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Synthesis and preliminary evaluation of an 18F-labeled oleic acid analog for PET imaging of fatty acid uptake and metabolism
Synthesis and preliminary evaluation of an 18 F-labeled oleic acid analog for PET imaging of fatty acid uptake and metabolism Zhengxin Cai, N. Scott Mason, Carolyn J. Anderson, W. Barry Edwards PII: DOI: Reference:
S0969-8051(15)00149-3 doi: 10.1016/j.nucmedbio.2015.08.005 NMB 7762
To appear in:
Nuclear Medicine and Biology
Received date: Revised date: Accepted date:
14 May 2015 24 August 2015 28 August 2015
Please cite this article as: Cai Zhengxin, Mason N. Scott, Anderson Carolyn J., Edwards W. Barry, Synthesis and preliminary evaluation of an 18 F-labeled oleic acid analog for PET imaging of fatty acid uptake and metabolism, Nuclear Medicine and Biology (2015), doi: 10.1016/j.nucmedbio.2015.08.005
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ACCEPTED MANUSCRIPT Synthesis and preliminary evaluation of an 18F-labeled oleic acid analog
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for PET imaging of fatty acid uptake and metabolism
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Zhengxin Cai1, N. Scott Mason1, Carolyn J. Anderson1,2,3, and W. Barry Edwards1* 1
Department of Radiology
2
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Department of Pharmacology and Chemical Biology
3
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Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15219, USA
Conflict of Interest
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No conflicts of interest were reported regarding to this article.
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* W. Barry Edwards, PhD Department of Radiology Molecular Imaging Laboratory University of Pittsburgh 100 Technology Drive, Bridgeside Point 1, Suite 452 Pittsburgh, PA 15219, USA Email: [email protected] Phone: 412-624-6873
Running title: Synthesis and preliminary evaluation of 18F-labeled clicked oleic acid analog Key words: 18F, oleic acid, fatty acid, PET, metabolism
ACCEPTED MANUSCRIPT Abstract Introduction: Imaging fatty acid uptake and utilization has broad impact in investigating
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myocardial diseases, hepatic functions, tumor progression, and the metabolic state of adipose tissue. The SPECT tracer 123I-15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) is a
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clinically used nuclear medicine tracer to image myocardial uptake of fatty acid. Although FTHA (18F-5) has been in clinical use for PET imaging of adipose tissue as well as the
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myocardium, here we developed a click oleate analog to compare to FTO, with the goal of
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improved stability to defluorination and suitability for imaging myocardial uptake and oxidation of fatty acids.
18
F-labeled oleate
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Methods: A rapid and convenient synthetic approach for a precursor to a
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analog using click chemistry was developed and evaluated for PET imaging in fasted mice. Results: The overall yield for the preparation of the labeling precursor of the clicked oleate
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analog was 12%. This precursor was efficiently radiolabeled with F-18 in 17% non-decay-
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corrected radiochemical yield. PET/CT imaging and biodistribution results show that this fatty acid analog had reasonable heart uptake (0.94 ± 0.28 %ID/g at 0.5 h p.i.) and heart-to-muscle ratio (2.05 ± 0.39 at 0.5 h p.i.) and is a potential lead for developing new PET tracers to image fatty acid uptake and utilization using click chemistry methodologies. The synthetic route to FTO was optimized to three steps from known starting materials. Conclusion: While the uptake of the clicked oleic acid analog was sufficient for visualizing the myocardium in mice, the preliminary metabolism data suggest only a fraction of the uptake was due to fatty acid beta-oxidation. Studies are underway to explore the uptake/oxidation mechanism and kinetics.
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Introduction
perfusion imaging (MPI) employs
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Tl-, and
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Myocardial nuclear medicine includes imaging of perfusion viability. Traditionally, myocardial 99m
Tc-based tracers for single photon emission 123
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computed tomography (SPECT) imaging. Although SPECT imaging with
I-15-(p-
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iodophenyl)-3-(R,S)-methylpentadecanoic acid (1, BMIPP, Figure 1) has been broadly used in myocardial imaging, positron emission tomography (PET) with a molecularly-targeted PET
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tracer coupled with CT or MRI could provide more rigorous quantitative information. Currently, the clinical PET tracers for MPI are based on the short half-lived positron emitters, 15
O (15O-H2O; T1/2 = 2 min), and
82
N (13N-
Rb (82Rb-chloride; T1/2 = 1.27
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ammonia; T1/2 = 9.97 min),
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min). Nitrogen-13 and O-15 are cyclotron-produced, which due to their short half-lives requires
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an on-site cyclotron, limiting their widespread usage. Generator-produced 82Rb-chloride provides
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blood flow information and has the advantage of ease of production and lower radiation dose
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(0.7-1.3 mSv/GBq) for the patients. Additionally,
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in early clinical trials [3]. Commercially available
F-Flurpiridaz has shown promise as in MPI
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F-FDG has clinical utility for determining
myocardial viability. A fatty acid analog would be a good compliment with a MPI tracer and 18FFDG for viability, since the heart uses fatty acids as a major energy source. An imaging tracer based on fatty acid uptake/metabolism has the potential to report on the viability of myocardial tissue, as the adult heart typically uses fatty acid β-oxidation to produce most of its ATP [6]. To this end, efforts have been devoted to the development of fatty acid analogs radiolabeled with either
11
C (T1/2 = 20.4 min) or
18
F (T1/2 = 109.8 min), resulting in
several agents that have shown promise [7-10]. 11C-Palmitate (2, Figure 1) has been investigated
ACCEPTED MANUSCRIPT in humans to quantify myocardial fatty acid uptake and metabolism, but the extensive metabolism requires complex modeling to derive fatty acid oxidation rates. Furthermore, due to
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the short half-life of 11C, the production of 11C-palmitate requires an on-site cyclotron. Fluorine-
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18-labeled FAs are amenable to commercialization since the half-life enables shipping from a
To obtain high target tissue uptake,
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central location.
F-labeled fatty acid tracers have incorporated either alkyl
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branching or the introduction of heteroatoms to prevent rapid metabolism and influence uptake
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towards fatty acid beta-oxidation. For example, methyl-branched 18F-fatty acids generally have higher myocardial uptake than their non-branched analogs owing to diminished metabolism as a
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result of methylation; however, the uptake appears to be largely within the cytosol through esterification [11, 12]. The incorporation of sulfur atoms, predominantly at the 4-position (18F-
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FTO, 18F-4, Figure 1), results in greater incorporation into cellular proteins, greater sensitivity to
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the effects of carnitine palmitoyl transporter inhibitors, and greater reduction in hypoxic
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myocardial tissue, all of which are putative indications of sensitivity to reduced FAO [8, 11-14]. Since thia-substituted fatty acid analogs are trappable in the myocardium of rodents [8, 15-17], we decided to investigate sulfur at the 4-position as the possible trapping surrogate. A clickable palmitate analog that was synthesized and evaluated by Kim, et al. showed promise in myocardial imaging, albeit as a 6-thia analog (3, 17-[4-(2-[18F]Fluoroethyl)-1H-1,2,3-triazol-1yl]-6-thia-heptadecanoic acid, clicked-FHA, Figure 1) [18]. The blood clearance of the compound was significantly slower than its non-clicked analog, 18F-FTHA (18F-5), possibly due to the added triazole functional group. To the best of our knowledge there are no reports on the corresponding clicked oleate analogs. Thus, we designed a 4-thia-oleic acid analog to mimic the fate of the natural fatty acid and enable the imaging of myocardial fatty acid uptake and
ACCEPTED MANUSCRIPT oxidation. Through this process, we developed a more efficient synthesis of the FTO precursor 18 (Scheme 2) that requires only three steps compared to nine for the previously published 18
F-6
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synthesis. Herein, we report the radio-synthesis of a radiofluorinated 4-thia-oleate analog
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and its biological evaluation in mice in comparison to 18F-FTO (18F-4).
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Materials and Methods
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Reagents and Instrumentation
All reagents and solvents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO),
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unless otherwise specified. Reactions were monitored by TLC on 0.25 mm silica gel glass plates containing F-254 indicator. Visualization by TLC was monitored by UV light, KMnO4, or
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radioactivity. Flash chromatography was performed using 200-mesh silica gel. 1H-NMR spectra
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were recorded on a Bruker DRX 400 MHz NMR spectrometer (Billerica, MA). 13C NMR spectra were obtained at 100 MHz. ESI-MS were obtained on a Waters LCT-Premier XE LC-MS station
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(Milford, MA). A microwave cavity (Resonance Instruments, Inc. Model 521) was used for
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radiolabeling and hydrolysis. Reversed-phase high-performance liquid chromatography (HPLC) was performed either on a Waters 600E (Milford, MA) chromatography system with a Waters 991 photodiode array detector and an Ortec model 661 (EG&G Instruments, Oak Ridge, TN) radioactivity detector, or a Waters 1525 Binary HPLC pump with a Waters 2489 UV/visible detector and a model 105-S-1 (Carroll& Ramsey Associates; Berkeley, CA) radioactivity detector. HPLC samples were analyzed on an analytical C18 column (Phenomenex, Torrance, CA) and purified on a semi-preparative C18 column (Phenomenex, Torrance, CA) with H2O (0.1% TFA; solvent A) and acetonitrile (0.1% TFA; solvent B) as the mobile phase. All final compounds were at least 95% pure by NMR or HPLC. Radioactive samples were counted using
ACCEPTED MANUSCRIPT an automated well-type gamma-counter (8000; Beckman, Irvine, CA). PET/CT data were acquired using an Inveon preclinical PET scanner (Siemens Medical Solutions, Knoxville, TN). F-fluoride was produced by the (p, n) reaction from H218O (ABX GmbH, Germany isotopic
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18
The semi-prep system for purification of the
18
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purity >97%) using an Eclipse HP cyclotron (Siemens Medical Solutions, Erlangen, Germany). F-labeled fatty acids was comprised of a Waters
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610 pump with 600E controller, remotely actuated injection valve (VICI), PIN diode radiation
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detector and Bioscan hot cell base unit and Waters 481 variable wavelength UV detector. The analytical system is comprised of a Waters 510 pump, Rheodyne 7125 manual injection valve,
330
photodiode
array
detector.
