Brief Article pubs.acs.org/molecularpharmaceutics
One-Pot Multi-Tracer Synthesis of Novel Agents
18
F‑Labeled PET Imaging
Anna Haslop,† Lisa Wells,§ Antony Gee,‡ Christophe Plisson,§ and Nicholas Long*,† †
Department of Chemistry, Imperial College London, South Kensington, London SW7 2AZ, U.K. Division of Imaging Sciences and Biomedical Engineering, St. Thomas’ Hospital, The Rayne Institute, King’s College London, London SE1 7EH, U.K. § Imanova, Ltd., Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K. ‡
S Supporting Information *
ABSTRACT: 18F labeled phosphonium salts are increasingly important molecular probes for targeting the mitochondrial membrane potential depletion during apoptosis and for detecting myocardial perfusion deficit. Here, we introduce three new tracers, [18F]MitoPhos_04, [18F]MitoPhos_05, and [18F]MitoPhos_07, that have the potential to act as mitochondrial imaging agents. Moreover, they have the added advantage of being synthesized in the same reaction vial from one radiolabeled synthon, demonstrating a new approach to synthesizing multiple tracers in one-pot, which is a highly useful means for increasing the throughput of radiotracer development. The radiosynthesis of the tracers was carried out on a fully automated system via a facile two-step reaction. Utilizing the radiolabeling of an ethyl azide, a copper-mediated 1,3-cycloaddition reaction and isolation via semiprep high-performance liquid chromatography (HPLC) allowed for the simultaneous synthesis of two or three tracers with a total synthesis time of less than 1 h. KEYWORDS: fluorine-18, click chemistry, mitochondria imaging, phosphonium cations
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INTRODUCTION The key role of mitochondria to cell function and survival has become clearer following the discovery of its importance in much common pathology.1,2 Being able to detect mitochondrial dysfunction by targeting the membrane potential could allow for early detection of different diseases.3−5 It is commonly known that delocalized lipophilic cations, such as simple phosphonium salts, can enter and accumulate in the matrix of the mitochondria dependent on this membrane potential.6−8 These compounds therefore also have the potential to detect malignant cells/tumors over healthy tissue due to the known increase in membrane potential observed in cancerous cells.9−11 Finally, the increased myocardium uptake enables cardiac imaging and the potential to detect myocardial perfusion deficits, thus demonstrating the diverse application of this type of tracer.12−14 Previous research has shown that detection of apoptosis was possible by combining lipophilic phosphonium cations with an imaging modality such as PET.15,16 Madar et al. have carried out an in-depth study on [18F]FBnTP ([18F]p-fluorobenzyltriphenyl phosphonium bromide) to determine its ability to not only act as a molecular probe for the detection of apoptosis and cancer but also as a successful myocardial imaging agent.16−18 Following this research, other 18F-labeled phosphonium cations have been reported in the literature, which lends weight and increasing importance to this type of imaging probe.19−21,14 © XXXX American Chemical Society
One of the highlighted characteristics that appears to aid increased tumor uptake and nonspecific clearance is the lipophilicity of the tracer.19,22 Being able to easily alter the lipophilicity of a tracer design is advantageous in designing a tracer with suitable biokinetics. With this in mind, we modified our previously reported tracer [18F]MitoPhos_01 in order to achieve a tracer with a suitable log P value to give favorable biodistribution results.23 By focusing on functionalizing the phenyl rings present around the phosphorus center, the site of radiolabeling remained unchanged meaning that the same automated radiolabeling method could be utilized. Herein, we report the synthesis of three novel phosphonium cations, [18F]MitoPhos_04, [18F]MitoPhos_05, and [18F]MitoPhos_07, which can be synthesized using the common radiolabeled synthon, [ 18 F]fluoroethylazide ([18F]FEA), in the same reaction vial.24,25 This one-pot synthesis of multiple compounds allows for the rapid synthesis of different tracers and enables direct comparison. Successful isolation has been achieved for the tracers with high radiochemical purities. Special Issue: Positron Emission Tomography: State of the Art Received: April 30, 2014 Revised: August 22, 2014 Accepted: August 26, 2014
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Figure 1. Reaction scheme to show the synthesis of labeled and unlabeled MitoPhos_04 and MitoPhos_07.
