Special Issue Article Received 23 September 2014,
Revised 19 January 2015,
Accepted 20 January 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3274
No-carrier-added labeling of the neuroprotective Ebselen with selenium-73 and selenium-75†,‡ Andreas Helfer, Johannes Ermert, Sven Humpert, and Heinz H. Coenen* Selenium-73 is a positron emitting non-standard radionuclide, which is suitable for positron emission tomography. A copper-catalyzed reaction allowed no-carrier-added labeling of the anti-inflammatory seleno-organic compound Ebselen with 73Se and 75Se under addition of sulfur carrier in a one-step reaction. The new authentically labeled radioselenium molecule is thus available for preclinical evaluation and positron emission tomography studies. Keywords: radioselenium; n.c.a. labeling; Ebselen; positron emission tomography
Introduction
J. Label Compd. Radiopharm 2015, 58 141–145
Institut für Neurowissenschaften und Medizin, Forschungszentrum Jülich, 52425 Jülich, Germany
INM-5:
Nuklearchemie,
*Correspondence to: Heinz H. Coenen, Institut für Neurowissenschaften und Medizin, INM-5: Nuklearchemie, Forschungszentrum Jülich, Jülich, Germany. E-mail: [email protected] †
This article is published in Journal of Labelled Compounds and Radiopharmaceuticals as a special issue on ‘Bengt Långström’, edited by Antony Gee, Department of Chemistry and Biology, Division of Imaging Sciences and Bioengineering, Kings College London, UK and Albert Windhorst, Department of Radiology and Nuclear Medicine, VU University Medical Center, Amsterdam, The Netherlands.
‡ Dedicated to Prof. Dr. Bengt Långström, with deepest appreciation, to celebrate his outstanding life-long contribution to the field of radiochemistry and on the occasion of his retirement as editor of the Journal of Labelled Compounds and Radiopharmaceuticals.
Copyright © 2015 John Wiley & Sons, Ltd.
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Even being highly toxic, selenium is known as an essential trace element for mammals.1 Its physiological role in mammals could not have been elucidated without the use of radioselenium.2 Because of the lack of suitable isotopes of sulfur for in vivo molecular imaging and the chemical homology of sulfur and selenium, the positron emitter selenium-73 (t1/2 = 7.1 h) may serve as a possible substitute for sulfur to obtain analogous radiotracers for in vivo application. A possible disadvantage of selenium-73 is its radioactive, rather long-lived decay product 73As (t1/2 = 80 days). Its contribution to the radiation dose must of course be carefully determined. This radiation dose depends on the individual kinetics of the 73Se-tracer applied, as well as on those of the decaygenerated chemical forms of arsenic at the no-carrier-added (n.c.a.) level, which has a strong influence on the excretion.3 However, no study on the speciation of radioarsenic formed by the decay of 73Se in vivo has been performed so far. Development of synthesis methods for 73Se-labeled radio tracers is for convenience generally carried out with the long-lived radioisotope selenium-75.4,5 Earlier, selenium-75 was even used as a gamma ray emitting nuclide in a few radiopharmaceuticals6 despite its decay characteristics that are in principle unsuitable for its in vivo medical application.7 Nowadays, the use of selenium-75 is almost restricted to biochemical in vitro studies.8 Its half-life of 120 days allows to perform multi-step reactions with a single production batch. 75Se can easily be produced via the 75As(p,n)75Se nuclear reaction on small cyclotrons with a proton energy of below 18 MeV. With the growing importance of positron emission tomography (PET) for in vivo imaging, there is great interest to expand the list of usable positron emitters such as selenium-73.9 Its nuclear properties with Eβ+ = 1.3 MeV and a β+ branching of 65% make this isotope attractive for PET application, and the half-life of 7.1 h allows for the implementation of extended syntheses and imaging protocols. However, the production via the 75As(p,3n)73Se nuclear reaction necessitates proton energies of 30 to 40 MeV.10 The nuclear reactions used for generation of both selenium isotopes allow their production in n.c.a. form.
So far, most 75Se-labeled compounds were synthesized by carrier-added (c.a.) methods. In consideration of the possible toxicity of selenium compounds, however, some labeling methods for the preparation of radioseleno compounds have also been developed at the n.c.a. level during the last three decades. The following methods were described, which allow the synthesis of n.c.a. radioselenium compounds: Starting from n.c.a. methyl[75Se] selenide, which is available using sulfur as non-isotopic carrier,4 alkylation of disubstituted radio selenoureas11 and conversion of alkyl[75Se]selenocyanates with alkyl and aryl lithium or Grignard compounds were realized.12 Only the latter method allows the synthesis of aromatic radioselenoethers. Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (Figure 1), is an organoselenium compound with glutathione peroxidase-like, thiol-dependent, hydroperoxide reducing activity and neuroprotective and cytoprotective properties.13,14 It inhibits radically induced apoptosis while exhibiting low toxicity. Furthermore, the aromatically bound selenium is highly stable with respect to degradation in vivo. Ebselen was investigated as a therapeutic agent for injury and stroke.15–17 Quite recently, it was found that Ebselen also inhibits inositol monophosphatase and induces lithium-like effects on mouse behavior.
A. Helfer et al. Biography Heinz H. Coenen received his PhD in nuclear chemistry at the University of Cologne in 1979 and began a career in radiopharmaceutical chemistry with a PostDoc position at the Research Centre Jülich (FZJ). Since 1996, he is a full professor at the University of Cologne and a director at the Institute of Nuclear Chemistry at FZJ. His research interests focus especially on the development of radionuclides and radiotracers for in vivo molecular imaging and their translation into clinical application. Besides other duties, he acted as President of the Society of Radiopharmaceutical Sciences.
