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Radiosynthesis of [thiocarbonyl-11C]disulfiram and its first PET study in mice
Journal Pre-proofs Radiosynthesis of [thiocarbonyl- 11 C]disulfir am and its fir st PET study in mice Hideki Ishii, Tomoteru Yamasaki, Joji Yui, Yiding Zhang, Masayuki Hanyu, Masanao Ogawa, Nobuki Nengaki, Atsushi B. Tsuji, Yuya Terashima, Kouji Matsushima, Ming-Rong Zhang PII: DOI: Reference:
S0960-894X(20)30061-5 https://doi.org/10.1016/j.bmcl.2020.126998 BMCL 126998
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
28 November 2019 15 January 2020 24 January 2020
Please cite this article as: Ishii, H., Yamasaki, T., Yui, J., Zhang, Y., Hanyu, M., Ogawa, M., Nengaki, N., Tsuji, A.B., Terashima, Y., Matsushima, K., Zhang, M-R., Radiosynthesis of [thiocarbonyl- 11 C]disulfir am and its fir st PET study in mice, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl. 2020.126998
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Radiosynthesis of [thiocarbonyl11C]disulfiram and its first PET study in mice
Hideki Ishii, Tomoteru Yamasaki, Joji Yui, Yiding Zhang, Masayuki Hanyu, Masanao Ogawa, Nobuki Nengaki, Atsushi B Tsuji, Yuya Terashima, Kouji Matsushima and Ming-Rong Zhang 11 11
CO2
11
CS2
N
S C
S S S
N
[11C]Disulfiram
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com
Radiosynthesis of [thiocarbonyl-11C]disulfiram and its first PET study in mice Hideki Ishiia, *, Tomoteru Yamasakia, Joji Yuia, Yiding Zhanga, Masayuki Hanyua, Masanao Ogawaa, Nobuki Nengakia, Atsushi B Tsujib, Yuya Terashimac, Kouji Matsushimac and Ming-Rong Zhanga, * Department of Advanced Nuclear Medicine Sciences, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan bDepartment of Molecular Imaging and Theranostics, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan cDivision of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute for Biomedical Science (RIBS), Tokyo University of Science, Chiba 278-0022, Japan a
ARTICLE INFO
ABSTRACT
Article history: Received Revised Accepted Available online
[Thiocarbonyl-11C]disulfiram ([11C]DSF) was synthesized via iodine oxidation of [11C]diethylcarbamodithioic acid ([11C]DETC), which was prepared from [11C]carbon disulfide and diethylamine. The decay-corrected isolated radiochemical yield (RCY) of [11C]DSF was greatly affected by the addition of unlabeled carbon disulfide. In the presence of carbon disulfide, the RCY was increased up to 22% with low molar activity (Am, 0.27 GBq/mol). On the other hand, [11C]DSF was obtained in 0.4% RCY with a high Am value (95 GBq/mol) in the absence of carbon disulfide. The radiochemical purity of [11C]DSF was always >98%. The first PET study on [11C]DSF was performed in mice. A high uptake of radioactivity was observed in the liver, kidneys, and gallbladder. The uptake level and distribution pattern in mice were not significantly affected by the Am value of the [11C]DSF sample used. In vivo metabolite analysis showed the rapid decomposition of [11C]DSF in mouse plasma.