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Bioscan Flow Count base unit fitted with a PIN diode radiation detector and a Varian ProStar Dec-9-yn-1-ol,
1,5-dibromopentane,
2-azidoethyl
4-
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methylbenzenesulfonate, 1-azido-2-fluoroethane, and 9-bromo-nonanal are commercially
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available (Sigma-Aldrich).
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Animal Model. All animal studies were conducted according to the procedures outlined by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC). Male CD-1
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mice, 4-8 weeks old, 38 ± 3 g, (Charles River; Malvern, Pennsylvania) were fasted by removing food overnight and used for biodistribution of 18F-4 and
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F-6, and PET/CT imaging of 18F-6. A
male nude mouse, (Charles River; (Malvern, Pennsylvania)) was fasted overnight and used for PET/CT imaging studies of 18F-4. Synthesis of intermediates, cold standard, precursors, and radiolabeling. Bromo(5-bromopentyl)triphenylphosphorane (8): Compound 8 was prepared following a published procedure from 1,5-dibromopentane (7) in 65% yield [19]: 1H NMR (CDCl3, 400 MHz) δ 7.90-7.78 (m, 9 H), 7.74-7.69 (m, 6 H), 3.93-3.86 (m, 2 H), 3.37 (t, 2 H, J = 6.4 Hz),
ACCEPTED MANUSCRIPT 1.94-1.82 (m, 4 H), 1.74-1.64 (m, 2 H); 13C NMR (CDCl3, 100 MHz) δ 135.0 (d, J = 3 Hz), 133.7 (d, J = 9 Hz), 130.5 (d, J = 12 Hz), 118.5 (d, J = 85 Hz), 33.5, 32.0, 28.8 (d, J = 6 Hz), 22.7 (d, J
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= 50 Hz), 21.9 (d, J = 5 Hz); 31P (CDCl3, 162 MHz) δ 24.4; HRMS calcd for C23H25BrP [M -
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Br]+ 411.0877, found 411.0867.
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3-((5-(Bromotriphenylphosphoranyl)pentyl)thio)propanoate (10): To a solution of methyl 3mercaptopropanoate (9, 2.44 g, 20.3 mmol) in 40 mL THF was added sodium hydride (0.77 g, 60%
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in mineral oil, 19.3 mmol) at 0 °C in three portions. Then 8 (5 g, 10.2 mmol) in 20 mL THF was
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added slowly to the reaction mixture at 0 °C. Then it was allowed to reach rt slowly. After 12 h at rt, it was concentrated at reduced pressure, and purified by flash chromatography, using 10%
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methanol/dichloromethane to yield the pure product (3.2 g, 59%): 1H NMR (CDCl3, 400 MHz) δ 7.89-7.77 (m, 9 H), 7.74-7.68 (m, 6 H), 3.90-3.82 (m, 2 H), 3.68 (s, 3 H), 2.73 (t, 2 H, J = 8 Hz),
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2.57 (t, 2 H, J = 8 Hz), 2.48 (t, 2 H, J = 7.2 Hz), 1.81-1.73 (m, 2 H), 1.70-1.55 (m, 4 H);
13
C
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NMR (CDCl3, 100 MHz) δ 172.2, 135.0 (d, J = 3 Hz), 133.7 (d, J = 10 Hz), 130.4 (d, J = 13 Hz), 118.4 (d, J = 86 Hz), 51.7, 34.7, 31.6, 29.4 (d, J = 16 Hz), 29.0, 27.0, 22.8 (d, J = 49 Hz), 22.3 (d, 31
P (CDCl3, 162 MHz) δ 24.4; HRMS calcd for C27H32O2PS [M - Br]+ 451.1861,
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J = 5 Hz);
found 451.1847.
(Z)-Methyl 3-(pentadec-5-en-14-yn-1-ylthio)propanoate (12): To a solution of methyl 3-((5(bromotriphenylphosphoranyl)pentyl)thio)propanoate (10, 1.8 g, 3.39 mmol) in 6.8 mL anhydrous THF was added NaOMe (0.37 g, 6.78 mmol) at rt. The solution turned yellow with a white suspension. After stirring vigorously for 5 min at rt, dec-9-ynal (11, 0.52 g, 3.39 mmol) was added drop wise at rt. The reaction mixture turned colorless with a white suspension and was quenched with saturated NH4Cl aqueous solution after stirring for 9 h at rt. The reaction mixture was extracted with Et2O (20 mL × 3), washed with brine (20 mL), dried with anhydrous
ACCEPTED MANUSCRIPT MgSO4, filtered, and concentrated at reduced pressure to get a pale yellow oil as the crude product. It was purified with flash chromatography using 10% EtOAc/hexane to yield the pure
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product (0.46 g, 42%): 1H NMR (CDCl3, 400 MHz) δ 5.41-5.30 (m, 2 H), 3.70 (s, 3 H), 2.79 (t, 2
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H, J = 7.2 Hz), 2.62 (t, 2 H, J = 7.2 Hz), 2.54 (t, 2 H, J = 7.2 Hz), 2.18 (dt, 2 H, J = 2.8, 6.8 Hz), 2.08-1.99 (m, 4 H), 1.95 (t, 1 H, J = 2.4 Hz), 1.66-1.27 (m, 14 H); 13C NMR (CDCl3, 100 MHz)
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δ 172.4, 130.3, 129.2, 84.7, 68.1, 51.8, 34.7, 32.1, 29.6, 29.1, 29.0, 28.9, 28.7, 28.5, 27.2, 27.0,
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26.7, 18.4; ESI-MS: 324.95 [M + H]+; HRMS calcd for C19H33O2S [M + H]+ 325.2203, found
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325.2191.
(Z)-Methyl 3-((13-(1-(2-(tosyloxy)ethyl)-1H-1,2,3-triazol-4-yl)tridec-5-en-1-yl)thio)propanoate
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(14): To a solution of (Z)-methyl 3-(pentadec-5-en-14-yn-1-ylthio)propanoate (12, 142 mg, 0.438 mmol) and 2-azidoethyl 4-methylbenzenesulfonate (13, 106 mg, 0.438 mmol) in CHCl3 (1
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mL) was added copper (I) iodide (8 mg, 0.044 mmol), followed by N,N-diisopropylethylamine
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(DIEA, 282 mg, 2.19 mmol) at rt. After stirring overnight, the reaction mixture was filtered through a short silica plug. It was rinsed with dichloromethane (100 mL), concentrated at
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reduced pressure, dissolved in MeCN, and purified with a pre-packed C18 column (30 g) using a gradient from 10% MeCN/H2O (0.1% TFA) to 90% MeCN/H2O (0.1% TFA) over 15 min with a flow of 35 mL/min. The eluent was monitored with a UV lamp at 230 nm and 254 nm. The fraction eluting at 15 min was collected and concentrated at reduced pressure to yield the pure product (178 mg, 72%). 1H NMR (CDCl3, 400 MHz) δ 7.68 (d, 2 H, J = 8 Hz), 7.48 (s, 1 H), 7.34 (d, 2 H, J = 8 Hz), 5.41-5.31 (m, 2 H), 4.65 (t, 2 H, J = 5.2 Hz), 4.42 (t, 2 H, J = 4.8 Hz), 3.71 (s, 3 H), 2.79 (t, 2 H, J = 7.2 Hz), 2.74 (t, 2 H, J = 8 Hz), 2.62 (t, 2 H, J = 7.2 Hz), 2.54 (t, 2 H, J = 7.2 Hz), 2.46 (s, 3 H), 2.08-2.00 (m, 4 H), 1.69-1.57 (m, 4 H), 1.48-1.28 (m, 10 H); 13C NMR (CDCl3, 100 MHz) δ 172.6, 145.7, 131.8, 130.3, 130.1, 129.2, 127.8, 67.3, 51.8, 49.8, 34.7,
ACCEPTED MANUSCRIPT 32.0, 29.6, 29.2, 29.1, 29.07, 29.06, 28.8, 27.2, 27.0, 26.7, 24.7, 21.6; ESI-MS: 566.80 [M + H]+; HRMS calcd for C28H44N3O5S2 [M + H]+ 566.2724, found 566.2711.
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(Z)-methyl 3-((14-bromotetradec-5-en-1-yl)thio)propanoate (18): To a solution of 10 (0.213 g, 0.4 mmol) in THF (9.4 mL, 0.2 M) was added sodium methoxide (22 mg, 0.4 mmol). After 10
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min at rt, aldehyde 17 (71 mg, 0.32 mmol) was added dropwise. After 1.5 h, the suspension was filtered through a short silica plug, rinsed with DCM, and concentrated at reduced pressure. The
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crude product was purified with 5% EtOAc/hexane on a silica column to yield the product (19)
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as colorless oil. The 1H NMR is consistent with published data (98 mg, 78%). 1H NMR (CDCl3, 400 MHz) δ 5.41-5.30 (m, 2 H), 3.73 (s, 3 H), 3.43 (t, 2 H, J = 6.8 Hz), 2.78 (dt, 2 H, J = 1.2, 8
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Hz), 2.62 (t, 2 H, J = 7.6 Hz), 2.54 (t, 2 H, J = 7.6 Hz), 2.07-1.99 (m, 4 H), 1.89-1.82 (m, 2 H), 1.64-1.56 (m, 2 H), 1.48-1.39 (m, 4 H), 1.35-1.27 (m, 8 H).