Therefore, Ebselen can be considered as possible lithium mimetic for the treatment of bipolar disorder.18 An earlier authentic labeling of the molecule with selenium-75 was published, however, in c.a. form only.19 In order to avoid pharmacodynamic effects, new labeling methods had to be developed for the production of the n.c.a. 73Se-labeled radiopharmaceuticals.
Results and discussion For convenience, the development and optimization of all radiosyntheses described here were performed using the longerlived isotope selenium-75. The optimum reaction conditions found were then adapted to the corresponding radiosynthesis using the short-lived positron emitter selenium-73. Synthesis of Ebselen and derivatives thereof was conventionally performed via three routes. The bis(ortho-benzoic acid) diselenide route is a method in which anthranilic acid is converted into a heterocycle by a series of reactions. The first step is the conversion into the diselenide derivative using Na2Se2. This method was also used for the synthesis of c.a. [75Se]Ebselen (Scheme 1).19 However, it cannot be employed for the n.c.a. synthesis of [73Se]Ebselen and [75Se]Ebselen because of the necessity to form a diselenide as intermediate that is not achievable without addition of carrier. The second method is based on lithiation of benzanilide with n-butyllithium, subsequent reaction with selenium powder, and finally, oxidative cyclization by copper
Figure 1. Molecular structure of Ebselen.
(Scheme 2).20 A new concept for synthesizing Ebselen on a macroscopic scale uses a copper-catalyzed one-step reaction resulting in good yields (cf. Scheme 4).21 N.c.a. [75Se]Ebselen via ortho-lithiation of N-phenylbenzamide The cyclotron-production of radioselenium isotopes by irradiation of a Cu3As target yielded n.c.a. elemental selenium as solution in benzene after thermochromatographic isolation as described in detail before.10,11,22,23 [Attention must be paid if the volume of this solution is reduced as it will lead to a loss of reactive selenium]. The preparation of Ebselen as reference material was performed as originally,20 using two equivalents of n-butyllithium in tetrahydrofuran, followed by the addition of elemental selenium powder at 0 °C each, and oxidative ring closure with CuBr2 at 78 °C (Scheme 2). The radiosynthesis according to Scheme 1 was attempted using a mixture of THF and the selenium containing benzene. This solvent mixture had a melting point just below 0 °C, and thus, it was much higher than the temperature ( 78 °C) of the oxidative ring closure described in the original method. To perform the n.c.a. radiosynthesis, N-phenylbenzamide was therefore first lithiated in a separate vial using THF at 0 °C and then transferred to the benzene solution containing the radioselenium. The subsequent oxidation step with CuBr2 was also carried out at the same reaction temperature. The organic product was separated from water-soluble products by solid phase extraction and was eluted with methanol. However, using these conditions, no formation of n.c.a. [75Se]Ebselen was observed. Neither the addition of sulfur as non-isotopic carrier nor the addition of selenium as carrier gave the desired product. [75Se]Ebselen via copper-catalyzed one-pot radiosynthesis According to the recently published synthetic method,21 the transition metal-catalyzed coupling of selenium with 2-iodo-Nphenylbenzamide was performed with 20 mol % of CuI and 1,10-phenanthroline and selenium powder in the presence of K2CO3 in DMF at 110 °C within 8 h. For work up, the reaction mixture was treated with an aqueous solution. The proposed mechanism of the Cu-catalyzed reaction is given in Scheme 3. A base-promoted reaction forms a Cu-amide complex followed by insertion of selenium into the Cu–N bond, and finally, reductive elimination of CuL leads to cyclization by forming the Se–C bond.21 The first attempts to synthesize radiolabeled Ebselen by this procedure were performed using n.c.a. selenium-75 dissolved in 1 mL of benzene and 0.6 mL of DMF containing the copper catalyst. However, no product was formed under these conditions, and even increasing both the reaction temperature up to 150 °C and the reaction time up to 14 h did not lead to any formation of
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75
Scheme 1. Radiosynthesis of c.a. [ Se]Ebselen starting from anthranilic acid.19
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J. Label Compd. Radiopharm 2015, 58 141–145
A. Helfer et al.
Scheme 2. Synthesis of Ebselen by ortho-lithiation and subsequent oxidative ring closure.20
[75Se]Ebselen. Thus, the direct transfer of literature conditions21 for labeling failed again under n.c.a. conditions. Therefore, as a further attempt, selenium carrier was added to the reaction mixture. A 0.8 equiv. of selenium relative to the amount of 2-iodo-N-phenylbenzamide led to the formation of c.a. [75Se]Ebselen with 41% ± 11% radiochemical yield (RCY) at 110 °C (Scheme 4). As shown in Figure 2, the RCY of c.a. [75Se]Ebselen can be increased to 60% ± 18% at 150 °C, but with very limited reproducibility. With regard to the relatively short half-life of selenium-73, the reaction times of the coupling reaction and the water treatment were reduced to 3 and 1 h, respectively, so that the total reaction time was within one half-life of selenium-73. In contrast to the literature,21 where brine was used to treat the reaction mixture for work up, diluted acetic acid gave more reliable results.