Keywords: disulfiram PET 11C-carbon disulfide radiosynthesis diethylcarbamodithioic acid
1,1',1'',1'''-[Disulfanediylbis(carbonothioylnitrilo)]tetraethane (disulfiram, DSF, also sold as Antabuse) has been widely used for the treatment of alcoholism for more than 60 years.1 Recent studies also suggest that DSF may have potential therapeutic effects for cocaine dependence,2 several cancers (lung, melanoma, breast, and prostate cancers) and other applications (see citations in reference 5),3,4,5 as well as the prevention of cataract development,6 treatment of Menkes disease as its copper-complex,7 and Alzheimer’s disease.8 In addition, many types of biological activity including the inhibition of dopamine--hydroxylase,9 carboxylesterase,10 cholinesterase,11 ubiquitin E3 ligase activity,12 cytochrome P450 2E1,13 and P-glycoprotein ATPase14 have been reported. Recently, our co-authors reported that DSF binds strongly to FROUNT15 (also known as NUP85), a chemokine receptor-associating cytoplasmic protein, which governs both macrophage accumulation in tumors and their intratumoral properties, and reduces tumor progression via inhibition of the FROUNT-CCR2 interaction.16 The detailed metabolic pathway of DSF has been already investigated by many research groups. The most probable metabolic fate of DSF is its rapid degradation in human plasma upon reaction with protein (serum albumin) to produce diethylcarbamodithioic acid (DETC) and its covalently
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bonded adduct (protein-DETC), followed by the further degradation of DETC to give S-methyl-N,N-diethylthiocarbamate sulfoxide (DETC-MeSO), S-methyl-N,N-diethylthiocarbamate sulfone (DETC-MeSO2), diethylamine, and carbon disulfide (Figure 1).17-25 Thus, most of the biological actions of DSF, especially the inhibition of aldehyde dehydrogenase, are believed to be caused by the metabolites of DSF, such as DETC-MeSO and DETC-MeSO2. Although the anti-tumor activity of DSF has been previously reported to date,26,27 there are only a few successful reports on the clinical use of DSF.28,29 This may be due to its instability in the bloodstream.30 Recently, several research groups have reported that the anticancer activity of DSF can be increased using encapsulation technology, which prevents the degradation of DSF in the bloodstream.31-33 Thus, the direct delivery of DSF into cancer cells appears to be needed for effective cancer therapy.
Figure 1. Disulfiram and its metabolites.
On the other hand, recent advances in positron emission tomography (PET) using a short-lived positron emitter (e.g., 11C, half-life of 20.4 min or 18F, half-life of 109.8 min) not only serves as a diagnosis imaging biomarker for several diseases, but also as an assessment tool for pharmacokinetics/pharmacodynamics in living animals.34,35 Therefore, the development of 11C-labelled DSF is of great help to clarify the biological action and evaluate the pharmacokinetics of DSF, especially when newly developed techniques such as encapsulation are employed. Herein, we report the first synthesis of [11C]DSF using [11C]carbon disulfide as a radiolabeling precursor and its first application in a PET study using mice. DSF is a C2-symmetric molecule, which has three different carbon atoms. Therefore, three possible 11C-labeled compounds [11C]DSF, [11C]DSF', and [11C]DSF'' may be prepared from two precursors, as shown in Fig. 2 and Scheme 1. However, DSF is generally synthesized via the oxidation of DETC, which is prepared from carbon disulfide and diethylamine (Scheme 1).12, 3638 Although a same precursor (N,N,N'-triethylthiuram disulfide) can be used to prepare [11C]DSF' and [11C]DSF'', there are no reports on the synthesis of DSF using N,N,N'-triethylthiuram disulfide. Thus, we planned the synthesis of [11C]DSF via route A (Scheme 1). Fortunately, the 11C-labeled analog of the [11C]DETC intermediate (or its complex with diethylamine, [11C]DETC·NHEt2) has already been synthesized by Miller et al. using [11C]carbon disulfide and diethylamine, as shown in Scheme 2.39 However, to the best of our knowledge, there are no reports on the synthesis of [11C]DSF published to date.
solution containing carbon disulfide and diethylamine without the need to isolate the DETC intermediate. Thus, we examined the synthesis of [11C]DSF using the I2 oxidation of [11C]DETC, which was prepared from [11C]carbon disulfide and diethylamine.
Scheme 3. Synthesis of disulfiram.
The preparation of [11C]carbon disulfide was carried out using the method reported by Miller et al.39 with some modification. [11C]Methyl iodide gas produced in the reaction was passed through a heated glass column packed with phosphorus pentasulfide–silica gel (1:2, w/w) at 400 C and the resulting [11C]carbon disulfide product was trapped in acetonitrile at room temperature. To the solution of [11C]carbon disulfide was added a solution of diethylamine in acetonitrile. After leaving the reaction mixture for 2 min at room temperature, a solution of iodine in acetonitrile was added and allowed to stand for 2 min at room temperature (Scheme 4). However, only a trace amount of [11C]DSF was formed accompanied with unknown radiochemical products (Figure 3). A comparison of the retention time observed for the [11C]DETC intermediate suggested that most of the [11C]carbon disulfide had been converted into the [11C]DETC intermediate (Figure 4, retention time = 1.2 min). Thus, we speculated that the oxidation process using iodine may be hampered by the lack of the [11C]DETC intermediate.
Figure 2. 11C-Labeled disulfiram candidates.
Scheme 4. Iodine oxidation of [11C]diethyldithiocarbamate.