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EXPERIMENTAL SECTION Chemistry. Full synthetic details of precursors and reference compounds can be found in the Supporting Information. Radiochemistry. Two Tracer Production. [ 18 F]MitoPhos_04 and [18F]MitoPhos_07 were synthesized together using a fully automated platform with the radiolabeled synthon, [18F]fluoroethylazide ([18F]FEA), being synthesized on the Advion NanoTek. The remaining synthesis was carried out on an in-house developed automated system consisting of a valve-tower made of 16 valves, a mass flow controller, oven, and a 6-port/2-way high-performance liquid chromatography (HPLC) valve. 18F-fluoride was produced by an 18O(p,n)18F reaction on a Siemens Eclipse cyclotron. Aqueous [18F]fluoride (∼1.8 mL) was passed through an anion exchange resin cartridge, and the activity then eluted using a mixture of Kryptofix-222 (5 mg, 13.3 μmol), potassium carbonate (1 mg, 7.2 μmol, dissolved in 50 μL water), and acetonitrile (1 mL). The solvent was removed by heating at 80 °C under a stream of nitrogen. Then, acetonitrile (0.5 mL, 3 repeats) was added, and the distillation was continued. After cooling to room temperature, a solution of 2-azidoethyl-4-tosylate (2 μL, 10 μmol) in acetonitrile (0.5 mL) was added. The reaction mixture was stirred for 15 min at 80 °C. After the addition of ethanol (0.1 mL), [18F]fluoroethyl azide was distilled at 130 °C under a flow of helium (20 mL/min) into a trapping vial containing the reaction mixture. The reaction mixture contained a premade mixture of CuSO4·5H2O (56 μL, 0.4 mM), tris(benzyltriazolylmethyl)amine (TBTA) (10 mg), and sodium ascorbate (40 mg) in water (300 μL) and ethanol (10 μL), which had been shaken until all the reagents had mixed together. To this, but-3-ynyl (tris-4-tert-butylphenyl)phosphonium bromide (0.5 mg) and 3but-3-ynyl (tris-3,5-dimethylphenyl)phosphonium bromide (0.5 mg) in ethanol (100 μL) were added. Following the distillation of [18f]fluoroethyl azide into this vial, it was left to mix under helium at room temperature for 10 min. Water (1 mL) was then added before being injected into the semipreparative HPLC (Agilent Eclipse C18 250 × 10 mm). The flow rate was 7 mL/min, with the mobile phase starting from 10% ethanol and 90% buffer (50 mM dihydrogen sodium phosphate buffer pH = 5.5) to 70% ethanol over 8 min and then increased to 80% ethanol following the elution of [18F]MitoPhos_07. The radioactive peaks for [18F]fluoroethyl azide, [18F]MitoPhos_04, and [18F]MitoPhos_07 were at 4.860, 9.052, and 12.794 min, respectively, and were collected
via manual isolation. The total reaction time from EOB to isolation was on average 56 min. The two tracers were found to be >99% radiochemically and >95% chemically pure as determined by analytical HPLC (4.6 × 250 mm Agilent Eclipse C18 column): flow rate, 2.0 mL/min; elution conditions consisted of a gradient starting from 70% AMF (50 mM, pH 4) and 30% acetonitrile to 20% AMF over 6 min followed by a plateau at 80% acetonitrile for the remainder of the analysis. Three Tracer Production. The methodology for the twotracer production was repeated exactly but with the addition of 3-but-3-ynyl (4-methylphenyl)phosphonium bromide (0.3 mg) to the CuAAC reaction mixture, alongside but-3-ynyl (tris-4tert-butylphenyl)phosphonium bromide (0.3 mg) and 3-but-3ynyl (tris-3,5-dimethylphenyl)phosphonium bromide (0.3 mg). A small adjustment was made to the semiprep HPLC conditions to enable good separation between all three tracers. The flow rate was 7 mL/min, with the mobile phase starting from 10% ethanol and 90% buffer (50 mM dihydrogen sodium phosphate buffer pH = 5.5) to 50% ethanol over 7 min and then increased to 70% ethanol following the elution of [18F]MitoPhos_05 and then to 80% following the elution of [18F]MitoPhos_07. The radioactive peaks for [18F]fluoroethyl azide, [18F]MitoPhos_04, [18F]MitoPhos_05, and [18F]MitoPhos_07 were at 4.997, 9.200, 11.575, and 15.190 min, respectively, and were collected via manual isolation. The total reaction time from EOB to isolation was on average 59 min. Partition Coefficient Determination. The log P values