and 1.31 at pH 7.0 and 7.4, respectively, showing no great difference between pH 7.0 and 7.4. The log P of the complex is within the range of values quoted for lipophilicity (0.9–2.5) that are suitable
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for crossing the blood brain barrier (BBB). Due to its high lipophilicity, the complex is easily adsorbed on the walls of the vials and syringes. In order to avoid this adsorption, 10 mg of gcyclodextrin was added to the final solution.
103
3.4. Paper electrophoresis In paper electrophoresis, almost all the radioactivity was found at the point of spotting (percentage of radioactivity: 97.9%), suggesting that it was neutral.
3.3. Stability of the complex 3.5. Biodistribution of the complex in mice The RCP of the product was nearly constant ( 490%) over the observed period of 6 h, suggesting that it was stable in the reaction mixture at room temperature.
491664 13.80
count
368748
245832
122916
0
0
5
10
15 20 t (min)
Fig. 1. The HPLC pattern of
Table 1 Biodistribution in mice of the
99m
25
30
99m
TcN(BZDTC)2.
TcN(BZDTC)2 complex (x7 s, n =3) (%ID/g).
t (min)
5
30
60
Brain Heart Liver Lung Kidney Blood Br/Bl ratio
1.87 7 0.18 12.11 7 3.47 47.28 7 8.48 11.95 7 1.96 11.91 7 0.10 2.50 7 0.38 0.75
1.21 70.08 1.61 70.72 31.97 77.43 3.39 70.65 3.23 71.03 0.78 70.13 1.55
0.85 70.40 1.23 70.07 34.31 72.81 2.05 70.47 2.24 70.16 0.76 70.12 1.12
Table 2 Comparison of biodistribution of recently reported
99m
Biological distribution pattern of the complex in mice is presented in Table 1, as a percentage of the injected dose per gram of organ (%ID/g). Results of biodistribution of recently reported 99mTcN dithiocarbamate complexes and 99mTc-ECD are presented in Table 2 for comparison. As described in Table 1, the 99mTcN(BZDTC)2 complex can penetrate the intact blood-brain barrier (BBB) and has a high brain uptake and good retention. The blood clearance is fast so that the brain/blood ratio is high as 0.75, 1.55 and 1.12 at 5, 30 and 60 min post-injection, respectively. The 99mTcN(BZDTC)2 complex also exhibits significant initial myocardial uptake, but the washout from the heart is rapid thus making it unsuitable to be a good myocardial perfusion imaging agent. The liver uptake was appreciable and remained high at 60 min post-injection, suggesting that the hepatobiliary system is the major route of excretion of the administered radioactivity. As can be seen in Table 2, among these reported 99mTcNdithiocarbamates for cerebral imaging, including [99mTcN(PREDTC)2 (PREDTC: N-2-(1-pyrrolidino) ethyl dithiocarbamate), 99mTcN(BZDTC)2, 99mTcN(PPEDTC)2 (PPEDTC: N-2-(1piperidyl) ethyl dithiocarbamate), 99mTcN(BDTC)2 (BDTC: N-butyl dithiocarbamate), 99mTcN(SBDTC)2 (SBDTC: N-sec-butyl dithio99m TcN(MEDTC)2 (MEDTC: N-2-methoxyethyl carbamate), dithiocarbamate), 99mTcN(MEMDTC)2 (MEMDTC: N-2-methoxyethyl-N-methyl dithiocarbamate), 99mTcN(EPDTC)2 (EPDTC: N-3-ethoxypropyl dithiocarbamate), 99mTcN(EPMDTC)2 (EPMDTC: N-3-ethoxypropyl-N-methyl dithiocarbamate)], 99mTcN(BZDTC)2 exhibits the least blood uptake so that it has a high brain/blood ratio. Their biodistributions are strongly dependent on the lateral groups bonded to the uncoordinated nitrogen atom of the dithiocarbamate ligand. A decrease of brain uptake in the order is observed: 99mTcN(SBDTC)2 4 99mTcN(BDTC)2 4 99mTcN(PPEDTC)2 4 99mTcN(BZDTC)2 4 99mTcN(MEDTC)2 4 99mTcN(MEMDTC)2 4 99m TcN(EPDTC)2 4 99mTcN(EPMDTC)2 4 99mTcN(PREDTC)2. In the limits of our study, if the lipophilicity of the complex (log P) is within the range of (0.9–1.6), interestingly, on increasing the lipophilicity of the complex (log P), one can observe an increase of the brain uptake. As compared with 99mTc-ECD (log P: 1.20) (Mang’era et al., 1996), 99mTcN(BZDTC)2 shows higher log P value (1.31) and higher brain uptake. However, if the lipophilicity of the complex(log P) is higher than 1.70, the brain uptake of the complex gradually decreases as the lipophilicity of the complex (log P) increases.