Scheme 1. Typical synthesis of disulfiram and possible routes for the preparation of 11C-labeled disulfiram.
Scheme 2. Synthesis of [11C]diethyldithiocarbamate ([11C]DETC).
Therefore, the synthesis of DSF via DETC was initially investigated. Scheme 3 shows the target disulfide, DSF, was easily obtained via I2 oxidation of DETC (diethylamine salt). This reaction was also achieved via the direct I2 oxidation of a mixed
Figure 3. RI chromatogram obtained for the reaction mixture of [11C]carbon disulfide, diethylamine, and iodine.
[11C]DSF product in a 12% radiochemical yield (RCY) with low molar activity (Am = 0.16 GBq/mol) (Table 1, line 1; Figure 4). Interestingly, the RCY was increased up to 22% when using an excess of diethylamine (Table 1, line 2). In order to increase the Am value, the relationship between carbon disulfide, diethylamine, and iodine was investigated. When using 5 L of carbon disulfide, the desired [11C]DSF product was formed with an improved RCY of 21% at a substrate/reagent (carbon disulfide/diethylamine/iodine) ratio of 1:2:1 (Table 1, entry 3–5). However, when the amount of carbon disulfide was decreased from 5 L to 1 L, the RCY was also decreased from 21 to 7.4% (Table 1, entry 3, 6, and 7). Finally, we attempted to prepare [11C]DSF in the absence of carbon disulfide using a small amount of diethylamine/iodine (2:1), which gave the best Am value, but poor RCY (0.4%) (Table 1, entry 9). Despite our extensive attempts to increase the RCY in the absence of carbon disulfide, only low RCY values were observed (data not shown)..
Figure 4. Typical UV and RI chromatograms obtained for (a) a solution of [11C]carbon disulfide in acetonitrile including unreacted [11C]methyl iodide (<1%), (b) the reaction mixture containing [11C]carbon disulfide and diethylamine, (c) the reaction mixture containing [11C]carbon disulfide, diethylamine, and iodine in the presence of carbon disulfide,, (d) the reference carbon disulfide sample, (e) a mixture of carbon disulfide and diethylamine, and (f) the reference DSF sample (COSMOSIL 5C18PAQ 4.6 mm I.D. x 250 mm, acetonitrile–H2O (6:4), flow rate 2 mL/min, UV 200 nm).
In order to promote the generation of [11C]DSF, we added 10 L (0.17 mmol) of unlabeled carbon disulfide into the reaction system prior to the addition of diethylamine. As expected, in the presence of carbon disulfide the oxidation reaction of [11C]DETC using iodine was successfully carried out to give the desired
Table 1. Isolated radiochemical yields and molar radioactivity of the [11C]disulfiram samples. Entry
CS2 (L)
Et2NH (L)
I2 (L)
Mol Ratio (CS2/Et2NH/I2)
RCY(%)a
Am (GBq/mmol)
1
10
10
30
1/0.6/0.7
12
0.16
2
10
50
35
1/2.9/0.8
22
0.27
3
5
10
30
1/1.2/1.4
14
0.41
4
5
20
20
1/2.3/0.9
21
0.34
5
5
40
30
1/4.6/1.4
10
0.56
6
4
15
20
1/2.2/1.2
9
0.70
7
2
6
8
1/1.7/0.9
15
0.86
8
1
3
4
1/1.7/0.9
7.4
2.1
9
0
1.5
2
-
0.4
95
Decay corrected isolated radiochemical yield (RCY) based on [11C]carbon dioxide.
a
After intravenous injection of [11C]DSF prepared with three different Am values (high: 95 GBq/mol; low: 0.56 GBq/µmol; ultra-low: 0.01 GBq/µmol) in mice, PET was conducted for 30 min. Figure 5 and 6 show the PET images and time-active curves in the heart, lungs, liver, kidneys, muscle, and brain.
Figure 6. Time-activity curves (TACs) observed between 0 and 30 min after injection of [11C]disulfiram with three different Am values.