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(Z)-3-(pentadec-5-en-14-yn-1-ylthio)propanoic acid (15): To a solution of 12 (8.2 gm, 0.025
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mmol) in 1.25 mL EtOH was added aqueous KOH solution (1.25 mL, 0.2 N, 0.25 mmol) at rt stirred for 5 h at rt, and concentrated on a roto-evaporator. The crude product was purified with a
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C18 column to yield pure product (6.1 mg, 78%). 1H NMR (CDCl3, 400 MHz) δ 5.41-5.30 (m 2 H), 2.79 (dt, 2 H, J = 0.8, 8.0 Hz), 2.67 (dt, 2 H, J = 0.8, 8.0 Hz), 2.55 (t, 2 H, J = 7.2 Hz), 2.18 (dt, 2 H, J = 2.4, 7.2 Hz), 2.07-1.99 (m, 4 H), 1.94 (t, 1 H, J = 2.8 Hz), 1.65-1.27 (m, 15 H); ESIMS: 311.07 [M+H]. HRMS calcd for C18H29O2S [M - H] – 309.1887, found 309.1893. (Z)-3-((13-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)tridec-5-en-1-yl)thio)propanoic acid (6): A solution of (Z)-3-(pentadec-5-en-14-yn-1-ylthio)propanoic acid (15) (9.4 µmol) and 1-azido-2fluoroethane (16, 9.4 µmol) was added to a solution containing copper (I) iodide (1.8 mg, 9.4 µmol) and DIEA (8.2 µl, 47 µmol) in CHCl3. The reaction mixture was filtered through a short
ACCEPTED MANUSCRIPT silica plug, rinsed with dichloromethane, and concentrated at reduced pressure. It was then purified by HPLC to yield the pure product (2.6 mg, 70%): 1H NMR (CDCl3, 400 MHz) δ 5.45-
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5.31 (m, 2 H), 4.87 (t, 1 H, J = 4.8 Hz), 4.75 (t, 1 H, J = 4 Hz), 4.70 (t, 1 H, J = 4 Hz), 4.63 (t, 1
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H, J = 4.8 Hz), 2.95-2.90 (b, 1 H), 2.82 (dd, 2 H, J = 7.2, 14.4 Hz), 2.76 (t, 2 H, J = 7.6 Hz), 2.68 (t, 2 H, J = 7.2 Hz), 2.58 (t, 2 H, J = 7.2 Hz), 2.14-2.02 (m, 4 H), 1.83-1.46 (m, 7 H), 1.41-1.28
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(m, 8 H); HRMS calcd for C20H35FN3O2S [M + H]+ 400.2436, found 400.2423.
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being consistent with the published data [7].
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The cold standard of 18F-FTO was synthesized following the published procedure, with H-NMR
(Z)-3-((13-(1-(2-[18F]fluoroethyl)-1H-1,2,3-triazol-4-yl)tridec-5-en-1-yl)thio)propanoic
acid
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(18F-6): Cyclotron-produced [18F] fluoride (~ 22.2 GBq) was dried down under argon at 110 °C in a 3 mL glass vial containing Kryptofix 2.2.2 (10 mg), MeCN (0.8 mL), and aqueous solution
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of K2CO3 (4 mg in 0.2 mL). The residue was further azeotropically dried with MeCN (2 × 1 mL)
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at 110 °C under a continuous stream of argon gas. One mL of MeCN was added to the final
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[18F]fluoride residue. The resultant solution was then transferred to a solution of labeling precursor (2 to 4 mg) in 250 µl MeCN. The V-vial was sealed and heated at 75 °C for 10 min under microwave heating conditions. After cooling on ice for 1 min, silica TLC plates were spotted with the reaction mixture and developed with ethyl acetate to determine the crude incorporation yields. The hydrolysis of the ester intermediate was carried out with aqueous KOH solution (0.15 mL, 0.2 N) at 75 °C for 5 min with microwave-assisted heating, followed by acidifying with 0.15 mL glacial acetic acid after cooling the mixture on ice for 1 min. The crude mixture was purified by semi-preparative HPLC. The fraction containing the desired product was diluted with 50 mL water and passed across a C18 SepPak cartridge. The cartridge was washed with 10 mL water and then eluted with 1 mL ethanol. The volume was concentrated at 110 °C
ACCEPTED MANUSCRIPT under a continuous stream of argon gas to a final volume of approximately 200 µL and then diluted with 1% BSA (lipid free) in saline for cell or animal studies.
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F-FTO (18F-4): The published protocol was followed for the synthesis of 18F-FTO (18F-4) with
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7% radiochemical yield (non-decay corrected), and > 90% radiochemical purity [7].
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Biodistribution and Folch-type extraction:
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The biodistribution studies were carried out as previously described [20]. The tracers (1.85 MBq) were injected into CD-1 mice (n = 4 for 18F-6 or 7 for 18F-4 per group) via the tail vein. The mice
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were sacrificed at 30 min or 2 h p.i., and selected organs were removed, weighed, and counted on a gamma counter (Perkin Elmer Wizard2, Waltham, MA). Groups of mice (n = 4) were injected
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intraperitoneally with etomoxir (40 mg/kg, in saline) 3 h before injection of the tracer and were
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sacrificed at 2 h post-injection. Folch-type extraction was done following the published protocol.
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PET/CT imaging:
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Small animal PET/CT imaging was performed with as previously described [20]. Briefly, the radiotracers (3.7 MBq) were injected via tail vein and the mice (n = 4) were imaged at 2 h p.i. Static imaging was performed on an Inveon PET/CT scanner [21, 22] with 10 min PET scanning followed by 5 min CT. The mice were anesthetized with isoflurane. Inveon Research Workplace (IRW) software (Siemens AG, Germany) was used for co-registration of PET/CT images and quantification of regions of interest (ROI). PET/CT images were reconstructed with maximum a posteriori (MAP), 3D ordered-subset expectation maximization (OSEM3D), 2D ordered-subset expectation maximization (OSEM2D), and filtered back projection (2DFBP). Standard uptake values (SUVs) were generated by measuring ROI from PET/CT images and calculated with the formula: SUV = [MBq/mL] × [animal weight (g)]/injected dose [MBq].
ACCEPTED MANUSCRIPT Statistical Methods. All of the data are presented as mean ± standard deviation. Two-tailed unpaired t-tests were performed using GraphPad Prism 5.04.
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Results
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Synthesis of labeling precursors, cold standards, and radiolabeling of 18F-4 and 18F-6: The dibromide 7 reacted with triphenylphosphine to yield bromide 8 in 65% yield (Scheme 1). 8
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was then reacted with compound 9 to generate compound 10 in 59% yield. Dec-9-ynal (11) was prepared from commercially available dec-9-yn-1-ol in 81% yield as the aldehyde for the Wittig
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reaction. This aldehyde (11) was converted to (Z)-methyl 3-(pentadec-5-en-14-yn-1ylthio)propanoate (12) via a (Z)-selective Wittig reaction in 42% yield. The propanoate 12 was
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reacted with 2-azidoethyl 4-methylbenzenesulfonate (13) to yield the desired (Z)-methyl 3-((13-
substrate for
18
F-labeling.
(14)
as
the
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(1-(2-(tosyloxy)ethyl)-1H-1,2,3-triazol-4-yl)tridec-5-en-1-yl)thio)propanoate 18
F-labeling was performed under microwave assisted heating for 10
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min at 75 °C. The intermediate methyl ester was then saponified with KOH under microwave
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assisted heating for 5 min at 75 °C to yield the final product, which was purified on reversedphase HPLC using a C18 column. The non-decay corrected radiochemical yield of 18F-6 was 17% (non-decay corrected RCY), with > 95% radiochemical purity (based upon radio-HPLC analysis). The cold standard 6 was synthesized from methyl ester 12 by saponification to get the acid 15 in 78% yield, which reacted with 2-fluoroethyl azide 16 to generate 6 in 70% yield. The synthesis of the precursor for
18
F-labeling of FTO (compound 18) involved a (Z)-selective
Wittig reaction between the bromoaldehyde 17 and the methyl ester 10, which generated the desired bromide 18 in 78% yield (Scheme 2). The
18
F-labeling of 18 was done following
ACCEPTED MANUSCRIPT published procedure, resulting in the 18F-FTO (18F-4) in 7% non-decay-corrected RCY. The cold standard 4 was synthesized following published literature from 18 [7].
18
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In order to evaluate
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PET/CT imaging and biodistribution:
F-6 as a myocardial imaging agent and determine its specificity for β-
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oxidation, PET/CT scanning and biodistribution studies of 18F-6 were performed in fasted CD-1
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mice to determine the extent of myocardial uptake of the tracer. Blocking of carnitine palmitoyltransferase I (CPT-I) was performed following pre-treatment with etomoxir in fasted
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mice. The PET/CT imaging and biodistribution studies of the oleate analog
18
F-6 revealed high
liver uptake (18.4 ± 6.2 %ID/g) with excretion through the gallbladder into the intestine, 18
F-6 into the liver.