were measured to compare the lipophilicities of [18F]MitoPhos_04, [18F]MitoPhos_05, and [18F]MitoPhos_07. A general methodology is as follows: Following the complete removal of all volatiles, the radiotracer (40 μL) was added to 1octanol (10 mL) and phosphate buffer (10 mL) in a 50 mL separatory funnel. After hand shaking for about 3 min, the bottom aqueous layer was discarded. Three aliquots (2.0 mL each) of octanol phase containing the washed radiotracer were pipetted into three 12 mL test tubes containing the phosphate buffer (2.0 mL). The test tubes were stoppered, mechanically shaken for 10 min, and centrifuged (5 min, 1000g). From each tube, buffer (0.2 mL, n = 3) was transferred into a test tube for counting. The remaining buffer was discarded, and octanol phase (0.2 mL, n = 3) was also transferred into a separate test tube. The samples were then placed into a carrier tray and transferred to an automatic gamma counter (PerkinElmer Wizard 1470) for counting. The log P value was reported as the average of data obtained in 3 independent measurements. B
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RESULTS The nonradioactive reference compounds of [ 18 F]MitoPhos_04 and [18F]MitoPhos_07 were synthesized from the functionalized triphenyl phosphonium alkyne with fluoroethylazide (Figure 1). The oily nature of the final products caused difficulties with isolation, and because of failed attempts to purify via column chromatography and recrystallization, washing the oil with different solvents lead to a pure product. Both products were isolated via a series of washings and the identity of the compounds confirmed by NMR and electrospray ionization mass spectrometry (ESI-MS). For the radiosynthesis, a copper catalyzed alkyne−azide cycloaddition (CuAAC) reaction was carried out with [18F]FEA and the two starting alkynes in the same reaction vial. A good separation of [18F]MitoPhos_04 and [18F]MitoPhos_07 was obtained using semipreparative HPLC as depicted in Figure 2. The products
Figure 3. Structure of [18F]MitoPhos_05.
Using the same method discussed previously, but with the addition of the alkyne precursor to MitoPhos_05, three tracers of varying lipophilicity were prepared. The radiochromatogram illustrates the three peaks corresponding to the different tracers in order of increasing lipophilicity (for this example, unreacted [18F]FEA is still present) (Figure 4). All three tracers were isolated in less than 1
Figure 4. Semiprep HPLC radiochromatogram of the crude mixture from the three-tracer one-pot synthesis.
h, and analytical HPLC confirmed both high chemical and radiochemical purity. As already mentioned, this result is preliminary but confirms that the method can be utilized for more than two tracers at a time. To determine the lipophilicity of [18F]MitoPhos_04, [18F]MitoPhos_05, and [18F]MitoPhos_07, the log P values were evaluated. These were found to be 2.03 ± 0.19 (n = 3), 1.312 ± 0.055 (n = 3), and 1.83 ± 0.04 (n = 3), respectively, and considered to be consistent with the number of carbon atoms.
Figure 2. Semiprep HPLC radiochromatogram of the crude mixture from the two-tracer one-pot synthesis.
were collected separately using online radiodetectors; a collection line was also washed following collection of the first tracer to ensure that there was no cross-contamination. Both tracers were obtained in equal quantities confirming similar reaction kinetics for each reaction. A radiochemical purity of >99% was confirmed by QC HPLC. It can also be noted that the conversion percentage of the labeled synthon, fluoroethylazide, to the labeled phosphonium cations was in the range of 80%. The specific activity of [18F]MitoPhos_04 and [18F]MitoPhos_07 was determined to be 49.6 ± 6.5 GBq/μmol (n = 3) and 68 ± 18.8 GBq/μmol (n = 3), respectively. Total reaction time was less than 1 h from EOB to the isolation of the two tracers. Furthermore, initial studies of a three-tracer one-pot synthesis have shown promising preliminary results. Alongside MitoPhos_04 and _07, MitoPhos_05 (Figure 3) was also isolated following a reaction with all three alkyne-phosphonium precursors in the same reaction vial.