TcN dithiocarbamate complexes and
99m
Tc-ECD in mice at 30 min post-injection (%ID/g, x 7s, n= 3).
Complexes
log P
Brain
Blood
Br/Bl ratio
Reference
99m
0.99 1.20 1.31 1.39 1.51 1.51 1.85 1.87 1.97 1.99
0.29 70.04 0.30 70.00 1.21 70.08 1.58 70.37 2.73 70.35 3.44 70.28 0.94 70.03 0.69 70.04 0.63 70.10 0.38 70.07
3.35 7 0.42 2.107 0.10 0.787 0.13 5.61 7 0.42 1.63 7 0.39 2.37 7 0.18 3.92 7 0.57 1.77 7 0.10 2.28 7 0.14 1.13 7 0.13
0.09 0.14 1.55 0.28 1.67 1.45 0.24 0.24 0.28 0.34
Zhang et al., (2007b) Mang’era et al., (1996) This study Zhang et al., (2007b) Zhang et al., (2007a) Zhang et al., (2005) Mallia et al. (2006) Mallia et al. (2006) Mallia et al. (2006) Mallia et al. (2006)
TcN(PREDTC)2 Tc-ECD TcN(BZDTC)2 99m TcN(PPEDTC)2 99m TcN(BDTC)2 99m TcN(SBDTC)2 99m TcN(MEDTC)2 99m TcN(MEMDTC)2 99m TcN(EPDTC)2 99m TcN(EPMDTC)2 99m 99m
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Among the nine complexes, only three complexes (99mTcN(BDTC)2,99mTcN(BZDTC)2 and 99mTcN(SBDTC)2) have a high brain uptake and higher brain/blood ratio (Br/Bl ratio 41), suggesting 99mTcN(BZDTC)2 would also be a potential brain perfusion imaging agent. The facts suggest the dithiocarbamate ligands containing the aromatic group seemed reasonable for preparing a novel brain tracer containing the [99mTcN]2 + core.
4. Conclusion To conclude, a novel 99mTc nitrido dithiocarbamate complex containing aromatic group 99mTcN(BZDTC)2 was prepared in high yields through a ligand-exchange reaction. As the complex has low molecular weight ( o600 Da), and is neutral and lipophilic, it has a tendency to cross the BBB. The biodistribution studies in mice showed that 99mTcN(BZDTC)2 accumulated in the brain with high uptake and good retention, suggesting it would be a potential brain imaging agent.
Acknowledgments The work was financially supported by National Natural Science Foundation of China (20771018) and by Beijing Normal University.