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presumably due to the high lipophilicity of the tracer or active transport of
Biodistribution studies show that the heart uptake of 18F-6 was moderate (0.94 ± 0.28 %ID/g at
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0.5 h p.i.), and decreased at 2 h p.i. (0.48 ± 0.05 %ID/g). The heart-to-muscle ratio of 18F-6 was 18
F-6 PET visualized myocardial tissue in
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2.05 ± 0.39 at 0.5 h p.i. and 1.85 ± 0.33 at 2 h p.i.
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fasted mice at 30 min post-injection (Figure 3). To determine whether the observed uptake was mediated by CPT-I transport, the CPT-I inhibitor etomoxir was injected to the mice 3 h before injection of
18
F-6, and organs were harvested (2 h p.i.). The heart uptake of
18
F-6 in etomoxir-
treated mice decreased relative to the control group (0.34 ± 0.07 %ID/g vs 0.48 ± 0.05 %ID/g, P = 0.0027, Table 1), which suggests that the uptake is mediated at least in part by CPT-I mediated mitochondrial uptake. To determine the metabolic fate of the
18
F-fatty acid, a group of mice were pre-injected with
etomoxir 3 h prior to the injection of the radiotracer and the hearts were harvested (30 min p.i.), and subjected to a Folch extraction (Figure 2). The highest activity fraction was the organic
ACCEPTED MANUSCRIPT phase, which was increased upon pre-treatment of etomoxir (0.66 ± 0.02 %ID/g vs. 0.76 ± 0.02 %ID/g, n = 4, P = 0.0068). At the same time, the aqueous fraction decreased upon etomoxir
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treatment (0.15 ± 0.01 %ID/g vs. 0.06 ± 0.01 %ID/g, n = 4, P = 0.0002). The pellet activity did
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not change significantly upon treatment of etomoxir (0.19 ± 0.02 %ID/g vs. 0.18 ± 0.04, n = 4, P
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= 0.66).
To our knowledge, there are no reported investigations of
18
F-4 (FTO) in mice; therefore, a
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biodistribution of 18F-4 in fasted CD-1 mice was also performed for the purpose of comparison
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to 18F-6 (Table 1). The heart uptake of 18F-4 at 30 min p.i. was 1.8 ± 0.61 %ID/g, decreasing to 1.7 ± 0.27 %ID/g at 2 h p.i. Etomoxir decreased the heart uptake of 18F-4 to 0.82 ± 0.23 %ID/g at
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2 h p.i. The high bone uptake of 18F-4 (3.19 ± 2.31 %ID/g at 30 min p.i., 7.5 ± 1.82 %ID/g at 2 h p.i.) indicated significant defluorination in vivo, which was not observed for
18
F-6 (0.80 ±
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0.54 %ID/g, P = 0.0233, at 30 min p.i.; 0.60 ± 0.23 %ID/g, P < 0.0001, at 2 h p.i.). PET imaging
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with 18F-4 in a nude mouse showed clear myocardial uptake (SUV = 1.1) and high bone uptake
Discussion
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as the result of in vivo defluorination (Figure 3).
Fatty acid metabolism is the main source of energy for the myocardium. Therefore, metabolically trapped PET tracers that are sensitive to changes in FAO could determine changes in myocardial FA metabolism. Quantification of these changes in myocardial diseases with altered FA metabolism such as diabetes mellitus or myocardial ischemia could have a major impact on therapeutic regimens [23]. While numerous
18
F-FAs have been introduced for FAO
imaging with promising results, there is still a need to develop new tracers for probing the FAO
ACCEPTED MANUSCRIPT mechanisms [15, 24, 25]. To date, FA analogs with 4-thia substitutions appear to be the most sensitive analogs, because their Co-A esters are substrates for CPT-I mediated transesterification
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prior to transport into the mitochondria [24]. Additionally, radiofluorinated oleate analogs
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showed superior imaging properties in the rat heart relative to radiofluorinated palmitate-based agents [8, 16, 24]. For these reasons, we chose 4-thia-oleate to contain the resultant triazole by
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utilizing click chemistry.
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Click chemistry is an efficient way to build bioactive small molecules and could enable modular
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synthesis of a library of FA analogs to probe myocardial FAO. Therefore, we adopted the copper-catalyzed alkyne azide cycloaddition reaction to synthesize a clicked 4-thia-oleate analog
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(18F-6) in order to explore the influence of the -terminal modification of the fatty acid with the triazole group. We also synthesized
18
F-FTO for comparison, because it has been previously
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shown in rat heart to have good sensitivity to FAO and have high target tissue uptake [24].
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In the process of working out the chemistry of 14, we developed a novel and convergent
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synthesis of the precursor for FTO (18), making it amenable for further structural modifications that might improve the pharmacokinetics, sensitivity to FAO, and the resultant image quality. We improved the synthesis of 18 by reducing the synthetic steps from 9 to 3 using readily available starting materials.
18
F-FTO was employed as a reference tracer for the newly
synthesized clicked oleate analog (Scheme 2) [24].
18
F-FTO shows promise as a myocardial
imaging tracer in spite of severe enzymatic defluorination in rodents; however, defluorination of -terminal FAs is not as severe in swine as in rodents [7]. When
18
F is incorporated at -n
carbons, the enzymatic defluorination is diminished, but it generates a stereocenter, resulting in an inseparable racemic mixture as the product [28]. For example, relative to FTO (2 h bone, ~7 %ID/g), FTHA, which is fluorinated at the -4 position, showed much lower bone uptake in
ACCEPTED MANUSCRIPT mice (2 h bone, 3.8 %ID/g) [15]. Previous work has shown that incorporation of a triazole into the aliphatic chain apparently reduces hepatic enzyme-mediated defluorination for the clicked
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FTHA (1 h bone, 1.2 %ID/g); however, there was no direct comparison to -fluorinated FAs
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[18]. When compared to 18F-FTO, 18F-6 showed relatively low bone uptake (7.5 ± 1.8 vs 0.60 ±
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0.23 %ID/g), confirming that the triazoles do inhibit hepatic enzymatic defluorination and that the inhibition is not dependent of the position of the triazole in the aliphatic chain.
18
F-4 and
18
F-6 in mouse heart in comparison to that of
F-FA analogs in mouse as well as in rat heart. For example, the uptake of
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other
18
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There are major differences between
18
F-6 was
sufficient to visualize the mouse myocardial tissue with PET, and was about half that of FTO 18
F-6 or
18
F-4
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(18F-4). In contrast, clicked-FHA (18F-3) demonstrated more uptake than either
(%ID/g = 3.16 ± 0.36, 0.5 h) [18]. Clicked-FHA (3) may be considered an analog of 14(R,S)-
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[18F]fluoro-6-thia-hepatadecanoic acid (FTHA), which showed extremely high uptake in mouse
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heart (%ID/g = 29.2 ± 0.3, 1 h) [15]; however, in rat heart, the uptake of FTHA dropped significantly (0.96 ± 0.18 %ID/g) [29]. The first step towards metabolism and intracellular
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trafficking of FAs is molecular transport across the across the sarcolemma into the cytoplasm of the myocardial cell. Therefore, these differences in myocardial uptake between species are likely attributable to the expression levels of FA transport proteins and transport kinetics of these FAs. FA transport proteins are a class of integral membrane proteins that mediate transport of FAs across the plasma membrane. When FATP6 was transfected in HEK293 cells, which do not express any FATPs or other transport proteins, FATP6 mediated transport of palmitate was 4fold that of oleate [30]. FATP6 is highly expressed in the mouse myocardial tissue compared to low expression of FATP1 [30]. However, in rat heart, FATP1 is expressed at higher levels than FATP6 [31]. The incorporation of fluorine and sulfur atoms, while being reasonable isoteres of
ACCEPTED MANUSCRIPT hydrogen and carbon respectively, could conceivably impair binding efficiency and transport. Moreover, the incorporation of a triazole in 5 could be deleterious to the molecular recognition 18
F-6 may
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by the FATPs leading to diminished transport rates. Thus, the structural features of
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account for the observed diminished myocardial uptake relative to FTO and clicked-FHA [32]. Taken together, the aforementioned uptakes of FTHA (18F-5), FTO (18F-4), clicked-FHA (18F-3),
18
F-6 revealed high uptake in the liver, kidney, and heart, consistent with the
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PET imaging of
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and 18F-6 could easily be influenced by the transporter levels and rates of transport.
than that of FTO
18
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biodistribution data. While the SUV of 18F-6 in the heart is 0.26 ± 0.12 (n = 4), which is lower F-4, it is still sufficient for visualization of the heart, albeit with increased
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background. Based on the biodistribution data, the heart-to-muscle ratio of p.i.) is lower than that of clicked-FHA
18
18
F-6 (2.05 at 0.5 h
F-3 (4.79 at 0.5 h p.i.), which may be due to the
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preferential expression of FATPs favoring the transportation of this heptanoic acid analog rather
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than oleate in mouse heart. The higher heart-to-muscle ratio of FTO relative to that the terminal triazole group interfered with the transportation of
18
F-6 suggests
F-6 into the myocardial
cells. Of note, the relatively high heart-to-muscle and heart-to-blood ratios of
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18
18
F-4 in rat were
not reproduced in mouse, which suggests distinct molecular processes are responsible for uptake between species and they are not confined to those processes related to -oxidation alone [24]. To determine whether the observed myocardial uptake of 18F-6 was mediated by -oxidation, the CPT-I inhibitor etomoxir was pre-administered with 18F-6. CPT-I is an enzyme that catalyzes the trans-esterification of palmitate and other fatty acid CoA esters with carnitine for subsequent transport into the mitochondria [33]. Myocardial uptake of 18F-6 was partially blocked by CPT-I inhibition, suggesting that the uptake of the tracer was due in part to its oxidization in the mitochondria (Table 1). Etomoxir was reported to reduce the CPT-I mediated -oxidation of
ACCEPTED MANUSCRIPT FTO by 50%, while we observed only a 29% reduction of 5 (Table 1) [24]. The remaining fraction of 18F-6 is presumably esterified into cellular lipids, consistent with the observed Folch
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extraction results showing the largest fraction of activity in the lipid fraction with low amounts
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of oxidized FA incorporated into cellular proteins (Figure 2). Etomoxir did not reduce the amount of radioactivity associated with the protein fraction [34]. Based on these data,
18
F-6
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would not be sensitive to reduced metabolism in myocardial tissues.