After the injection of [11C]DSF prepared with three different Am values, the injected dosing solution was carried through the vena cava to the heart and then its radioactivity was distributed throughout the body. The radioactivity initially displays high uptake levels in the heart and lungs depending on the blood Figure 5. PET imaging of [11C]disulfiram with three different Am values in mice.
concentration, exhibiting a quick washout. Subsequently, high radioactivity was observed in the liver and kidneys, which then rapidly decreased. Uptake in these organs peaked rapidly and was then eliminated. On the other hand, the enormous radioactive accumulation observed in the urinary bladder demonstrates its rapid and significant excretion in urine (see Figure 7). The high radioactivity observed in the liver, gall bladder, and small intestine during the initial time period also suggests biliary excretion of the radioactivity.
acetonitrile, and centrifuged. The supernatant was analyzed using high-performance liquid chromatography (HPLC) equipped with a radioactivity detector. The recovery of radioactivity obtained from HPLC analysis of both samples was >90%. Figure 8 shows the HPLC traces recorded for [11C]DSF in plasma and urine 1 min after the injection of the radioprobe. No unchanged [11C]DSF was observed in both samples. A radiolabelled metabolite (Rt = 2 min) was much more hydrophilic than the intact form. On the other hand, no significant effect was determined due to the Am values of the [11C]DSF sample used.
Figure 8. Metabolite analysis of [11C]disulfiram in plasma and urine samples 1 min after the injection of [11C]disulfiram.
Figure 7. Static 4-5 min whole-body PET imaging of [11C]disulfiram with three different Am values in mice.
The time-activity curves shown in Figure 6 indicate there was no difference in the radioactivity levels in almost all of the organs within 30 min after the injection of [11C]DSF with three different Am, although the initial uptake levels of radioactivity in these organs were highly dependent on unlabeled DSF contents. Thus, low molar activity combined with the use of a high dose of unlabeled DSF did not show any significant effects on the rapid kinetics of [11C]DSF prepared with a high Am value for at least 30 min after the injection. After injection of [11C]DSF in the mouse, arterial blood and urine samples were collected at 1 and 5 min, deproteinized with
In summary, [thiocarbonyl-11C]disulfiram ([11C]DSF) was successfully synthesized for the first time upon the reaction of [11C]carbon disulfide and diethylamine followed by iodine oxidation. Moreover, the first PET studies of [11C]DSF with three different Am were performed on mice. The uptake level and radioactivity distribution were not significantly affected by the Am values of [11C]DSF used. A high uptake of radioactivity in the liver, kidneys, and gallbladder were observed and metabolite analysis in the plasma showed the rapid metabolism of [11C]DSF. This is the first report on the pharmacokinetics of DSF at such an early stage20-22 in living animals. Recently, the tumor suppressing effect of DSF targeting p97 has been reported, but the mechanism of this in vivo is not yet fully understood.40 Therefore, we believe that PET with [11C]DSF may be a useful tool for evaluating the therapeutic effects and characterizing the mechanism of DSF. 6.
Acknowledgments We would like to thank the staff of the Cyclotron Operation Section and the Department of Advanced Nuclear Medicine Sciences of the National Institute of Radiological Sciences (NIRS), National Institutes for Quantum and Radiological Science and Technology for their support with the operation of the cyclotron and the production of the radioisotopes. This study was supported by a Grant-in-aid for P-DIRECT from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References and notes 1. 2. 3. 4.
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Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Radiosynthesis of [thiocarbonyl11C]disulfiram and its first PET study in mice
Hideki Ishii, Tomoteru Yamasaki, Joji Yui, Yiding Zhang, Masayuki Hanyu, Masanao Ogawa, Nobuki Nengaki, Atsushi B Tsuji, Yuya Terashima, Kouji Matsushima and Ming-Rong Zhang 11 11
CO2
11
CS2
N
S C
S S S
N
[11C]Disulfiram 30. 31. 32. 33. 34.
35. 36. 37. 38. 39. 40.