Scheme 3. Proposed mechanism for the N–Se–C cyclization reaction.21
Thus, although the copper-catalyzed synthesis of [75Se]Ebselen failed at the n.c.a. level, it was possible with the addition of selenium carrier which, however, decreases the specific activity considerably. Bhakuni et al.24 reported that the coppercatalyzed reaction is also applicable to the sulfur homologue of Ebselen, and even within much shorter reaction time. They further reported that an excess of K2CO3 would lower the yield of the corresponding benzoisothiazolone.24 Therefore, the addition of sulfur as non-isotopic carrier was also examined in order to obtain [75Se]Ebselen in n.c.a. labeled form. First experiments using sulfur as non-isotopic carrier under the same conditions as explored in the c.a. reactions at 110 °C (cf. aforementioned discussion) did not show any product formation. Increasing both the amount of starting material and K2CO3 from 25 to 37.5 μmol, however, led to the formation of the desired product (Scheme 5). A study of the influence of the reaction time showed that a maximum RCY of 55% ± 7% of n.c.a. [75Se]Ebselen could be achieved within 3 h, which is far below one half-life of selenium-73 (Figure 3). The time dependence exhibited a decrease of the RCY with longer reaction times up to 5 h, most probably because of decomposition reactions. Final treatment of the reaction solution with diluted acetic acid was necessary for about 1 h, which extended the total synthesis time to 4 h. The sulfur analog exhibits a much higher lipophilicity than Ebselen. Thus, it was easily possible to develop a reverse phase HPLC separation as depicted in Figure 4, and n.c.a. [75Se]Ebselen was obtained in a radiochemical and chemical purity of >99%. N.c.a. radiosynthesis of Ebselen with the positron emitter selenium-73
75
Scheme 4. Copper-catalyzed synthesis of n.c.a. [ Se]Ebselen according to ref. 21 with addition of selenium as isotopic carrier.
75
J. Label Compd. Radiopharm 2015, 58 141–145
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Figure 2. Influence of the reaction temperature on the RCY of [ Se]Ebselen formed by the copper-catalyzed reaction and addition of selenium carrier. Conditions: 25 μmol of catalyst, 25 μmol of 2-iodo-N-phenylbenzamide, 20 μmol 75 of selenium, 0.6 mL of DMF, 1 mL of benzene solution of n.c.a. Se, 37.5 μmol of K2CO3, and 3 h reaction time.
When transferring the reaction conditions optimized with selenium-75 (cf. Scheme 4) to the radiosyntheses with the positron emitter 73Se, the maximum RCY of n.c.a. [73Se]Ebselen was limited to approximately 22%. The differences in the RCY of the same reaction using the two isotopes cannot be explained by an isotopic effect, but it is presumably caused by changes of the chemical form of radioselenium in the benzene solution after separation from the target. Indeed, a dependence of the labeling yield on the time lapse between the benzene extraction of radioselenium and the performance of the labeling step was observed. Thus, an RCY of about 15% was obtained with labeling reactions immediately following the extraction of selenium into benzene, but approximately 50% RCY was obtained with the same benzene solution used for radiolabeling after 8 days. Comparable yields of radiolabeling with selenium-75 and selenium-73 were demonstrated independent of the addition of Se carrier. Therefore, it can only be concluded that the reactive species in the benzene solution is formed rather slowly from an unreactive chemical species, although only one radioactive peak could be observed in the HPLC analysis of the benzene solution (cf. Experimental).
A. Helfer et al.
75
Scheme 5. Copper-catalyzed one-pot radiosynthesis of n.c.a. [ Se]Ebselen with sulfur as non-isotopic carrier.
chromatography was carried out with Fluka silica gel 60 (220–440 mesh). The syntheses of 2-phenyl-1,2-benzisoselenazol-3(2H)-one (Ebselen) and 2-phenyl-1,2-benzisothiozol-3(2H)-one were carried out according to literature methods.20 Analytical radio-HPLC was performed on a system consisting of a Hitachi L-6000 pump and a Sykam UV/vis photometer S3300 with a detector wavelength of 220 nm. Sample injection was accomplished by a Rheodyne-injector block 7125. For measurement of radioactivity, the outlet of the UV detector was connected to a NaI(Tl) well-type scintillation detector (EG&G ACE Mate Ortec AMETEK GmbH, Germany), and the recorded data were processed by the software system Raytest Gina (Raytest, Germany). Radio-TLC was performed on Merck silica gel plates. The developed TL-chromatograms were measured for radioactivity on an Instant Imager® (Packard, USA).
75
Figure 3. Influence of the reaction time on the RCY of [ Se]Ebselen formed by the copper-catalyzed reaction and addition of sulfur carrier. Conditions: 25 μmol of catalyst; 37 μmol of 2-iodo-N-phenylbenzamide; 35 μmol of sulfur; 1 mL of 75 benzene solution of n.c.a. Se; 37.5 μmol of K2CO3, 110 °C, and 2 h hydrolysis.
Production of n.c.a. [75Se]selenium Selenium-75 was produced using 17 MeV protons at the JSW cyclotron 75 75 BC-1710 of the Forschungszentrum Jülich via the As(p,n) Se reaction in a solid Cu3As-target.22 Isolation of the radionuclide was performed as reported earlier.23 After thermochromatographic separation, n.c.a. 75 2 selenium-75 is obtained in oxidized form as [ Se]SeO3 in hydrochloric acid. It was then reduced with sulfur dioxide to obtain n.c.a. selenium-75, which was extracted into benzene.22 HPLC analysis showed one uniform peak in the extraction solution (Luna® 5-μm phenyl–hexyl 100-Å (250 × 10 mm) column (Phenomenex, Germany); mobile phase consists of MeOH/H2O/ trifluoracetic acid 40:60:0.01 (given as V/V ratios) at a flow rate of 1.0 mL/min).