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DISCUSSION Having previously discussed the radiosynthesis of [18F]MitoPhos_01, which utilized the favorable conditions of the CuAAC reaction to allow for rapid labeling of a phosphonium cation via a triazole moiety, this report explores this methodology further.23 In this work, we have designed structural compound changes to alter the lipophilicity of the phosphonium cation by functionalization of the phenyl rings while maintaining the same radiolabeling synthetic route. The other main focus of this research was to simultaneously synthesize multiple tracers, to not only make time and financial C
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cells by the use of ionic probes. Biochim. Biophys. Acta, Biomembr. 1984, 771 (2), 217−227. (7) Murphy, M. P. Targeting lipophilic cations to mitochondria. Biochim. Biophys. Acta, Bioenerg. 2008, 1777 (7−8), 1028−1031. (8) Porteous, C. M.; Logan, A.; Evans, C.; Ledgerwood, E. C.; Menon, D. K.; Aigbirhio, F.; Smith, R. A. J.; Murphy, M. P. Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: Implications for mitochondriaspecific therapies and probes. Biochim. Biophys. Acta, Gen. Subj. 2010, 1800 (9), 1009−1017. (9) Ross, M. F.; Kelso, G. F.; Blaikie, F. H.; James, A. M.; Cocheme, H. M.; Filipovska, A.; Da Ros, T.; Hurd, T. R.; Smith, R. A. J.; Murphy, M. P. Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology. Biochemistry (Moscow) 2005, 70 (2), 222−230. (10) Kroemer, G. Mitochondria in cancer. Oncogene 2006, 25, 4630− 4632. (11) Modica-Napolitano, J. S.; Aprille, J. R. Delocalized lipophilic cations selectively target the mitochondria of carcinoma cells. Adv. Drug Delivery Rev. 2001, 49 (1−2), 63−70. (12) Higuchi, T.; Fukushima, K.; Rischpler, C.; Isoda, T.; Javadi, M. S.; Ravert, H.; Holt, D. P.; Dannals, R. F.; Madar, I.; Bengel, F. M. Stable delineation of the ischemic area by the PET perfusion tracer 18F-fluorobenzyl triphenyl phosphonium after transient coronary occlusion. J. Nucl. Med. 2011, 52 (6), 965−969. (13) Fukuda, H.; Syrota, A.; Charbonneau, P.; Vallois, J.; Crouzel, M.; Prenant, C.; Sastre, J.; Crouzel, C. Use of 11C-triphenylmethylphosphonium for the evaluation of membrane potential in the heart by positron-emission tomography. Eur. J. Nucl. Med. Mol. Imaging 1986, 11 (12), 478−483. (14) Zhao, Z.; Yu, Q.; Mou, T.; Liu, C.; Yang, W.; Fang, W.; Peng, C.; Lu, J.; Liu, Y.; Zhang, X. Highly efficient one-pot labeling of new phosphonium cations with fluorine-18 as potential PET agents for myocardial perfusion imaging. Mol. Pharmaceutics 2014, DOI: 10.1021/mp500216g. (15) Heinrich, T. K.; Gottumukkala, V.; Snay, E.; Dunning, P.; Fahey, F. H.; Ted Treves, S.; Packard, A. B. Synthesis of fluorine-18 labeled rhodamine B: A potential PET myocardial perfusion imaging agent. Appl. Radiat. Isot. 2010, 68 (1), 96−100. (16) Madar, I.; Ravert, H.; Nelkin, B.; Abro, M.; Pomper, M.; Dannals, R.; Frost, J. J. Characterization of membrane potentialdependent uptake of the novel PET tracer 18F-fluorobenzyl triphenylphosphonium cation. Eur. J. Nucl. Med. Mol. Imaging 2007, 34 (12), 2057−65. (17) Madar, I.; Ravert, H. T.; Du, Y.; Hilton, J.; Volokh, L.; Dannals, R. F.; Frost, J. J.; Hare, J. M. Characterization of uptake of the new PET imaging compound 18F-fluorobenzyl triphenyl phosphonium in dog myocardium. J. Nucl. Med. 2006, 47 (8), 1359−1366. (18) Madar, I.; Huang, Y.; Ravert, H.; Dalrymple, S. L.; Davidson, N. E.; Isaacs, J. T.; Dannals, R. F.; Frost, J. J. Detection and quantification of the evolution dynamics of apoptosis using the PET voltage sensor 18F-fluorobenzyl triphenyl phosphonium. J. Nucl. Med. 2009, 50 (5), 774−80. (19) Kim, D.; Kim, H.; Yu, K.; Min, J. Synthesis of [18F]-labeled (2(2-fluoroethoxy)ethyl)triphenylphosphonium cation as a potential agent for myocardial imaging using positron emission tomography. Bioorg. Med. Chem. Lett. 2012, 22 (1), 319−22. (20) Kim, D. Y.; Kim, H. J.; Yu, K. H.; Min, J. J. Synthesis of [18F]labeled (6-fluorohexyl)triphenylphosphonium cation as a potential agent for myocardial imaging using positron emission tomography. Bioconjugate Chem. 2012, 23 (3), 431−7. (21) Yuan, H.; Cho, H.; Chen, H. H.; Panagia, M.; Sosnovik, D. E.; Josephson, L. Fluorescent and radiolabeled triphenylphosphonium probes for imaging mitochondria. Chem. Commun. 2013, 49 (88), 10361−10363. (22) Tait, J. F. Imaging of apoptosis. J. Nucl. Med. 2008, 49 (10), 1573−1576. (23) Haslop, A.; Gee, A.; Plisson, C.; Long, N. Fully automated radiosynthesis of [1-(2-[18F]fluoroethyl),1H[1,2,3]triazole 4-ethyl-