References Baldas, J., Bonnyman, J., Pojer, P.M., Williams, G.A., Mackay, M.F., 1981. Synthesis and structure of bis(diethyldithiocarbamato) nitrido technetium(V): a technetium nitrogen triple bond. J. Chem. Soc. Dalton. Trans. , 1798–1801. Baldas, J., Bonnyman, J., 1985. Effect of the Tc-nitrido group on the behaviour of 99m Tc-radiopharmaceuticals. Int. J. Appl. Radiat. Isot. 36, 919–923. Mallia, M.B., Mathur, A., Subramanian, S., Banerjee, S., Kothari, K., Koiry, S.P., Sarma, H.D., Venkatesh, M., 2006. Synthesis and evaluation of ether containing 99mTcnitrido dithiocarbamate complexes as brain perfusion imaging agent. Appl. Radiat. Isot. 64, 361–367. Mang’era, K.O., Vanbilloen, H.P., Bellande, E., Pasqualini, R., Verbruggen, A.M., 1996. Influence of a 99mTcN core on the biological and physicochemical behavior of 99m Tc complexes of L,L-EC and L,L-ECD. Nucl. Med. Biol. 23, 987–993. Nowotnik, D.P., Canning, L.R., Cumming, S.A., Harrison, R.C., Higley, B., Nechvatal, G., Pickett, R.D., Piper, I.M., Bayne, V.J., 1985. Development of a 99Tcm-labelled radiopharmaceutical for cerebral blood flow imaging. Nucl. Med. Commun. 6, 499–506. Pasqualini, R., Duatti, A., Bellande, E., Comazzi, V., Brucato, V., Hoffschir, D., Fagret, D., Comet, M., 1994. Bis(dithiocarbamato) nitrido technetium-99m radiopharmaceuticals: a class of neutral myocardial imaging agents. J. Nucl. Med. 35, 334–341. Vallabhajosula, S., Zimmerman, R.E., Picard, M., Stritzke, P., Mena, I., Hellman, R.S., Tikofsky, R.S., Stabin, M.G., Morgan, R.A., Goldsmith, S.J., 1989. Technetium99m ECD: a new brain imaging agent: in vivo kinetics and biodistribution studies in normal human subjects. J. Nucl. Med. 30, 599–604. Zhang, J., Wang, X., Liu, J., 2005. Synthesis of a bis-(N-sec-butyl-dithiocarbamato) nitrido 99mTc complex: a potential new radiopharmaceutical for brain perfusion studies. Appl. Radiat. Isot. 62, 33–37. Zhang, J., Wang, X., Tian, C., 2007a. Synthesis of a bis-(N-butyl- dithiocarbamato)nitrido 99mTc complex: a potential new brain imaging agent. J. Radioanal. Nucl. Chem. 273, 15–17. Zhang, J., Yu, T., Wang, Z., Lin, Y., Wen, F., Xing, M., Wang, X., 2007b. Synthesis and biodistribution of two novel 99mTc nitrido dithiocarbamate complexes containing heterocyclic linkage as potential brain perfusion imaging agents. J. Radioanal. Nucl. Chem. 274, 195–197.
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Synthesis and biodistribution of a novel 99mTc nitrido dithiocarbamate complex containing aromatic group for cerebral imaging JunBo Zhang , Xiao Lin, Jialei Ren, Jing Liu, XueBin Wang Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China
a r t i c l e in fo
abstract
Article history: Received 7 April 2009 Received in revised form 19 June 2009 Accepted 30 August 2009
In the present study, the N-benzyl dithiocarbamate (BZDTC) was synthesized and radiolabeled with [99mTcN]2 + intermediate to form the bis(N-benzyl dithiocarbamato) nitrido technetium-99m complex [99mTcN(BZDTC)2]. The radiochemical purity of the complex was over 90% by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). It was stable over 6 h at room temperature. The partition coefficient and electrophoresis results indicated that this complex was lipophilic and neutral. Biodistribution in mice showed that the complex accumulated in the brain with high uptake. The brain uptake (ID%/g) was 1.87, 1.21 and 0.85 and the brain/blood ratio was 0.75, 1.55 and 1.12 at 5, 30 and 60 min post-injection, respectively. These results suggest that this complex could be a potential brain perfusion imaging agent. & 2009 Elsevier Ltd. All rights reserved.