Production of n.c.a. [73Se]selenium 75
Selenium-73 was produced through the As(p,3n) process on a solid Cu3As-target using 45 MeV protons at the high energy isochronous cyclotron (JULIC) of the Forschungszentrum Jülich GmbH.10 Thermochromatographic work up and the following reduction as described earlier provided an n.c.a. solution of selenium-73 in benzene.
Figure 4. Separation of Ebselen and its sulfur analog by HPLC. Conditions: Luna®5 μm phenyl–hexyl 100 Å (150 × 21.2 mm) with MeOH/H2O/trifluoroacetic acid 40:60:0.1 (v/v/v).
In the earlier developmental work, the selenium activity extracted into benzene was considered to be bona fide elemental radioselenium, because oxidized species were not taken up in the organic phase after dissolving and reducing the target material. An identification of the definite chemical form has not been carried out so far. 22,25 Further radioanalytical work on radionuclide development was out of the scope of this study but appears mandatory to identify the chemical form of n.c.a. selenium present in benzene after extraction from the aqueous solution of the dissolved target.
Experimental Material and methods
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All chemicals and solvents were purchased from Aldrich (Germany), Fluka (Switzerland), and Merck (Germany). They were reagent grade or better and used without further purification. All selenocompounds were prepared under a slightly positive pressure of argon. Flash
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Radiosynthesis of n.c.a. [73,75Se]Ebselen In a 5-mL Wheaton V-Vial, 20 μmol of CuI and 20 μmol of 1,10phenanthroline were added in 0.6 mL of dry DMF and stirred for 20 min at room temperature under argon atmosphere. An orangebrown copper complex was formed that was added to a mixture of 37.5 μmol of 2-iodo-N-phenylbenzamide, 35 μmol of sulfur, and 37.5 μmol of dry K2CO3 powder in a 5-mL round-bottom flask. Thereafter, the benzene solution with n.c.a. radioselenium was added to the mixture, the flask equipped with a Dimroth cooler and flushed with argon. The solution was stirred for 3 h at 110 °C. Afterwards, 15 mL of H2O and 2 mL of 1% acetic acid were poured into the reaction mixture and stirred at room temperature for 1 h. In order to prevent the loading of HPLC column with particles and copper salts, an solid phase extraction using SepPak C-18 cartridge was performed. The cartridge was preconditioned with methanol and water, and the raw products were eluted with 4 mL of methanol. Radio-HPLC analysis and separations were performed using a Luna® 5 μm phenyl–hexyl 100 Å (250 × 10 mm) column; mobile phase consists of MeOH/H2O/trifluoroacetic acid 40:60:0.01 (given as V/V ratios) at a flow rate of 1.0 mL/min. The organic solvent of the product fraction was finally removed by evaporation and neutralized with phosphate-buffered saline to a pH of 7.4.
Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 141–145
A. Helfer et al.
Conclusions In an initial study, the preparation of [75Se]Ebselen by a coppercatalyzed one-pot radiosynthesis could only be achieved under c.a. conditions but in RCYs of 60% ± 18%. The n.c.a. product could then be obtained using sulfur as non-isotopic carrier in the reaction mixture and its separation from its co-produced sulfur analog by HPLC. After optimization of reaction parameters, n.c.a. [75Se]Ebselen could be synthesized at 110 °C with RCYs of 55% ± 7% within 3 h. On transferring the conditions to the radiosyntheses with the positron emitter 73Se, however, n.c.a. [73Se]Ebselen could only be obtained with an RCY of 22% ± 1%. Nevertheless, the compound can now be used as a potential radiotracer in preclinical evaluation studies aiming at tracer application with PET.
Conflict of Interest The authors did not report any conflict of interest.
References [1] K. Ekambaramgnanadesigan, E. Balumahendran, N. Gnanagurudasan, S. Kumar, Int J Pharma Bio Sci 2013, 4, B1. [2] C. Galinha, M. C. Freitas, A. M. G. Pacheco, J. Coutinho, B. Maçãs, A. S. Almeida, J Radioanal Nucl Chem 2012, 291, 193. [3] T. W. Gebel, Int J Hyg Environ Health 2002, 205, 505. [4] J. Ermert, T. Blum, K. Hamacher, H. H. Coenen, Radiochim Acta 2001, 89, 863.