Cancer P. D. 2013;16:357-361. .Johansson B. Acta Psychiatr. Scand. 1992;86:15-26. Liu P, Wang ZP, Brown S, Kannappan V, Tawari PE, Jiang WG, Irache JM, Tang JZ, Britland S, Armesilla AL, Darling JL, Tang X, Wang W. Oncotarget 2014;5:7471-7485. Duan X, Xiao J, Yin Q, Zhang Z, Yu H, Mao S, Li Y. Nanotechnology 2014;25:125102. Fasehee H, Dinarvand R, Ghavamzadeh A, Esfandyari-Manesh M, Moradian H, Faghihi S, Ghaffari SH. J. Nanobiotechnology 2016;14:32. Abanades S, van der Aart J, Barletta JAR, Marzano C, Searle GE, Salinas CA, Ahmad JJ, Reiley RR, Pampols-Maso S, Zamuner S, Cunningham VJ, Rabiner EA, Laruelle MA, Gunn RN. J. Cerebr. Blood F. Met. 2011;31:944-952. Nakatani Y, Suzuki M, Tokunaga M, Maeda J, Sakai M, Ishihara H, Yoshinaga T, Takenaka O, Zhang MR, Suhara T, Higuchi M. Plos One 2013;8:1-13. Kapanda CN, Mucci GG, Labar G, Poupaert JH, Lambert DM. J. Med. Chem. 2009;52:7310-7314. Milosavljevic MM, Marinkovic AD, Markovic JM, Brkovic DV, Misavljevic MM. Chem. Ind. Eng. Q. 2012;18:73-81. Liang F, Tan J, Piao C, Liu Q. Synthesis 2008;22:3579-3584. Miller PW, Bender D. Chem. Eur. J. 2012;18:433-436. Skrott Z, Mistrik M, Andersen KK, Friis S, Majera D, Gursky J, Ozdian T, Bartkova J, Turi Z, Moudry P, Kraus M, Michalova M, Vaclavkova J, Dzubak P, Vrobel I, Pouckova P, Sedlacek J, Miklovicova A, Kutt A, Li J, Mattova J, Driessen C, Dou QP, Olsen J, Hajduch M, Cvek B, Deshaies RJ, Bartek J. Nature 2017;552:194-199.
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S0960-894X(20)30061-5 https://doi.org/10.1016/j.bmcl.2020.126998 BMCL 126998
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
28 November 2019 15 January 2020 24 January 2020
Please cite this article as: Ishii, H., Yamasaki, T., Yui, J., Zhang, Y., Hanyu, M., Ogawa, M., Nengaki, N., Tsuji, A.B., Terashima, Y., Matsushima, K., Zhang, M-R., Radiosynthesis of [thiocarbonyl- 11 C]disulfir am and its fir st PET study in mice, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl. 2020.126998
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.
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To create your abstract, type over the instructions in the template box below. Fonts or abstract dimensions should not be changed or altered.
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Radiosynthesis of [thiocarbonyl11C]disulfiram and its first PET study in mice
Hideki Ishii, Tomoteru Yamasaki, Joji Yui, Yiding Zhang, Masayuki Hanyu, Masanao Ogawa, Nobuki Nengaki, Atsushi B Tsuji, Yuya Terashima, Kouji Matsushima and Ming-Rong Zhang 11 11
CO2
11
CS2
N
S C
S S S
N
[11C]Disulfiram
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com
Radiosynthesis of [thiocarbonyl-11C]disulfiram and its first PET study in mice Hideki Ishiia, *, Tomoteru Yamasakia, Joji Yuia, Yiding Zhanga, Masayuki Hanyua, Masanao Ogawaa, Nobuki Nengakia, Atsushi B Tsujib, Yuya Terashimac, Kouji Matsushimac and Ming-Rong Zhanga, * Department of Advanced Nuclear Medicine Sciences, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan bDepartment of Molecular Imaging and Theranostics, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan cDivision of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute for Biomedical Science (RIBS), Tokyo University of Science, Chiba 278-0022, Japan a
ARTICLE INFO
ABSTRACT
Article history: Received Revised Accepted Available online
[Thiocarbonyl-11C]disulfiram ([11C]DSF) was synthesized via iodine oxidation of [11C]diethylcarbamodithioic acid ([11C]DETC), which was prepared from [11C]carbon disulfide and diethylamine. The decay-corrected isolated radiochemical yield (RCY) of [11C]DSF was greatly affected by the addition of unlabeled carbon disulfide. In the presence of carbon disulfide, the RCY was increased up to 22% with low molar activity (Am, 0.27 GBq/mol). On the other hand, [11C]DSF was obtained in 0.4% RCY with a high Am value (95 GBq/mol) in the absence of carbon disulfide. The radiochemical purity of [11C]DSF was always >98%. The first PET study on [11C]DSF was performed in mice. A high uptake of radioactivity was observed in the liver, kidneys, and gallbladder. The uptake level and distribution pattern in mice were not significantly affected by the Am value of the [11C]DSF sample used. In vivo metabolite analysis showed the rapid decomposition of [11C]DSF in mouse plasma.