Keywords: [99mTcN]2 + core Aromatic group Dithiocarbamate Cerebral imaging
1. Introduction The search of 99mTc complex for brain imaging agents which accurately reflects regional cerebral blood flow in single-photon emission computed tomography (SPECT) remains the subject of great interest in the radiopharmaceutical field. Now, 99mTc-d, l-HMPAO (hexamethyl-propylene amine oxime) (Nowotnik et al., 1985) and 99mTc-l,l-ECD(ethyl cysteinate dimer) (Vallabhajosula et al., 1989) are accepted as valuable tracer agents for the determination of regional cerebral blood flow, but they are complexes containing the [99mTcQO]3 + core. The [TcRN]2 + core, which is isoelectronic with [TcQO]3 + and [OQTcQO] + , exhibits a very high chemical stability towards oxidation– reduction reactions and pH variations and the presence of the [99mTcN]2 + core in the molecular structure of a radiopharmaceutical may dramatically alter its biological behavior (Baldas and Bonnyman, 1985). Therefore, it may be of great interest to probe the 99mTc nitrido chemistry with some ligands to develop novel diagnostic agents. In the development of 99mTc radiopharmaceuticals for brain imaging, recently, several 99mTcN dithiocarbamate complexes containing aliphatic group on the nitrogen atom on the dithiocarbamate ligand have been reported (Zhang et al., 2005; Mallia et al., 2006; Zhang et al., 2007a, b). Bearing in mind that containing the aromatic group on the nitrogen atom on the dithiocarbamate ligand possibly alters the lipophilicity and biodistribution of
Corresponding author. Tel.: + 86 10 62208126; fax: + 86 10 62205562.
E-mail address: [email protected] (J. Zhang). 0969-8043/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2009.08.019
the complex (Pasqualini et al., 1994), our attention was drawn to study the 99mTcN(BZDTC)2 complex for finding a good imaging tracer. In this study, we report the synthesis and biodistribution in mice of a novel 99mTc nitrido complex with dithiocarbamate ligand containing the aromatic group in the ligating framework.
2. Materials and methods Succinic dihydrazide (SDH), propylenediamine tetraacetic acid (PDTA) and stannous chloride dihydrate were purchased from Aldrich Chemical Company, USA. 99Mo/99mTc generator was obtained from the China Institute of Atomic Energy (CIAE). All other chemicals were of reagent grade and were used without further purification. 2.1. Synthesis of sodium salt of N-benzyl dithiocarbamate (BZDTC) Benzylamine (0.1 mol) was dissolved in dried diethyl ether and the resulting solution was cooled in an ice–salt bath. Sodium hydroxide (0.1 mol) was added to this solution under stirring, followed by the addition of carbon disulfide (0.1 mol). The mixture was stirred for 2 h in an ice–salt bath. Most of the solvent were removed under reduced pressure, and the precipitate was collected by filtration. The crude product was recrystallized from isopropanol/diethyl ether to give white crystals of sodium N-benzyl dithiocarbamate (yield, 40%). HPLC analysis was carried out with a reversed-phase column (Kromasil 100-5C, 250 4.6 mm), Shimadzu SCL-10AVP series, by using methanol/ water (50:50, V/V) as a mobile phase, working at a flow rate of
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1.0 ml/min. HPLC chromatogram showed the impurities (about 5%) are small peaks right after the main peak (95.04%), indicating the purity of the product was over 95%. IR (KBr)/cm 1: 3314(nN–H); 3084, 3054, 3024 (nAr–H); 2970 (nC–H); 1505, 1454 (nCQC); 1047 (nCQS). 1 H NMR(D2O, 400 MHz) d (ppm): 7.24–7.13 (m, 5H, Ar–H); 4.59(s, 2H, C–H). 13 C NMR(D2O, 400 MHz) d (ppm): 211.60, 137.70, 128.62, 128.62, 127.13, 126.86, 126.86, 50.83. ESI-MS: 182 ([M–Na] ).