[5] T. Blum, J. Ermert, W. Wutz, D. Bier, H. H. Coenen, J Label Compd Radiopharm 2004, 47, 415. [6] R. Riemsma, M. Al, I. Corro Ramos, S. Deshpande, N. Armstrong, Y. C. Lee, S. Ryder, C. Noake, M. Krol, M. Oppe, J. Kleijnen, H. Severens, Health Technol Assess 2013, 17, 1. [7] M. Neves, A. Kling, A. Oliveira, J Radioanal Nucl Chem 2005, 266, 377. [8] A. Graebert, D. Schmidt, A. Kyriakopoulos, Trace Elem Electrolytes 2010, 27, 140. [9] S. M. Qaim, Radiochim Acta 2011, 99, 611. [10] A. Mushtaq, S. M. Qaim, G. Stöcklin, Int J Appl Radiat Isot 1988, 39, 1085. [11] T. Blum, J. Ermert, H. H. Coenen, Appl Radiat Isot 2002, 57, 51. [12] T. Blum, J. Ermert, H. H. Coenen, Nucl Med Biol 2003, 30, 361. [13] M. Parnham, H. Sies, Expert Opin Investig Drugs 2000, 9, 607. [14] M. J. Parnham, H. Sies, Biochem Pharmacol 2013, 86, 1248. [15] T. Yamaguchi, K. Sano, K. Takakura, I. Saito, Y. Shinohara, T. Asano, H. Yasuhara, Stroke 1998, 29, 12. [16] H. Kaur, A. Prakash, B. Medhi, Pharmacology 2013, 92, 324. [17] S. Park, S. Kang, D. S. Kim, B. K. Shin, N. R. Moon, J. W. Daily Iii, Free Radic Res 2014, 48, 864. [18] N. Singh, A. C. Halliday, J. M. Thomas, O. V. Kuznetsova, R. Baldwin, E. C. Y. Woon, P. K. Aley, I. Antoniadou, T. Sharp, S. R. Vasudevan, G. C. Churchill, Nat Commun 2013, 4, 1332. [19] R. Cantineau, G. Tihange, A. Plenevaux, J Label Compd Radiopharm 1986, 23, 59. [20] L. Engman, A. Hallberg, J Org Chem 1989, 54, 2964. [21] S. J. Balkrishna, B. S. Bhakuni, D. Chopra, S. Kumar, Org Lett 2010, 12, 5394. [22] G. Blessing, N. Lavi, S. M. Qaim, Int J Appl Radiat Isot 1992, 43, 455. [23] G. Blessing,N.Lavi, K. Hashimoto, S. M. Qaim,Radiochim Acta 1994, 65, 93. [24] B. S. Bhakuni, S. J. Balkrishna, A. Kumar, S. Kumar, Tetrahedron Lett 2012, 53, 1354. [25] A. Plenevaux, M. Guillaume, C. Brihaye, C. Lemaire, R. Cantineau, Appl Radiat Isot 1990, 41, 829.
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J. Label Compd. Radiopharm 2015, 58 141–145
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org
Revised 19 January 2015,
Accepted 20 January 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3274
No-carrier-added labeling of the neuroprotective Ebselen with selenium-73 and selenium-75†,‡ Andreas Helfer, Johannes Ermert, Sven Humpert, and Heinz H. Coenen* Selenium-73 is a positron emitting non-standard radionuclide, which is suitable for positron emission tomography. A copper-catalyzed reaction allowed no-carrier-added labeling of the anti-inflammatory seleno-organic compound Ebselen with 73Se and 75Se under addition of sulfur carrier in a one-step reaction. The new authentically labeled radioselenium molecule is thus available for preclinical evaluation and positron emission tomography studies. Keywords: radioselenium; n.c.a. labeling; Ebselen; positron emission tomography
Introduction
J. Label Compd. Radiopharm 2015, 58 141–145
Institut für Neurowissenschaften und Medizin, Forschungszentrum Jülich, 52425 Jülich, Germany
INM-5:
Nuklearchemie,
*Correspondence to: Heinz H. Coenen, Institut für Neurowissenschaften und Medizin, INM-5: Nuklearchemie, Forschungszentrum Jülich, Jülich, Germany. E-mail: [email protected] †
This article is published in Journal of Labelled Compounds and Radiopharmaceuticals as a special issue on ‘Bengt Långström’, edited by Antony Gee, Department of Chemistry and Biology, Division of Imaging Sciences and Bioengineering, Kings College London, UK and Albert Windhorst, Department of Radiology and Nuclear Medicine, VU University Medical Center, Amsterdam, The Netherlands.
‡ Dedicated to Prof. Dr. Bengt Långström, with deepest appreciation, to celebrate his outstanding life-long contribution to the field of radiochemistry and on the occasion of his retirement as editor of the Journal of Labelled Compounds and Radiopharmaceuticals.
Copyright © 2015 John Wiley & Sons, Ltd.
141
Even being highly toxic, selenium is known as an essential trace element for mammals.1 Its physiological role in mammals could not have been elucidated without the use of radioselenium.2 Because of the lack of suitable isotopes of sulfur for in vivo molecular imaging and the chemical homology of sulfur and selenium, the positron emitter selenium-73 (t1/2 = 7.1 h) may serve as a possible substitute for sulfur to obtain analogous radiotracers for in vivo application. A possible disadvantage of selenium-73 is its radioactive, rather long-lived decay product 73As (t1/2 = 80 days). Its contribution to the radiation dose must of course be carefully determined. This radiation dose depends on the individual kinetics of the 73Se-tracer applied, as well as on those of the decaygenerated chemical forms of arsenic at the no-carrier-added (n.c.a.) level, which has a strong influence on the excretion.3 However, no study on the speciation of radioarsenic formed by the decay of 73Se in vivo has been performed so far. Development of synthesis methods for 73Se-labeled radio tracers is for convenience generally carried out with the long-lived radioisotope selenium-75.4,5 Earlier, selenium-75 was even used as a gamma ray emitting nuclide in a few radiopharmaceuticals6 despite its decay characteristics that are in principle unsuitable for its in vivo medical application.7 Nowadays, the use of selenium-75 is almost restricted to biochemical in vitro studies.8 Its half-life of 120 days allows to perform multi-step reactions with a single production batch. 75Se can easily be produced via the 75As(p,n)75Se nuclear reaction on small cyclotrons with a proton energy of below 18 MeV. With the growing importance of positron emission tomography (PET) for in vivo imaging, there is great interest to expand the list of usable positron emitters such as selenium-73.9 Its nuclear properties with Eβ+ = 1.3 MeV and a β+ branching of 65% make this isotope attractive for PET application, and the half-life of 7.1 h allows for the implementation of extended syntheses and imaging protocols. However, the production via the 75As(p,3n)73Se nuclear reaction necessitates proton energies of 30 to 40 MeV.10 The nuclear reactions used for generation of both selenium isotopes allow their production in n.c.a. form.