Keywords: disulfiram PET 11C-carbon disulfide radiosynthesis diethylcarbamodithioic acid
1,1',1'',1'''-[Disulfanediylbis(carbonothioylnitrilo)]tetraethane (disulfiram, DSF, also sold as Antabuse) has been widely used for the treatment of alcoholism for more than 60 years.1 Recent studies also suggest that DSF may have potential therapeutic effects for cocaine dependence,2 several cancers (lung, melanoma, breast, and prostate cancers) and other applications (see citations in reference 5),3,4,5 as well as the prevention of cataract development,6 treatment of Menkes disease as its copper-complex,7 and Alzheimer’s disease.8 In addition, many types of biological activity including the inhibition of dopamine--hydroxylase,9 carboxylesterase,10 cholinesterase,11 ubiquitin E3 ligase activity,12 cytochrome P450 2E1,13 and P-glycoprotein ATPase14 have been reported. Recently, our co-authors reported that DSF binds strongly to FROUNT15 (also known as NUP85), a chemokine receptor-associating cytoplasmic protein, which governs both macrophage accumulation in tumors and their intratumoral properties, and reduces tumor progression via inhibition of the FROUNT-CCR2 interaction.16 The detailed metabolic pathway of DSF has been already investigated by many research groups. The most probable metabolic fate of DSF is its rapid degradation in human plasma upon reaction with protein (serum albumin) to produce diethylcarbamodithioic acid (DETC) and its covalently
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bonded adduct (protein-DETC), followed by the further degradation of DETC to give S-methyl-N,N-diethylthiocarbamate sulfoxide (DETC-MeSO), S-methyl-N,N-diethylthiocarbamate sulfone (DETC-MeSO2), diethylamine, and carbon disulfide (Figure 1).17-25 Thus, most of the biological actions of DSF, especially the inhibition of aldehyde dehydrogenase, are believed to be caused by the metabolites of DSF, such as DETC-MeSO and DETC-MeSO2. Although the anti-tumor activity of DSF has been previously reported to date,26,27 there are only a few successful reports on the clinical use of DSF.28,29 This may be due to its instability in the bloodstream.30 Recently, several research groups have reported that the anticancer activity of DSF can be increased using encapsulation technology, which prevents the degradation of DSF in the bloodstream.31-33 Thus, the direct delivery of DSF into cancer cells appears to be needed for effective cancer therapy.
Figure 1. Disulfiram and its metabolites.
On the other hand, recent advances in positron emission tomography (PET) using a short-lived positron emitter (e.g., 11C, half-life of 20.4 min or 18F, half-life of 109.8 min) not only serves as a diagnosis imaging biomarker for several diseases, but also as an assessment tool for pharmacokinetics/pharmacodynamics in living animals.34,35 Therefore, the development of 11C-labelled DSF is of great help to clarify the biological action and evaluate the pharmacokinetics of DSF, especially when newly developed techniques such as encapsulation are employed. Herein, we report the first synthesis of [11C]DSF using [11C]carbon disulfide as a radiolabeling precursor and its first application in a PET study using mice. DSF is a C2-symmetric molecule, which has three different carbon atoms. Therefore, three possible 11C-labeled compounds [11C]DSF, [11C]DSF', and [11C]DSF'' may be prepared from two precursors, as shown in Fig. 2 and Scheme 1. However, DSF is generally synthesized via the oxidation of DETC, which is prepared from carbon disulfide and diethylamine (Scheme 1).12, 3638 Although a same precursor (N,N,N'-triethylthiuram disulfide) can be used to prepare [11C]DSF' and [11C]DSF'', there are no reports on the synthesis of DSF using N,N,N'-triethylthiuram disulfide. Thus, we planned the synthesis of [11C]DSF via route A (Scheme 1). Fortunately, the 11C-labeled analog of the [11C]DETC intermediate (or its complex with diethylamine, [11C]DETC·NHEt2) has already been synthesized by Miller et al. using [11C]carbon disulfide and diethylamine, as shown in Scheme 2.39 However, to the best of our knowledge, there are no reports on the synthesis of [11C]DSF published to date.
solution containing carbon disulfide and diethylamine without the need to isolate the DETC intermediate. Thus, we examined the synthesis of [11C]DSF using the I2 oxidation of [11C]DETC, which was prepared from [11C]carbon disulfide and diethylamine.
Scheme 3. Synthesis of disulfiram.