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The incorporation of structural features in FAs to block -oxidation and trap the metabolite(s) in
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the myocardial cells must be balanced against the need to maintain affinity and trafficking of the FA by proteins involved in the metabolic process. Our results suggest that molecular recognition
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of FAs by membrane associated and cytosolic proteins is an important determinant of uptake. Therefore, future work will entail moving the triazole group away from the -terminus toward
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the acyl terminus in an attempt to increase the myocardial uptake of the clicked oleate analogs.
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Previous work demonstrated that moving polar modifications away from the -terminus increased myocardial uptake of 99mTc-labeled fatty acid analogs [35]. Since small changes in the
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structure of the fatty acid backbone could have significant impact on the myocardial uptake, it is foreseeable that an instructive structure-activity relationship could be obtained with this new synthetic route that allows one to generate a library of clicked fatty acid analogs. In addition to their utility as potential myocardial PET imaging agents, the generation of such a library of 18Flabeled fatty acids that retain affinity for their transport proteins and are substrates for oxidation could be used to probe a variety of other disease conditions and organ functionalities including hepatic functions [36, 37], diabetes [38, 39], neuroinflammation, and a range of tumor types that utilize fatty acids as a major energy source [41, 42].
ACCEPTED MANUSCRIPT Conclusion Here we report a novel clicked oleic acid analog (14) that was synthesized via click chemistry
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convergently in good yield (16% overall), and was efficiently labeled with 18F-fluoride (90 min; 17% non-decay corrected radiochemical yield). We also optimized the synthetic route to FTO
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requiring only three steps from commercially available materials, which is a significant improvement over the former synthetic route (nine steps) that required extensive protecting
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group manipulations. The incorporation of a triazole group (18F-6) significantly reduced the in
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vivo defluorination compared with FTO (18F-4). While the uptake of analog 18F-6 was sufficient for visualizing the myocardium in mice, the preliminary metabolism data suggest only a fraction
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of the uptake (~29%) was due to fatty acid beta-oxidation. Studies are underway to explore the
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uptake/oxidation mechanism and kinetics. Acknowledgements
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The authors thank Joseph Latoche, Kathryn Day Elizabeth, Nicole DeBlasio, and Jalpa Modi for
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the excellent technique assistance. The small animal PET/CT imaging at UPCI was supported in part by P30CA047904 (UPCI CCSG).
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Table 1. Tissue biodistribution of 18F-4 and 18F-6 in fasted CD-1 mice Mean ± SD %ID/g of 18F-4
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Mean ± SD %ID/g of 18F-6 30 min
2h
2 h, etomoxir a
30 min
2h
2 h, etomoxir a
blood
0.91 ± 0.22
0.43 ± 0.09
0.24 ± 0.13
0.64 ± 0.19
0.24 ± 0.03
0.33 ± 0.17
lung
1.45 ± 0.51
0.57 ± 0.09
0.52 ± 0.08
0.78 ± 0.28
0.40 ± 0.09
0.37 ± 0.11
liver
18.37 ± 6.19
9.87 ± 3.22
6.45 ± 0.58
2.96 ± 1.38
1.59 ± 0.19
1.36 ± 0.15
kidney
7.34 ± 2.33
5.92 ± 2.73
4.27 ± 1.31
3.96 ± 1.92
2.38 ± 0.23
0.89 ± 0.29
muscle 0.46 ± 0.14
0.27 ± 0.06
0.17 ± 0.06
0.37 ± 0.22
0.26 ± 0.07
0.23 ± 0.09
heart
0.94 ± 0.28
0.48 ± 0.05b
0.34 ± 0.07b
1.8 ± 0.61
1.69 ± 0.27b
0.82 ± 0.23b
brain
0.34 ± 0.12
0.18 ± 0.04
0.11 ± 0.03
0.22 ± 0.14
0.2 ± 0.04
0.18 ± 0.03
Bone
0.80 ± 0.54
0.60 ± 0.23
0.82 ± 0.33
3.19 ± 2.31
7.5 ± 1.82
8.52 ± 2.14
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tissue
Mice were pretreated with etomoxir 3 h before injecting 18F-4 or 18F-6, n = 4.
b
P = 0.0027.
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a
ACCEPTED MANUSCRIPT Legends for figures
F-FTO (18F-4); 18F-FTHA (18F-5); 18F-clicked FTO (18F-6).
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18
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Figure 1. Structures of 123I-BMIPP (123I-1); 11C-Palmitate (11C-2); and 18F-Clicked FHA (18F-3);
Figure 2. Representative midventricular trans-axial PET images of 18
F-4 in fasted nude mouse
F-6 in fasted CD-1 mouse at 30 minutes p.i. with different scale bars due to significantly
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and
18
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different SUV in the heart. SUV of 18F-4 in the heart is 1.1 (n = 1). SUV of 18F-6 in the heart is 0.26 ± 0.12 (n = 4). The location of spine and fat is identified based on the co-registration of CT
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images. The radiotracers (3.7 MBq) were injected via tail vein and the mice were imaged at 2 h p.i. Static imaging was performed on an Inveon PET/CT scanner with 10 min PET scanning
18
F-6 and its metabolites in the hearts of fasted mice by Folch-
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Figure 3. Total distribution of
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followed by 5 min CT. The mice were anesthetized with isoflurane during the time of scanning.
type extraction, with and without etomoxir pre-treatment. (Statistical significant difference: *P <
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0.05, n = 4, 7 for etomoxir pre-treated group and untreated group respectively).
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Legends for schemes
Scheme 1. Synthesis of 18F-clicked-FTO (18F-6) and the cold standard, 19F-clicked-FTO (6). Scheme 2. Improved Synthesis of 18F-FTO (18F-4).
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ACCEPTED MANUSCRIPT
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F-FTO (18F-4); 18F-FTHA (18F-5); 18F-clicked FTO (18F-6).
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18
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Figure 1. Structures of 123I-BMIPP (123I-1); 11C-Palmitate (11C-2); and 18F-Clicked FHA (18F-3);
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ACCEPTED MANUSCRIPT
18
F-4
F-6
Heart
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3E-1 SUV
18
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Heart
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Spine
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Fat
and
18
1E-1 SUV
18
F-4 in fasted nude mouse
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Figure 2. Representative midventricular trans-axial PET images of
Fat
F-6 in fasted CD-1 mouse at 30 minutes p.i. with different scale bars due to significantly
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different SUV in the heart. SUV of 18F-4 in the heart is 1.1 (n = 1). SUV of 18F-6 in the heart is
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0.26 ± 0.12 (n = 4). The location of spine and fat is identified based on the co-registration of CT images. The radiotracers (3.7 MBq) were injected via tail vein and the mice were imaged at 2 h
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p.i. Static imaging was performed on an Inveon PET/CT scanner with 10 min PET scanning followed by 5 min CT. The mice were anesthetized with isoflurane during the time of scanning.
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0.6 0.4
*
0.2 0.0
Pellets
Aqueous
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Organic
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0.5 h 0.5 h+etomoxir
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*
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0.8
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fraction of total ctivity
1.0
18
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Figure 3. Total distribution of
F-6 and its metabolites in the hearts of fasted mice by Folch-
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type extraction, with and without etomoxir pre-treatment. (Statistical significant difference: *P <
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0.05, n = 4, 7 for etomoxir pre-treated group and untreated group respectively).
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Scheme 1. Synthesis of 18F-clicked-FTO (18F-6) and the cold standard, 19F-clicked-FTO (6).
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Scheme 2. Improved synthesis of 18F-FTO.
S0969-8051(15)00149-3 doi: 10.1016/j.nucmedbio.2015.08.005 NMB 7762
To appear in:
Nuclear Medicine and Biology
Received date: Revised date: Accepted date:
14 May 2015 24 August 2015 28 August 2015
Please cite this article as: Cai Zhengxin, Mason N. Scott, Anderson Carolyn J., Edwards W. Barry, Synthesis and preliminary evaluation of an 18 F-labeled oleic acid analog for PET imaging of fatty acid uptake and metabolism, Nuclear Medicine and Biology (2015), doi: 10.1016/j.nucmedbio.2015.08.005
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ACCEPTED MANUSCRIPT Synthesis and preliminary evaluation of an 18F-labeled oleic acid analog
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for PET imaging of fatty acid uptake and metabolism
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Zhengxin Cai1, N. Scott Mason1, Carolyn J. Anderson1,2,3, and W. Barry Edwards1* 1
Department of Radiology
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Department of Pharmacology and Chemical Biology
3
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Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15219, USA
Conflict of Interest
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No conflicts of interest were reported regarding to this article.