2.2. Preparation of the
99m
TcN(BZDTC)2 complex
The preparation of the complex was carried out using the following procedure: 1 ml of saline containing [99mTcO4] (15 MBq) was added to a kit containing 0.05 mg of stannous chloride dihydrate, 5.0 mg of succinic dihydrazide (SDH) and 5.0 mg of propylenediamine tetraacetic acid (PDTA). The mixture was kept at room temperature for 15 min.
buffer, in an electrophoresis bath. Across 12 cm of the strip, 150 V was applied for 1.5 h. The strips were dried, and the distribution of radioactivity on the strip was determined. 2.6. Biodistribution study A solution of 99mTcN(BZDTC)2 (0.1 ml, 740 KBq) was administered via a tail vein to Kunming mice (18–20 g) and the injected radioactivity measured with a well-type NaI(Tl) detector. The mice were sacrificed at 5, 30 and 60 min after injection. The organs of interest and the blood were collected, weighed and measured for radioactivity. All biodistribution studies were carried out in compliance with the national laws related to the conduct of animal experimentation.
3. Results and discussion 3.1. Chemistry of the
99m
TcN(BZDTC)2 complex
The preparation of the 99mTcN(BZDTC)2 complex can be carried out by the following method:
SDH kit 99m
[
99m
-
[
TcO4]
r.t., 15 min
2+
CH2NHCS2Na
TcN]
99m
TcN(
CH2NHCS2)2
r.t., 10 min
Successively, 5.0 mg of the sodium salt of N-benzyl dithiocarbamate dissolved in 1.0 ml of ethanol was added and the reaction was allowed to proceed for 10 min at room temperature. The radiochemical purity of the products was evaluated by TLC and HPLC ranged from 90–99%. The TLC was performed on a polyamide strip and was eluted with saline and CH2Cl2: CH3OH= 9:1(V/V). HPLC analysis was carried out with a reversed-phase column (Kromasil 100-5C, 250 4.6 mm), Shimadzu SCL-10AVP series, by using methanol/water (70:30, V/V) as a mobile phase, working at a flow rate of 1.0 ml/min. 2.3. Determination of the partition coefficient for the complex The partition coefficient was determined by mixing the complexes with an equal volume of 1-octanol and phosphate buffer (0.025 M, pH 7.0 and 7.4) in a centrifuge tube. The mixture was vortexed at room temperature for 1 min and then centrifuged at 5000 rpm for 5 min. From each phase, 0.1 ml of the aliquot was pipetted and counted in a well g-counter. The measurement was repeated three times. Care was taken to avoid cross contamination between the phases. The partition coefficient, P, was calculated using the following equation: P ¼ ðcpm in octanol cpm in backgroundÞ=ðcpm in buffer cpm in backgroundÞ
Usually the final partition coefficient value was expressed as log P. 2.4. Stability study The stability of the complex was assayed by measuring the radiochemical purity at different times after preparation. 2.5. Paper electrophoresis One ml sample was spotted on a piece of Whatman 1 chromatography paper, saturated with 0.05 M pH 7.4 phosphate
The method is based on the reaction of [99mTcO4] with succinic dihydrazide (SDH) in the presence of stannous chloride as reducing agent to form a technetium-99m nitrido intermediate. The [99mTcRN]2 + is a suitable substrate for the substitution reaction with the sodium salt of N-benzyl dithiocarbamate at room temperature to give the final complex 99m TcN(BZDTC)2. The radiochemical purity of the complex was routinely checked by TLC. In saline, 99mTcO4 , 99mTcO2 nH2O and 99m TcN(BZDTC)2 remained at the origin while [99mTcRN]2int+ migrated with the front. In CH2Cl2: CH3OH= 9:1 (V/V), 99m TcN(BZDTC)2 migrated with the front while 99mTcO4 , 99m TcO2 nH2O and [99mTcRN]2int+ remained at the origin. HPLC retention times (Rt) for some selected complexes were reported as 99m TcO4 : 3.2 min, [99mTcRN]2int+ : 2.6 min, follows: 99m TcN(BZDTC)2: 13.8 min. The HPLC pattern of 99mTcN(BZDTC)2 is shown in Fig. 1. Single peak suggested only one product was formed. The mean radiochemical purity of the product was over 90% immediately after the preparation. Bis(diethyldithiocarbamato) nitrido technetium-99m [99mTcN(DDC)2] is neutral and has a square pyramidal geometry with an apical TcRN bond and two monoanionic dithiocarbamate ligands spanning the four positions in the basal plane through the four sulfur atoms (Baldas et al., 1981). It can be presumed that 99mTcN(BZDTC)2 would also have a similar structure. Clearly, further studies should be performed, using macroscopic levels of the long-lived 99Tc, to determine and characterize the structure of the novel complex.