savings but also to allow for all tracers to be analyzed in in vitro models simultaneously eliminating any adverse factors of using different cells. This is the first demonstration in the literature of synthesizing two, and even three, PET tracers in one reaction vial via one radiolabeled synthon. We have shown that both [18F]MitoPhos_04 and [18F]MitoPhos_07, followed by the introduction of a third tracer[18F]MitoPhos_05, can be synthesized in the same reaction vial, and, because of differences in lipophilicity, can be easily separated via semiprep HPLC without any cross contamination. The total synthesis, apart from the manual tracer isolation after HPLC, was carried out on an automated system, which could be used to increase the MitoPhos library further. By simply changing the phosphonium alkyne precursor in the CuAAC reaction vial, the reaction steps remain the same, but a different tracer is easily achieved. In conclusion, two or three novel tracers that have the potential to act as apoptosis or myocardium imaging agents due to the lipophilic nature were synthesized in the same reaction vial and separated with high radiochemical purity via HPLC. This enables rapid, parallel, and comparable in vitro evaluation of either both [18F]MitoPhos_04 and [18F]MitoPhos_07 or all three tracers together. These data indicate that this methodology could be widely extended to other analogous radiolabeled compounds and to the synthesis of multiple radiotracers in one pot.
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ASSOCIATED CONTENT
S Supporting Information *
Full synthetic details of precursors and reference compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(N.L.) E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to thank the BBSRC, GSK, and Imanova, Ltd., for financial assistance, in particular for funding a CASE award for A.H.
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REFERENCES
(1) Gupta, S.; Kass, G. E. N.; Szegezdi, E.; Joseph, B. The mitochondrial death pathway: a promising therapeutic target in diseases. J. Cell. Mol. Med. 2009, 13 (6), 1004−1033. (2) Wang, J.; Yang, C.-T.; Kim, Y.-S.; Sreerama, S. G.; Cao, Q.; Li, Z.B.; He, Z.; Chen, X.; Liu, S. 64Cu-labeled triphenylphosphonium and triphenylarsonium cations as highly tumor-selective imaging agents. J. Med. Chem. 2007, 50 (21), 5057−5069. (3) Modica-Napolitano, J. S.; Singh, K. Mitochondria as targets for detection and treatment of cancer. Expert Rev. Mol. Med. 2002, 4 (09), 1−19. (4) Blankenberg, F. G. In vivo detection of apoptosis. J. Nucl. Med. 2008, 49 Suppl 2 (Suppl_2), 81S−95S. (5) Vander Heiden, M. G.; Chandel, N. S.; Williamson, E. K.; Schumacker, P. T.; Thompson, C. B. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 1997, 91 (5), 627−637. (6) Gallo, R. L.; Finkelstein, J. N.; Notter, R. H. Characterization of the plasma and mitochondrial membrane potentials of alveolar type II D
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ene] triphenylphosphonium bromide as a potential positron emission tomography tracer for imaging apoptosis. J. Labelled Compd. Radiopharm. 2013, 56 (6), 313−316. (24) Glaser, M.; Arstad, E. ″Click labeling″ with 2-[18f]fluoroethylazide for positron emission tomography. Bioconjugate Chem. 2007, 18 (3), 989−93. (25) Glaser, M.; Robins, E. G. ‘Click labelling’ in PET radiochemistry. J. Labelled Comp. Radiopharm. 2009, 52 (10), 407−414.
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