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* W. Barry Edwards, PhD Department of Radiology Molecular Imaging Laboratory University of Pittsburgh 100 Technology Drive, Bridgeside Point 1, Suite 452 Pittsburgh, PA 15219, USA Email: [email protected] Phone: 412-624-6873
Running title: Synthesis and preliminary evaluation of 18F-labeled clicked oleic acid analog Key words: 18F, oleic acid, fatty acid, PET, metabolism
ACCEPTED MANUSCRIPT Abstract Introduction: Imaging fatty acid uptake and utilization has broad impact in investigating
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myocardial diseases, hepatic functions, tumor progression, and the metabolic state of adipose tissue. The SPECT tracer 123I-15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) is a
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clinically used nuclear medicine tracer to image myocardial uptake of fatty acid. Although FTHA (18F-5) has been in clinical use for PET imaging of adipose tissue as well as the
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myocardium, here we developed a click oleate analog to compare to FTO, with the goal of
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improved stability to defluorination and suitability for imaging myocardial uptake and oxidation of fatty acids.
18
F-labeled oleate
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Methods: A rapid and convenient synthetic approach for a precursor to a
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analog using click chemistry was developed and evaluated for PET imaging in fasted mice. Results: The overall yield for the preparation of the labeling precursor of the clicked oleate
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analog was 12%. This precursor was efficiently radiolabeled with F-18 in 17% non-decay-
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corrected radiochemical yield. PET/CT imaging and biodistribution results show that this fatty acid analog had reasonable heart uptake (0.94 ± 0.28 %ID/g at 0.5 h p.i.) and heart-to-muscle ratio (2.05 ± 0.39 at 0.5 h p.i.) and is a potential lead for developing new PET tracers to image fatty acid uptake and utilization using click chemistry methodologies. The synthetic route to FTO was optimized to three steps from known starting materials. Conclusion: While the uptake of the clicked oleic acid analog was sufficient for visualizing the myocardium in mice, the preliminary metabolism data suggest only a fraction of the uptake was due to fatty acid beta-oxidation. Studies are underway to explore the uptake/oxidation mechanism and kinetics.
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Introduction
perfusion imaging (MPI) employs
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Tl-, and
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Myocardial nuclear medicine includes imaging of perfusion viability. Traditionally, myocardial 99m
Tc-based tracers for single photon emission 123
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computed tomography (SPECT) imaging. Although SPECT imaging with
I-15-(p-
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iodophenyl)-3-(R,S)-methylpentadecanoic acid (1, BMIPP, Figure 1) has been broadly used in myocardial imaging, positron emission tomography (PET) with a molecularly-targeted PET
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tracer coupled with CT or MRI could provide more rigorous quantitative information. Currently, the clinical PET tracers for MPI are based on the short half-lived positron emitters, 15
O (15O-H2O; T1/2 = 2 min), and
82
N (13N-
Rb (82Rb-chloride; T1/2 = 1.27
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ammonia; T1/2 = 9.97 min),
13
min). Nitrogen-13 and O-15 are cyclotron-produced, which due to their short half-lives requires
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an on-site cyclotron, limiting their widespread usage. Generator-produced 82Rb-chloride provides
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blood flow information and has the advantage of ease of production and lower radiation dose
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(0.7-1.3 mSv/GBq) for the patients. Additionally,
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in early clinical trials [3]. Commercially available
F-Flurpiridaz has shown promise as in MPI
18
F-FDG has clinical utility for determining
myocardial viability. A fatty acid analog would be a good compliment with a MPI tracer and 18FFDG for viability, since the heart uses fatty acids as a major energy source. An imaging tracer based on fatty acid uptake/metabolism has the potential to report on the viability of myocardial tissue, as the adult heart typically uses fatty acid β-oxidation to produce most of its ATP [6]. To this end, efforts have been devoted to the development of fatty acid analogs radiolabeled with either
11
C (T1/2 = 20.4 min) or
18
F (T1/2 = 109.8 min), resulting in
several agents that have shown promise [7-10]. 11C-Palmitate (2, Figure 1) has been investigated
ACCEPTED MANUSCRIPT in humans to quantify myocardial fatty acid uptake and metabolism, but the extensive metabolism requires complex modeling to derive fatty acid oxidation rates. Furthermore, due to
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the short half-life of 11C, the production of 11C-palmitate requires an on-site cyclotron. Fluorine-
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18-labeled FAs are amenable to commercialization since the half-life enables shipping from a
To obtain high target tissue uptake,
18
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central location.
F-labeled fatty acid tracers have incorporated either alkyl
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branching or the introduction of heteroatoms to prevent rapid metabolism and influence uptake
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towards fatty acid beta-oxidation. For example, methyl-branched 18F-fatty acids generally have higher myocardial uptake than their non-branched analogs owing to diminished metabolism as a
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result of methylation; however, the uptake appears to be largely within the cytosol through esterification [11, 12]. The incorporation of sulfur atoms, predominantly at the 4-position (18F-
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FTO, 18F-4, Figure 1), results in greater incorporation into cellular proteins, greater sensitivity to
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the effects of carnitine palmitoyl transporter inhibitors, and greater reduction in hypoxic
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myocardial tissue, all of which are putative indications of sensitivity to reduced FAO [8, 11-14]. Since thia-substituted fatty acid analogs are trappable in the myocardium of rodents [8, 15-17], we decided to investigate sulfur at the 4-position as the possible trapping surrogate. A clickable palmitate analog that was synthesized and evaluated by Kim, et al. showed promise in myocardial imaging, albeit as a 6-thia analog (3, 17-[4-(2-[18F]Fluoroethyl)-1H-1,2,3-triazol-1yl]-6-thia-heptadecanoic acid, clicked-FHA, Figure 1) [18]. The blood clearance of the compound was significantly slower than its non-clicked analog, 18F-FTHA (18F-5), possibly due to the added triazole functional group. To the best of our knowledge there are no reports on the corresponding clicked oleate analogs. Thus, we designed a 4-thia-oleic acid analog to mimic the fate of the natural fatty acid and enable the imaging of myocardial fatty acid uptake and
ACCEPTED MANUSCRIPT oxidation. Through this process, we developed a more efficient synthesis of the FTO precursor 18 (Scheme 2) that requires only three steps compared to nine for the previously published 18
F-6
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synthesis. Herein, we report the radio-synthesis of a radiofluorinated 4-thia-oleate analog
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and its biological evaluation in mice in comparison to 18F-FTO (18F-4).
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Materials and Methods
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Reagents and Instrumentation
All reagents and solvents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO),
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unless otherwise specified. Reactions were monitored by TLC on 0.25 mm silica gel glass plates containing F-254 indicator. Visualization by TLC was monitored by UV light, KMnO4, or
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radioactivity. Flash chromatography was performed using 200-mesh silica gel. 1H-NMR spectra
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were recorded on a Bruker DRX 400 MHz NMR spectrometer (Billerica, MA). 13C NMR spectra were obtained at 100 MHz. ESI-MS were obtained on a Waters LCT-Premier XE LC-MS station
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(Milford, MA). A microwave cavity (Resonance Instruments, Inc. Model 521) was used for
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radiolabeling and hydrolysis. Reversed-phase high-performance liquid chromatography (HPLC) was performed either on a Waters 600E (Milford, MA) chromatography system with a Waters 991 photodiode array detector and an Ortec model 661 (EG&G Instruments, Oak Ridge, TN) radioactivity detector, or a Waters 1525 Binary HPLC pump with a Waters 2489 UV/visible detector and a model 105-S-1 (Carroll& Ramsey Associates; Berkeley, CA) radioactivity detector. HPLC samples were analyzed on an analytical C18 column (Phenomenex, Torrance, CA) and purified on a semi-preparative C18 column (Phenomenex, Torrance, CA) with H2O (0.1% TFA; solvent A) and acetonitrile (0.1% TFA; solvent B) as the mobile phase. All final compounds were at least 95% pure by NMR or HPLC. Radioactive samples were counted using
ACCEPTED MANUSCRIPT an automated well-type gamma-counter (8000; Beckman, Irvine, CA). PET/CT data were acquired using an Inveon preclinical PET scanner (Siemens Medical Solutions, Knoxville, TN). F-fluoride was produced by the (p, n) reaction from H218O (ABX GmbH, Germany isotopic
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18
The semi-prep system for purification of the
18
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purity >97%) using an Eclipse HP cyclotron (Siemens Medical Solutions, Erlangen, Germany). F-labeled fatty acids was comprised of a Waters
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610 pump with 600E controller, remotely actuated injection valve (VICI), PIN diode radiation
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detector and Bioscan hot cell base unit and Waters 481 variable wavelength UV detector. The analytical system is comprised of a Waters 510 pump, Rheodyne 7125 manual injection valve,
330
photodiode
array
detector.
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Bioscan Flow Count base unit fitted with a PIN diode radiation detector and a Varian ProStar Dec-9-yn-1-ol,
1,5-dibromopentane,
2-azidoethyl
4-
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methylbenzenesulfonate, 1-azido-2-fluoroethane, and 9-bromo-nonanal are commercially
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available (Sigma-Aldrich).