So far, most 75Se-labeled compounds were synthesized by carrier-added (c.a.) methods. In consideration of the possible toxicity of selenium compounds, however, some labeling methods for the preparation of radioseleno compounds have also been developed at the n.c.a. level during the last three decades. The following methods were described, which allow the synthesis of n.c.a. radioselenium compounds: Starting from n.c.a. methyl[75Se] selenide, which is available using sulfur as non-isotopic carrier,4 alkylation of disubstituted radio selenoureas11 and conversion of alkyl[75Se]selenocyanates with alkyl and aryl lithium or Grignard compounds were realized.12 Only the latter method allows the synthesis of aromatic radioselenoethers. Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (Figure 1), is an organoselenium compound with glutathione peroxidase-like, thiol-dependent, hydroperoxide reducing activity and neuroprotective and cytoprotective properties.13,14 It inhibits radically induced apoptosis while exhibiting low toxicity. Furthermore, the aromatically bound selenium is highly stable with respect to degradation in vivo. Ebselen was investigated as a therapeutic agent for injury and stroke.15–17 Quite recently, it was found that Ebselen also inhibits inositol monophosphatase and induces lithium-like effects on mouse behavior.
A. Helfer et al. Biography Heinz H. Coenen received his PhD in nuclear chemistry at the University of Cologne in 1979 and began a career in radiopharmaceutical chemistry with a PostDoc position at the Research Centre Jülich (FZJ). Since 1996, he is a full professor at the University of Cologne and a director at the Institute of Nuclear Chemistry at FZJ. His research interests focus especially on the development of radionuclides and radiotracers for in vivo molecular imaging and their translation into clinical application. Besides other duties, he acted as President of the Society of Radiopharmaceutical Sciences.
Therefore, Ebselen can be considered as possible lithium mimetic for the treatment of bipolar disorder.18 An earlier authentic labeling of the molecule with selenium-75 was published, however, in c.a. form only.19 In order to avoid pharmacodynamic effects, new labeling methods had to be developed for the production of the n.c.a. 73Se-labeled radiopharmaceuticals.
Results and discussion For convenience, the development and optimization of all radiosyntheses described here were performed using the longerlived isotope selenium-75. The optimum reaction conditions found were then adapted to the corresponding radiosynthesis using the short-lived positron emitter selenium-73. Synthesis of Ebselen and derivatives thereof was conventionally performed via three routes. The bis(ortho-benzoic acid) diselenide route is a method in which anthranilic acid is converted into a heterocycle by a series of reactions. The first step is the conversion into the diselenide derivative using Na2Se2. This method was also used for the synthesis of c.a. [75Se]Ebselen (Scheme 1).19 However, it cannot be employed for the n.c.a. synthesis of [73Se]Ebselen and [75Se]Ebselen because of the necessity to form a diselenide as intermediate that is not achievable without addition of carrier. The second method is based on lithiation of benzanilide with n-butyllithium, subsequent reaction with selenium powder, and finally, oxidative cyclization by copper
Figure 1. Molecular structure of Ebselen.
(Scheme 2).20 A new concept for synthesizing Ebselen on a macroscopic scale uses a copper-catalyzed one-step reaction resulting in good yields (cf. Scheme 4).21 N.c.a. [75Se]Ebselen via ortho-lithiation of N-phenylbenzamide The cyclotron-production of radioselenium isotopes by irradiation of a Cu3As target yielded n.c.a. elemental selenium as solution in benzene after thermochromatographic isolation as described in detail before.10,11,22,23 [Attention must be paid if the volume of this solution is reduced as it will lead to a loss of reactive selenium]. The preparation of Ebselen as reference material was performed as originally,20 using two equivalents of n-butyllithium in tetrahydrofuran, followed by the addition of elemental selenium powder at 0 °C each, and oxidative ring closure with CuBr2 at 78 °C (Scheme 2). The radiosynthesis according to Scheme 1 was attempted using a mixture of THF and the selenium containing benzene. This solvent mixture had a melting point just below 0 °C, and thus, it was much higher than the temperature ( 78 °C) of the oxidative ring closure described in the original method. To perform the n.c.a. radiosynthesis, N-phenylbenzamide was therefore first lithiated in a separate vial using THF at 0 °C and then transferred to the benzene solution containing the radioselenium. The subsequent oxidation step with CuBr2 was also carried out at the same reaction temperature. The organic product was separated from water-soluble products by solid phase extraction and was eluted with methanol. However, using these conditions, no formation of n.c.a. [75Se]Ebselen was observed. Neither the addition of sulfur as non-isotopic carrier nor the addition of selenium as carrier gave the desired product. [75Se]Ebselen via copper-catalyzed one-pot radiosynthesis According to the recently published synthetic method,21 the transition metal-catalyzed coupling of selenium with 2-iodo-Nphenylbenzamide was performed with 20 mol % of CuI and 1,10-phenanthroline and selenium powder in the presence of K2CO3 in DMF at 110 °C within 8 h. For work up, the reaction mixture was treated with an aqueous solution. The proposed mechanism of the Cu-catalyzed reaction is given in Scheme 3. A base-promoted reaction forms a Cu-amide complex followed by insertion of selenium into the Cu–N bond, and finally, reductive elimination of CuL leads to cyclization by forming the Se–C bond.21 The first attempts to synthesize radiolabeled Ebselen by this procedure were performed using n.c.a. selenium-75 dissolved in 1 mL of benzene and 0.6 mL of DMF containing the copper catalyst. However, no product was formed under these conditions, and even increasing both the reaction temperature up to 150 °C and the reaction time up to 14 h did not lead to any formation of
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Scheme 1. Radiosynthesis of c.a. [ Se]Ebselen starting from anthranilic acid.19
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Scheme 2. Synthesis of Ebselen by ortho-lithiation and subsequent oxidative ring closure.20
[75Se]Ebselen. Thus, the direct transfer of literature conditions21 for labeling failed again under n.c.a. conditions. Therefore, as a further attempt, selenium carrier was added to the reaction mixture. A 0.8 equiv. of selenium relative to the amount of 2-iodo-N-phenylbenzamide led to the formation of c.a. [75Se]Ebselen with 41% ± 11% radiochemical yield (RCY) at 110 °C (Scheme 4). As shown in Figure 2, the RCY of c.a. [75Se]Ebselen can be increased to 60% ± 18% at 150 °C, but with very limited reproducibility. With regard to the relatively short half-life of selenium-73, the reaction times of the coupling reaction and the water treatment were reduced to 3 and 1 h, respectively, so that the total reaction time was within one half-life of selenium-73. In contrast to the literature,21 where brine was used to treat the reaction mixture for work up, diluted acetic acid gave more reliable results.