The preparation of [11C]carbon disulfide was carried out using the method reported by Miller et al.39 with some modification. [11C]Methyl iodide gas produced in the reaction was passed through a heated glass column packed with phosphorus pentasulfide–silica gel (1:2, w/w) at 400 C and the resulting [11C]carbon disulfide product was trapped in acetonitrile at room temperature. To the solution of [11C]carbon disulfide was added a solution of diethylamine in acetonitrile. After leaving the reaction mixture for 2 min at room temperature, a solution of iodine in acetonitrile was added and allowed to stand for 2 min at room temperature (Scheme 4). However, only a trace amount of [11C]DSF was formed accompanied with unknown radiochemical products (Figure 3). A comparison of the retention time observed for the [11C]DETC intermediate suggested that most of the [11C]carbon disulfide had been converted into the [11C]DETC intermediate (Figure 4, retention time = 1.2 min). Thus, we speculated that the oxidation process using iodine may be hampered by the lack of the [11C]DETC intermediate.
Figure 2. 11C-Labeled disulfiram candidates.
Scheme 4. Iodine oxidation of [11C]diethyldithiocarbamate.
Scheme 1. Typical synthesis of disulfiram and possible routes for the preparation of 11C-labeled disulfiram.
Scheme 2. Synthesis of [11C]diethyldithiocarbamate ([11C]DETC).
Therefore, the synthesis of DSF via DETC was initially investigated. Scheme 3 shows the target disulfide, DSF, was easily obtained via I2 oxidation of DETC (diethylamine salt). This reaction was also achieved via the direct I2 oxidation of a mixed
Figure 3. RI chromatogram obtained for the reaction mixture of [11C]carbon disulfide, diethylamine, and iodine.
[11C]DSF product in a 12% radiochemical yield (RCY) with low molar activity (Am = 0.16 GBq/mol) (Table 1, line 1; Figure 4). Interestingly, the RCY was increased up to 22% when using an excess of diethylamine (Table 1, line 2). In order to increase the Am value, the relationship between carbon disulfide, diethylamine, and iodine was investigated. When using 5 L of carbon disulfide, the desired [11C]DSF product was formed with an improved RCY of 21% at a substrate/reagent (carbon disulfide/diethylamine/iodine) ratio of 1:2:1 (Table 1, entry 3–5). However, when the amount of carbon disulfide was decreased from 5 L to 1 L, the RCY was also decreased from 21 to 7.4% (Table 1, entry 3, 6, and 7). Finally, we attempted to prepare [11C]DSF in the absence of carbon disulfide using a small amount of diethylamine/iodine (2:1), which gave the best Am value, but poor RCY (0.4%) (Table 1, entry 9). Despite our extensive attempts to increase the RCY in the absence of carbon disulfide, only low RCY values were observed (data not shown)..
Figure 4. Typical UV and RI chromatograms obtained for (a) a solution of [11C]carbon disulfide in acetonitrile including unreacted [11C]methyl iodide (<1%), (b) the reaction mixture containing [11C]carbon disulfide and diethylamine, (c) the reaction mixture containing [11C]carbon disulfide, diethylamine, and iodine in the presence of carbon disulfide,, (d) the reference carbon disulfide sample, (e) a mixture of carbon disulfide and diethylamine, and (f) the reference DSF sample (COSMOSIL 5C18PAQ 4.6 mm I.D. x 250 mm, acetonitrile–H2O (6:4), flow rate 2 mL/min, UV 200 nm).
In order to promote the generation of [11C]DSF, we added 10 L (0.17 mmol) of unlabeled carbon disulfide into the reaction system prior to the addition of diethylamine. As expected, in the presence of carbon disulfide the oxidation reaction of [11C]DETC using iodine was successfully carried out to give the desired
Table 1. Isolated radiochemical yields and molar radioactivity of the [11C]disulfiram samples. Entry
CS2 (L)
Et2NH (L)
I2 (L)
Mol Ratio (CS2/Et2NH/I2)
RCY(%)a
Am (GBq/mmol)
1
10
10
30
1/0.6/0.7
12
0.16
2
10
50
35
1/2.9/0.8
22
0.27
3
5
10
30
1/1.2/1.4
14
0.41
4
5
20
20
1/2.3/0.9
21
0.34
5
5
40
30
1/4.6/1.4
10
0.56
6
4
15
20
1/2.2/1.2
9
0.70
7
2
6
8
1/1.7/0.9
15
0.86
8
1
3
4
1/1.7/0.9
7.4
2.1
9
0
1.5
2
-
0.4
95
Decay corrected isolated radiochemical yield (RCY) based on [11C]carbon dioxide.
a
After intravenous injection of [11C]DSF prepared with three different Am values (high: 95 GBq/mol; low: 0.56 GBq/µmol; ultra-low: 0.01 GBq/µmol) in mice, PET was conducted for 30 min. Figure 5 and 6 show the PET images and time-active curves in the heart, lungs, liver, kidneys, muscle, and brain.