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Animal Model. All animal studies were conducted according to the procedures outlined by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC). Male CD-1
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mice, 4-8 weeks old, 38 ± 3 g, (Charles River; Malvern, Pennsylvania) were fasted by removing food overnight and used for biodistribution of 18F-4 and
18
F-6, and PET/CT imaging of 18F-6. A
male nude mouse, (Charles River; (Malvern, Pennsylvania)) was fasted overnight and used for PET/CT imaging studies of 18F-4. Synthesis of intermediates, cold standard, precursors, and radiolabeling. Bromo(5-bromopentyl)triphenylphosphorane (8): Compound 8 was prepared following a published procedure from 1,5-dibromopentane (7) in 65% yield [19]: 1H NMR (CDCl3, 400 MHz) δ 7.90-7.78 (m, 9 H), 7.74-7.69 (m, 6 H), 3.93-3.86 (m, 2 H), 3.37 (t, 2 H, J = 6.4 Hz),
ACCEPTED MANUSCRIPT 1.94-1.82 (m, 4 H), 1.74-1.64 (m, 2 H); 13C NMR (CDCl3, 100 MHz) δ 135.0 (d, J = 3 Hz), 133.7 (d, J = 9 Hz), 130.5 (d, J = 12 Hz), 118.5 (d, J = 85 Hz), 33.5, 32.0, 28.8 (d, J = 6 Hz), 22.7 (d, J
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= 50 Hz), 21.9 (d, J = 5 Hz); 31P (CDCl3, 162 MHz) δ 24.4; HRMS calcd for C23H25BrP [M -
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Br]+ 411.0877, found 411.0867.
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3-((5-(Bromotriphenylphosphoranyl)pentyl)thio)propanoate (10): To a solution of methyl 3mercaptopropanoate (9, 2.44 g, 20.3 mmol) in 40 mL THF was added sodium hydride (0.77 g, 60%
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in mineral oil, 19.3 mmol) at 0 °C in three portions. Then 8 (5 g, 10.2 mmol) in 20 mL THF was
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added slowly to the reaction mixture at 0 °C. Then it was allowed to reach rt slowly. After 12 h at rt, it was concentrated at reduced pressure, and purified by flash chromatography, using 10%
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methanol/dichloromethane to yield the pure product (3.2 g, 59%): 1H NMR (CDCl3, 400 MHz) δ 7.89-7.77 (m, 9 H), 7.74-7.68 (m, 6 H), 3.90-3.82 (m, 2 H), 3.68 (s, 3 H), 2.73 (t, 2 H, J = 8 Hz),
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2.57 (t, 2 H, J = 8 Hz), 2.48 (t, 2 H, J = 7.2 Hz), 1.81-1.73 (m, 2 H), 1.70-1.55 (m, 4 H);
13
C
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NMR (CDCl3, 100 MHz) δ 172.2, 135.0 (d, J = 3 Hz), 133.7 (d, J = 10 Hz), 130.4 (d, J = 13 Hz), 118.4 (d, J = 86 Hz), 51.7, 34.7, 31.6, 29.4 (d, J = 16 Hz), 29.0, 27.0, 22.8 (d, J = 49 Hz), 22.3 (d, 31
P (CDCl3, 162 MHz) δ 24.4; HRMS calcd for C27H32O2PS [M - Br]+ 451.1861,
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J = 5 Hz);
found 451.1847.
(Z)-Methyl 3-(pentadec-5-en-14-yn-1-ylthio)propanoate (12): To a solution of methyl 3-((5(bromotriphenylphosphoranyl)pentyl)thio)propanoate (10, 1.8 g, 3.39 mmol) in 6.8 mL anhydrous THF was added NaOMe (0.37 g, 6.78 mmol) at rt. The solution turned yellow with a white suspension. After stirring vigorously for 5 min at rt, dec-9-ynal (11, 0.52 g, 3.39 mmol) was added drop wise at rt. The reaction mixture turned colorless with a white suspension and was quenched with saturated NH4Cl aqueous solution after stirring for 9 h at rt. The reaction mixture was extracted with Et2O (20 mL × 3), washed with brine (20 mL), dried with anhydrous
ACCEPTED MANUSCRIPT MgSO4, filtered, and concentrated at reduced pressure to get a pale yellow oil as the crude product. It was purified with flash chromatography using 10% EtOAc/hexane to yield the pure
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product (0.46 g, 42%): 1H NMR (CDCl3, 400 MHz) δ 5.41-5.30 (m, 2 H), 3.70 (s, 3 H), 2.79 (t, 2
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H, J = 7.2 Hz), 2.62 (t, 2 H, J = 7.2 Hz), 2.54 (t, 2 H, J = 7.2 Hz), 2.18 (dt, 2 H, J = 2.8, 6.8 Hz), 2.08-1.99 (m, 4 H), 1.95 (t, 1 H, J = 2.4 Hz), 1.66-1.27 (m, 14 H); 13C NMR (CDCl3, 100 MHz)
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δ 172.4, 130.3, 129.2, 84.7, 68.1, 51.8, 34.7, 32.1, 29.6, 29.1, 29.0, 28.9, 28.7, 28.5, 27.2, 27.0,
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26.7, 18.4; ESI-MS: 324.95 [M + H]+; HRMS calcd for C19H33O2S [M + H]+ 325.2203, found
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325.2191.
(Z)-Methyl 3-((13-(1-(2-(tosyloxy)ethyl)-1H-1,2,3-triazol-4-yl)tridec-5-en-1-yl)thio)propanoate
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(14): To a solution of (Z)-methyl 3-(pentadec-5-en-14-yn-1-ylthio)propanoate (12, 142 mg, 0.438 mmol) and 2-azidoethyl 4-methylbenzenesulfonate (13, 106 mg, 0.438 mmol) in CHCl3 (1
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mL) was added copper (I) iodide (8 mg, 0.044 mmol), followed by N,N-diisopropylethylamine
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(DIEA, 282 mg, 2.19 mmol) at rt. After stirring overnight, the reaction mixture was filtered through a short silica plug. It was rinsed with dichloromethane (100 mL), concentrated at
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reduced pressure, dissolved in MeCN, and purified with a pre-packed C18 column (30 g) using a gradient from 10% MeCN/H2O (0.1% TFA) to 90% MeCN/H2O (0.1% TFA) over 15 min with a flow of 35 mL/min. The eluent was monitored with a UV lamp at 230 nm and 254 nm. The fraction eluting at 15 min was collected and concentrated at reduced pressure to yield the pure product (178 mg, 72%). 1H NMR (CDCl3, 400 MHz) δ 7.68 (d, 2 H, J = 8 Hz), 7.48 (s, 1 H), 7.34 (d, 2 H, J = 8 Hz), 5.41-5.31 (m, 2 H), 4.65 (t, 2 H, J = 5.2 Hz), 4.42 (t, 2 H, J = 4.8 Hz), 3.71 (s, 3 H), 2.79 (t, 2 H, J = 7.2 Hz), 2.74 (t, 2 H, J = 8 Hz), 2.62 (t, 2 H, J = 7.2 Hz), 2.54 (t, 2 H, J = 7.2 Hz), 2.46 (s, 3 H), 2.08-2.00 (m, 4 H), 1.69-1.57 (m, 4 H), 1.48-1.28 (m, 10 H); 13C NMR (CDCl3, 100 MHz) δ 172.6, 145.7, 131.8, 130.3, 130.1, 129.2, 127.8, 67.3, 51.8, 49.8, 34.7,
ACCEPTED MANUSCRIPT 32.0, 29.6, 29.2, 29.1, 29.07, 29.06, 28.8, 27.2, 27.0, 26.7, 24.7, 21.6; ESI-MS: 566.80 [M + H]+; HRMS calcd for C28H44N3O5S2 [M + H]+ 566.2724, found 566.2711.
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(Z)-methyl 3-((14-bromotetradec-5-en-1-yl)thio)propanoate (18): To a solution of 10 (0.213 g, 0.4 mmol) in THF (9.4 mL, 0.2 M) was added sodium methoxide (22 mg, 0.4 mmol). After 10
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min at rt, aldehyde 17 (71 mg, 0.32 mmol) was added dropwise. After 1.5 h, the suspension was filtered through a short silica plug, rinsed with DCM, and concentrated at reduced pressure. The
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crude product was purified with 5% EtOAc/hexane on a silica column to yield the product (19)
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as colorless oil. The 1H NMR is consistent with published data (98 mg, 78%). 1H NMR (CDCl3, 400 MHz) δ 5.41-5.30 (m, 2 H), 3.73 (s, 3 H), 3.43 (t, 2 H, J = 6.8 Hz), 2.78 (dt, 2 H, J = 1.2, 8
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Hz), 2.62 (t, 2 H, J = 7.6 Hz), 2.54 (t, 2 H, J = 7.6 Hz), 2.07-1.99 (m, 4 H), 1.89-1.82 (m, 2 H), 1.64-1.56 (m, 2 H), 1.48-1.39 (m, 4 H), 1.35-1.27 (m, 8 H).