Scheme 3. Proposed mechanism for the N–Se–C cyclization reaction.21
Thus, although the copper-catalyzed synthesis of [75Se]Ebselen failed at the n.c.a. level, it was possible with the addition of selenium carrier which, however, decreases the specific activity considerably. Bhakuni et al.24 reported that the coppercatalyzed reaction is also applicable to the sulfur homologue of Ebselen, and even within much shorter reaction time. They further reported that an excess of K2CO3 would lower the yield of the corresponding benzoisothiazolone.24 Therefore, the addition of sulfur as non-isotopic carrier was also examined in order to obtain [75Se]Ebselen in n.c.a. labeled form. First experiments using sulfur as non-isotopic carrier under the same conditions as explored in the c.a. reactions at 110 °C (cf. aforementioned discussion) did not show any product formation. Increasing both the amount of starting material and K2CO3 from 25 to 37.5 μmol, however, led to the formation of the desired product (Scheme 5). A study of the influence of the reaction time showed that a maximum RCY of 55% ± 7% of n.c.a. [75Se]Ebselen could be achieved within 3 h, which is far below one half-life of selenium-73 (Figure 3). The time dependence exhibited a decrease of the RCY with longer reaction times up to 5 h, most probably because of decomposition reactions. Final treatment of the reaction solution with diluted acetic acid was necessary for about 1 h, which extended the total synthesis time to 4 h. The sulfur analog exhibits a much higher lipophilicity than Ebselen. Thus, it was easily possible to develop a reverse phase HPLC separation as depicted in Figure 4, and n.c.a. [75Se]Ebselen was obtained in a radiochemical and chemical purity of >99%. N.c.a. radiosynthesis of Ebselen with the positron emitter selenium-73
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Scheme 4. Copper-catalyzed synthesis of n.c.a. [ Se]Ebselen according to ref. 21 with addition of selenium as isotopic carrier.
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Figure 2. Influence of the reaction temperature on the RCY of [ Se]Ebselen formed by the copper-catalyzed reaction and addition of selenium carrier. Conditions: 25 μmol of catalyst, 25 μmol of 2-iodo-N-phenylbenzamide, 20 μmol 75 of selenium, 0.6 mL of DMF, 1 mL of benzene solution of n.c.a. Se, 37.5 μmol of K2CO3, and 3 h reaction time.
When transferring the reaction conditions optimized with selenium-75 (cf. Scheme 4) to the radiosyntheses with the positron emitter 73Se, the maximum RCY of n.c.a. [73Se]Ebselen was limited to approximately 22%. The differences in the RCY of the same reaction using the two isotopes cannot be explained by an isotopic effect, but it is presumably caused by changes of the chemical form of radioselenium in the benzene solution after separation from the target. Indeed, a dependence of the labeling yield on the time lapse between the benzene extraction of radioselenium and the performance of the labeling step was observed. Thus, an RCY of about 15% was obtained with labeling reactions immediately following the extraction of selenium into benzene, but approximately 50% RCY was obtained with the same benzene solution used for radiolabeling after 8 days. Comparable yields of radiolabeling with selenium-75 and selenium-73 were demonstrated independent of the addition of Se carrier. Therefore, it can only be concluded that the reactive species in the benzene solution is formed rather slowly from an unreactive chemical species, although only one radioactive peak could be observed in the HPLC analysis of the benzene solution (cf. Experimental).