3.2. Partition coefficient (log P) of the complex The partition coefficient (log P) of the complex was 1.33 and 1.31 at pH 7.0 and 7.4, respectively, showing no great difference between pH 7.0 and 7.4. The log P of the complex is within the range of values quoted for lipophilicity (0.9–2.5) that are suitable
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for crossing the blood brain barrier (BBB). Due to its high lipophilicity, the complex is easily adsorbed on the walls of the vials and syringes. In order to avoid this adsorption, 10 mg of gcyclodextrin was added to the final solution.
103
3.4. Paper electrophoresis In paper electrophoresis, almost all the radioactivity was found at the point of spotting (percentage of radioactivity: 97.9%), suggesting that it was neutral.
3.3. Stability of the complex 3.5. Biodistribution of the complex in mice The RCP of the product was nearly constant ( 490%) over the observed period of 6 h, suggesting that it was stable in the reaction mixture at room temperature.
491664 13.80
count
368748
245832
122916
0
0
5
10
15 20 t (min)
Fig. 1. The HPLC pattern of
Table 1 Biodistribution in mice of the
99m
25
30
99m
TcN(BZDTC)2.
TcN(BZDTC)2 complex (x7 s, n =3) (%ID/g).
t (min)
5
30
60
Brain Heart Liver Lung Kidney Blood Br/Bl ratio
1.87 7 0.18 12.11 7 3.47 47.28 7 8.48 11.95 7 1.96 11.91 7 0.10 2.50 7 0.38 0.75
1.21 70.08 1.61 70.72 31.97 77.43 3.39 70.65 3.23 71.03 0.78 70.13 1.55
0.85 70.40 1.23 70.07 34.31 72.81 2.05 70.47 2.24 70.16 0.76 70.12 1.12
Table 2 Comparison of biodistribution of recently reported
99m
Biological distribution pattern of the complex in mice is presented in Table 1, as a percentage of the injected dose per gram of organ (%ID/g). Results of biodistribution of recently reported 99mTcN dithiocarbamate complexes and 99mTc-ECD are presented in Table 2 for comparison. As described in Table 1, the 99mTcN(BZDTC)2 complex can penetrate the intact blood-brain barrier (BBB) and has a high brain uptake and good retention. The blood clearance is fast so that the brain/blood ratio is high as 0.75, 1.55 and 1.12 at 5, 30 and 60 min post-injection, respectively. The 99mTcN(BZDTC)2 complex also exhibits significant initial myocardial uptake, but the washout from the heart is rapid thus making it unsuitable to be a good myocardial perfusion imaging agent. The liver uptake was appreciable and remained high at 60 min post-injection, suggesting that the hepatobiliary system is the major route of excretion of the administered radioactivity. As can be seen in Table 2, among these reported 99mTcNdithiocarbamates for cerebral imaging, including [99mTcN(PREDTC)2 (PREDTC: N-2-(1-pyrrolidino) ethyl dithiocarbamate), 99mTcN(BZDTC)2, 99mTcN(PPEDTC)2 (PPEDTC: N-2-(1piperidyl) ethyl dithiocarbamate), 99mTcN(BDTC)2 (BDTC: N-butyl dithiocarbamate), 99mTcN(SBDTC)2 (SBDTC: N-sec-butyl dithio99m TcN(MEDTC)2 (MEDTC: N-2-methoxyethyl carbamate), dithiocarbamate), 99mTcN(MEMDTC)2 (MEMDTC: N-2-methoxyethyl-N-methyl dithiocarbamate), 99mTcN(EPDTC)2 (EPDTC: N-3-ethoxypropyl