Figure 6. Time-activity curves (TACs) observed between 0 and 30 min after injection of [11C]disulfiram with three different Am values.
After the injection of [11C]DSF prepared with three different Am values, the injected dosing solution was carried through the vena cava to the heart and then its radioactivity was distributed throughout the body. The radioactivity initially displays high uptake levels in the heart and lungs depending on the blood Figure 5. PET imaging of [11C]disulfiram with three different Am values in mice.
concentration, exhibiting a quick washout. Subsequently, high radioactivity was observed in the liver and kidneys, which then rapidly decreased. Uptake in these organs peaked rapidly and was then eliminated. On the other hand, the enormous radioactive accumulation observed in the urinary bladder demonstrates its rapid and significant excretion in urine (see Figure 7). The high radioactivity observed in the liver, gall bladder, and small intestine during the initial time period also suggests biliary excretion of the radioactivity.
acetonitrile, and centrifuged. The supernatant was analyzed using high-performance liquid chromatography (HPLC) equipped with a radioactivity detector. The recovery of radioactivity obtained from HPLC analysis of both samples was >90%. Figure 8 shows the HPLC traces recorded for [11C]DSF in plasma and urine 1 min after the injection of the radioprobe. No unchanged [11C]DSF was observed in both samples. A radiolabelled metabolite (Rt = 2 min) was much more hydrophilic than the intact form. On the other hand, no significant effect was determined due to the Am values of the [11C]DSF sample used.
Figure 8. Metabolite analysis of [11C]disulfiram in plasma and urine samples 1 min after the injection of [11C]disulfiram.
Figure 7. Static 4-5 min whole-body PET imaging of [11C]disulfiram with three different Am values in mice.
The time-activity curves shown in Figure 6 indicate there was no difference in the radioactivity levels in almost all of the organs within 30 min after the injection of [11C]DSF with three different Am, although the initial uptake levels of radioactivity in these organs were highly dependent on unlabeled DSF contents. Thus, low molar activity combined with the use of a high dose of unlabeled DSF did not show any significant effects on the rapid kinetics of [11C]DSF prepared with a high Am value for at least 30 min after the injection. After injection of [11C]DSF in the mouse, arterial blood and urine samples were collected at 1 and 5 min, deproteinized with
In summary, [thiocarbonyl-11C]disulfiram ([11C]DSF) was successfully synthesized for the first time upon the reaction of [11C]carbon disulfide and diethylamine followed by iodine oxidation. Moreover, the first PET studies of [11C]DSF with three different Am were performed on mice. The uptake level and radioactivity distribution were not significantly affected by the Am values of [11C]DSF used. A high uptake of radioactivity in the liver, kidneys, and gallbladder were observed and metabolite analysis in the plasma showed the rapid metabolism of [11C]DSF. This is the first report on the pharmacokinetics of DSF at such an early stage20-22 in living animals. Recently, the tumor suppressing effect of DSF targeting p97 has been reported, but the mechanism of this in vivo is not yet fully understood.40 Therefore, we believe that PET with [11C]DSF may be a useful tool for evaluating the therapeutic effects and characterizing the mechanism of DSF. 6.
Acknowledgments We would like to thank the staff of the Cyclotron Operation Section and the Department of Advanced Nuclear Medicine Sciences of the National Institute of Radiological Sciences (NIRS), National Institutes for Quantum and Radiological Science and Technology for their support with the operation of the cyclotron and the production of the radioisotopes. This study was supported by a Grant-in-aid for P-DIRECT from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References and notes 1. 2. 3. 4.
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Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Radiosynthesis of [thiocarbonyl11C]disulfiram and its first PET study in mice
Hideki Ishii, Tomoteru Yamasaki, Joji Yui, Yiding Zhang, Masayuki Hanyu, Masanao Ogawa, Nobuki Nengaki, Atsushi B Tsuji, Yuya Terashima, Kouji Matsushima and Ming-Rong Zhang 11 11
CO2
11
CS2
N
S C
S S S
N
[11C]Disulfiram 30. 31. 32. 33. 34.
35. 36. 37. 38. 39. 40.
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