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Scheme 5. Copper-catalyzed one-pot radiosynthesis of n.c.a. [ Se]Ebselen with sulfur as non-isotopic carrier.
chromatography was carried out with Fluka silica gel 60 (220–440 mesh). The syntheses of 2-phenyl-1,2-benzisoselenazol-3(2H)-one (Ebselen) and 2-phenyl-1,2-benzisothiozol-3(2H)-one were carried out according to literature methods.20 Analytical radio-HPLC was performed on a system consisting of a Hitachi L-6000 pump and a Sykam UV/vis photometer S3300 with a detector wavelength of 220 nm. Sample injection was accomplished by a Rheodyne-injector block 7125. For measurement of radioactivity, the outlet of the UV detector was connected to a NaI(Tl) well-type scintillation detector (EG&G ACE Mate Ortec AMETEK GmbH, Germany), and the recorded data were processed by the software system Raytest Gina (Raytest, Germany). Radio-TLC was performed on Merck silica gel plates. The developed TL-chromatograms were measured for radioactivity on an Instant Imager® (Packard, USA).
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Figure 3. Influence of the reaction time on the RCY of [ Se]Ebselen formed by the copper-catalyzed reaction and addition of sulfur carrier. Conditions: 25 μmol of catalyst; 37 μmol of 2-iodo-N-phenylbenzamide; 35 μmol of sulfur; 1 mL of 75 benzene solution of n.c.a. Se; 37.5 μmol of K2CO3, 110 °C, and 2 h hydrolysis.
Production of n.c.a. [75Se]selenium Selenium-75 was produced using 17 MeV protons at the JSW cyclotron 75 75 BC-1710 of the Forschungszentrum Jülich via the As(p,n) Se reaction in a solid Cu3As-target.22 Isolation of the radionuclide was performed as reported earlier.23 After thermochromatographic separation, n.c.a. 75 2 selenium-75 is obtained in oxidized form as [ Se]SeO3 in hydrochloric acid. It was then reduced with sulfur dioxide to obtain n.c.a. selenium-75, which was extracted into benzene.22 HPLC analysis showed one uniform peak in the extraction solution (Luna® 5-μm phenyl–hexyl 100-Å (250 × 10 mm) column (Phenomenex, Germany); mobile phase consists of MeOH/H2O/ trifluoracetic acid 40:60:0.01 (given as V/V ratios) at a flow rate of 1.0 mL/min).
Production of n.c.a. [73Se]selenium 75
Selenium-73 was produced through the As(p,3n) process on a solid Cu3As-target using 45 MeV protons at the high energy isochronous cyclotron (JULIC) of the Forschungszentrum Jülich GmbH.10 Thermochromatographic work up and the following reduction as described earlier provided an n.c.a. solution of selenium-73 in benzene.
Figure 4. Separation of Ebselen and its sulfur analog by HPLC. Conditions: Luna®5 μm phenyl–hexyl 100 Å (150 × 21.2 mm) with MeOH/H2O/trifluoroacetic acid 40:60:0.1 (v/v/v).
In the earlier developmental work, the selenium activity extracted into benzene was considered to be bona fide elemental radioselenium, because oxidized species were not taken up in the organic phase after dissolving and reducing the target material. An identification of the definite chemical form has not been carried out so far. 22,25 Further radioanalytical work on radionuclide development was out of the scope of this study but appears mandatory to identify the chemical form of n.c.a. selenium present in benzene after extraction from the aqueous solution of the dissolved target.
Experimental Material and methods
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All chemicals and solvents were purchased from Aldrich (Germany), Fluka (Switzerland), and Merck (Germany). They were reagent grade or better and used without further purification. All selenocompounds were prepared under a slightly positive pressure of argon. Flash
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Radiosynthesis of n.c.a. [73,75Se]Ebselen In a 5-mL Wheaton V-Vial, 20 μmol of CuI and 20 μmol of 1,10phenanthroline were added in 0.6 mL of dry DMF and stirred for 20 min at room temperature under argon atmosphere. An orangebrown copper complex was formed that was added to a mixture of 37.5 μmol of 2-iodo-N-phenylbenzamide, 35 μmol of sulfur, and 37.5 μmol of dry K2CO3 powder in a 5-mL round-bottom flask. Thereafter, the benzene solution with n.c.a. radioselenium was added to the mixture, the flask equipped with a Dimroth cooler and flushed with argon. The solution was stirred for 3 h at 110 °C. Afterwards, 15 mL of H2O and 2 mL of 1% acetic acid were poured into the reaction mixture and stirred at room temperature for 1 h. In order to prevent the loading of HPLC column with particles and copper salts, an solid phase extraction using SepPak C-18 cartridge was performed. The cartridge was preconditioned with methanol and water, and the raw products were eluted with 4 mL of methanol. Radio-HPLC analysis and separations were performed using a Luna® 5 μm phenyl–hexyl 100 Å (250 × 10 mm) column; mobile phase consists of MeOH/H2O/trifluoroacetic acid 40:60:0.01 (given as V/V ratios) at a flow rate of 1.0 mL/min. The organic solvent of the product fraction was finally removed by evaporation and neutralized with phosphate-buffered saline to a pH of 7.4.
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Conclusions In an initial study, the preparation of [75Se]Ebselen by a coppercatalyzed one-pot radiosynthesis could only be achieved under c.a. conditions but in RCYs of 60% ± 18%. The n.c.a. product could then be obtained using sulfur as non-isotopic carrier in the reaction mixture and its separation from its co-produced sulfur analog by HPLC. After optimization of reaction parameters, n.c.a. [75Se]Ebselen could be synthesized at 110 °C with RCYs of 55% ± 7% within 3 h. On transferring the conditions to the radiosyntheses with the positron emitter 73Se, however, n.c.a. [73Se]Ebselen could only be obtained with an RCY of 22% ± 1%. Nevertheless, the compound can now be used as a potential radiotracer in preclinical evaluation studies aiming at tracer application with PET.
Conflict of Interest The authors did not report any conflict of interest.
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