dithiocarbamate), 99mTcN(EPMDTC)2 (EPMDTC: N-3-ethoxypropyl-N-methyl dithiocarbamate)], 99mTcN(BZDTC)2 exhibits the least blood uptake so that it has a high brain/blood ratio. Their biodistributions are strongly dependent on the lateral groups bonded to the uncoordinated nitrogen atom of the dithiocarbamate ligand. A decrease of brain uptake in the order is observed: 99mTcN(SBDTC)2 4 99mTcN(BDTC)2 4 99mTcN(PPEDTC)2 4 99mTcN(BZDTC)2 4 99mTcN(MEDTC)2 4 99mTcN(MEMDTC)2 4 99m TcN(EPDTC)2 4 99mTcN(EPMDTC)2 4 99mTcN(PREDTC)2. In the limits of our study, if the lipophilicity of the complex (log P) is within the range of (0.9–1.6), interestingly, on increasing the lipophilicity of the complex (log P), one can observe an increase of the brain uptake. As compared with 99mTc-ECD (log P: 1.20) (Mang’era et al., 1996), 99mTcN(BZDTC)2 shows higher log P value (1.31) and higher brain uptake. However, if the lipophilicity of the complex(log P) is higher than 1.70, the brain uptake of the complex gradually decreases as the lipophilicity of the complex (log P) increases.
TcN dithiocarbamate complexes and
99m
Tc-ECD in mice at 30 min post-injection (%ID/g, x 7s, n= 3).
Complexes
log P
Brain
Blood
Br/Bl ratio
Reference
99m
0.99 1.20 1.31 1.39 1.51 1.51 1.85 1.87 1.97 1.99
0.29 70.04 0.30 70.00 1.21 70.08 1.58 70.37 2.73 70.35 3.44 70.28 0.94 70.03 0.69 70.04 0.63 70.10 0.38 70.07
3.35 7 0.42 2.107 0.10 0.787 0.13 5.61 7 0.42 1.63 7 0.39 2.37 7 0.18 3.92 7 0.57 1.77 7 0.10 2.28 7 0.14 1.13 7 0.13
0.09 0.14 1.55 0.28 1.67 1.45 0.24 0.24 0.28 0.34
Zhang et al., (2007b) Mang’era et al., (1996) This study Zhang et al., (2007b) Zhang et al., (2007a) Zhang et al., (2005) Mallia et al. (2006) Mallia et al. (2006) Mallia et al. (2006) Mallia et al. (2006)
TcN(PREDTC)2 Tc-ECD TcN(BZDTC)2 99m TcN(PPEDTC)2 99m TcN(BDTC)2 99m TcN(SBDTC)2 99m TcN(MEDTC)2 99m TcN(MEMDTC)2 99m TcN(EPDTC)2 99m TcN(EPMDTC)2 99m 99m
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Among the nine complexes, only three complexes (99mTcN(BDTC)2,99mTcN(BZDTC)2 and 99mTcN(SBDTC)2) have a high brain uptake and higher brain/blood ratio (Br/Bl ratio 41), suggesting 99mTcN(BZDTC)2 would also be a potential brain perfusion imaging agent. The facts suggest the dithiocarbamate ligands containing the aromatic group seemed reasonable for preparing a novel brain tracer containing the [99mTcN]2 + core.
4. Conclusion To conclude, a novel 99mTc nitrido dithiocarbamate complex containing aromatic group 99mTcN(BZDTC)2 was prepared in high yields through a ligand-exchange reaction. As the complex has low molecular weight ( o600 Da), and is neutral and lipophilic, it has a tendency to cross the BBB. The biodistribution studies in mice showed that 99mTcN(BZDTC)2 accumulated in the brain with high uptake and good retention, suggesting it would be a potential brain imaging agent.
Acknowledgments The work was financially supported by National Natural Science Foundation of China (20771018) and by Beijing Normal University.
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