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(Z)-3-(pentadec-5-en-14-yn-1-ylthio)propanoic acid (15): To a solution of 12 (8.2 gm, 0.025
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mmol) in 1.25 mL EtOH was added aqueous KOH solution (1.25 mL, 0.2 N, 0.25 mmol) at rt stirred for 5 h at rt, and concentrated on a roto-evaporator. The crude product was purified with a
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C18 column to yield pure product (6.1 mg, 78%). 1H NMR (CDCl3, 400 MHz) δ 5.41-5.30 (m 2 H), 2.79 (dt, 2 H, J = 0.8, 8.0 Hz), 2.67 (dt, 2 H, J = 0.8, 8.0 Hz), 2.55 (t, 2 H, J = 7.2 Hz), 2.18 (dt, 2 H, J = 2.4, 7.2 Hz), 2.07-1.99 (m, 4 H), 1.94 (t, 1 H, J = 2.8 Hz), 1.65-1.27 (m, 15 H); ESIMS: 311.07 [M+H]. HRMS calcd for C18H29O2S [M - H] – 309.1887, found 309.1893. (Z)-3-((13-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)tridec-5-en-1-yl)thio)propanoic acid (6): A solution of (Z)-3-(pentadec-5-en-14-yn-1-ylthio)propanoic acid (15) (9.4 µmol) and 1-azido-2fluoroethane (16, 9.4 µmol) was added to a solution containing copper (I) iodide (1.8 mg, 9.4 µmol) and DIEA (8.2 µl, 47 µmol) in CHCl3. The reaction mixture was filtered through a short
ACCEPTED MANUSCRIPT silica plug, rinsed with dichloromethane, and concentrated at reduced pressure. It was then purified by HPLC to yield the pure product (2.6 mg, 70%): 1H NMR (CDCl3, 400 MHz) δ 5.45-
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5.31 (m, 2 H), 4.87 (t, 1 H, J = 4.8 Hz), 4.75 (t, 1 H, J = 4 Hz), 4.70 (t, 1 H, J = 4 Hz), 4.63 (t, 1
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H, J = 4.8 Hz), 2.95-2.90 (b, 1 H), 2.82 (dd, 2 H, J = 7.2, 14.4 Hz), 2.76 (t, 2 H, J = 7.6 Hz), 2.68 (t, 2 H, J = 7.2 Hz), 2.58 (t, 2 H, J = 7.2 Hz), 2.14-2.02 (m, 4 H), 1.83-1.46 (m, 7 H), 1.41-1.28
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(m, 8 H); HRMS calcd for C20H35FN3O2S [M + H]+ 400.2436, found 400.2423.
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being consistent with the published data [7].
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The cold standard of 18F-FTO was synthesized following the published procedure, with H-NMR
(Z)-3-((13-(1-(2-[18F]fluoroethyl)-1H-1,2,3-triazol-4-yl)tridec-5-en-1-yl)thio)propanoic
acid
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(18F-6): Cyclotron-produced [18F] fluoride (~ 22.2 GBq) was dried down under argon at 110 °C in a 3 mL glass vial containing Kryptofix 2.2.2 (10 mg), MeCN (0.8 mL), and aqueous solution
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of K2CO3 (4 mg in 0.2 mL). The residue was further azeotropically dried with MeCN (2 × 1 mL)
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at 110 °C under a continuous stream of argon gas. One mL of MeCN was added to the final
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[18F]fluoride residue. The resultant solution was then transferred to a solution of labeling precursor (2 to 4 mg) in 250 µl MeCN. The V-vial was sealed and heated at 75 °C for 10 min under microwave heating conditions. After cooling on ice for 1 min, silica TLC plates were spotted with the reaction mixture and developed with ethyl acetate to determine the crude incorporation yields. The hydrolysis of the ester intermediate was carried out with aqueous KOH solution (0.15 mL, 0.2 N) at 75 °C for 5 min with microwave-assisted heating, followed by acidifying with 0.15 mL glacial acetic acid after cooling the mixture on ice for 1 min. The crude mixture was purified by semi-preparative HPLC. The fraction containing the desired product was diluted with 50 mL water and passed across a C18 SepPak cartridge. The cartridge was washed with 10 mL water and then eluted with 1 mL ethanol. The volume was concentrated at 110 °C
ACCEPTED MANUSCRIPT under a continuous stream of argon gas to a final volume of approximately 200 µL and then diluted with 1% BSA (lipid free) in saline for cell or animal studies.
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F-FTO (18F-4): The published protocol was followed for the synthesis of 18F-FTO (18F-4) with
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7% radiochemical yield (non-decay corrected), and > 90% radiochemical purity [7].
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Biodistribution and Folch-type extraction:
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The biodistribution studies were carried out as previously described [20]. The tracers (1.85 MBq) were injected into CD-1 mice (n = 4 for 18F-6 or 7 for 18F-4 per group) via the tail vein. The mice
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were sacrificed at 30 min or 2 h p.i., and selected organs were removed, weighed, and counted on a gamma counter (Perkin Elmer Wizard2, Waltham, MA). Groups of mice (n = 4) were injected
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intraperitoneally with etomoxir (40 mg/kg, in saline) 3 h before injection of the tracer and were
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sacrificed at 2 h post-injection. Folch-type extraction was done following the published protocol.
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PET/CT imaging:
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Small animal PET/CT imaging was performed with as previously described [20]. Briefly, the radiotracers (3.7 MBq) were injected via tail vein and the mice (n = 4) were imaged at 2 h p.i. Static imaging was performed on an Inveon PET/CT scanner [21, 22] with 10 min PET scanning followed by 5 min CT. The mice were anesthetized with isoflurane. Inveon Research Workplace (IRW) software (Siemens AG, Germany) was used for co-registration of PET/CT images and quantification of regions of interest (ROI). PET/CT images were reconstructed with maximum a posteriori (MAP), 3D ordered-subset expectation maximization (OSEM3D), 2D ordered-subset expectation maximization (OSEM2D), and filtered back projection (2DFBP). Standard uptake values (SUVs) were generated by measuring ROI from PET/CT images and calculated with the formula: SUV = [MBq/mL] × [animal weight (g)]/injected dose [MBq].
ACCEPTED MANUSCRIPT Statistical Methods. All of the data are presented as mean ± standard deviation. Two-tailed unpaired t-tests were performed using GraphPad Prism 5.04.
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Results
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Synthesis of labeling precursors, cold standards, and radiolabeling of 18F-4 and 18F-6: The dibromide 7 reacted with triphenylphosphine to yield bromide 8 in 65% yield (Scheme 1). 8
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was then reacted with compound 9 to generate compound 10 in 59% yield. Dec-9-ynal (11) was prepared from commercially available dec-9-yn-1-ol in 81% yield as the aldehyde for the Wittig
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reaction. This aldehyde (11) was converted to (Z)-methyl 3-(pentadec-5-en-14-yn-1ylthio)propanoate (12) via a (Z)-selective Wittig reaction in 42% yield. The propanoate 12 was
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reacted with 2-azidoethyl 4-methylbenzenesulfonate (13) to yield the desired (Z)-methyl 3-((13-
substrate for
18
F-labeling.
(14)
as
the
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(1-(2-(tosyloxy)ethyl)-1H-1,2,3-triazol-4-yl)tridec-5-en-1-yl)thio)propanoate 18
F-labeling was performed under microwave assisted heating for 10
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min at 75 °C. The intermediate methyl ester was then saponified with KOH under microwave
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assisted heating for 5 min at 75 °C to yield the final product, which was purified on reversedphase HPLC using a C18 column. The non-decay corrected radiochemical yield of 18F-6 was 17% (non-decay corrected RCY), with > 95% radiochemical purity (based upon radio-HPLC analysis). The cold standard 6 was synthesized from methyl ester 12 by saponification to get the acid 15 in 78% yield, which reacted with 2-fluoroethyl azide 16 to generate 6 in 70% yield. The synthesis of the precursor for
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F-labeling of FTO (compound 18) involved a (Z)-selective
Wittig reaction between the bromoaldehyde 17 and the methyl ester 10, which generated the desired bromide 18 in 78% yield (Scheme 2). The
18
F-labeling of 18 was done following
ACCEPTED MANUSCRIPT published procedure, resulting in the 18F-FTO (18F-4) in 7% non-decay-corrected RCY. The cold standard 4 was synthesized following published literature from 18 [7].
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In order to evaluate
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PET/CT imaging and biodistribution:
F-6 as a myocardial imaging agent and determine its specificity for β-
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oxidation, PET/CT scanning and biodistribution studies of 18F-6 were performed in fasted CD-1
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mice to determine the extent of myocardial uptake of the tracer. Blocking of carnitine palmitoyltransferase I (CPT-I) was performed following pre-treatment with etomoxir in fasted
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mice. The PET/CT imaging and biodistribution studies of the oleate analog
18
F-6 revealed high
liver uptake (18.4 ± 6.2 %ID/g) with excretion through the gallbladder into the intestine, 18
F-6 into the liver.
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presumably due to the high lipophilicity of the tracer or active transport of
Biodistribution studies show that the heart uptake of 18F-6 was moderate (0.94 ± 0.28 %ID/g at
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0.5 h p.i.), and decreased at 2 h p.i. (0.48 ± 0.05 %ID/g). The heart-to-muscle ratio of 18F-6 was 18
F-6 PET visualized myocardial tissue in
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2.05 ± 0.39 at 0.5 h p.i. and 1.85 ± 0.33 at 2 h p.i.
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fasted mice at 30 min post-injection (Figure 3). To determine whether the observed uptake was mediated by CPT-I transport, the CPT-I inhibitor etomoxir was injected to the mice 3 h before injection of
18
F-6, and organs were harvested (2 h p.i.). The heart uptake of
18
F-6 in etomoxir-
treated mice decreased relative to the control group (0.34 ± 0.07 %ID/g vs 0.48 ± 0.05 %ID/g, P = 0.0027, Table 1), which suggests that the uptake is mediated at least in part by CPT-I mediated mitochondrial uptake. To determine the metabolic fate of the
18
F-fatty acid, a group of mice were pre-injected with
etomoxir 3 h prior to the injection of the radiotracer and the hearts were harvested (30 min p.i.), and subjected to a Folch extraction (Figure 2). The highest activity fraction was the organic
ACCEPTED MANUSCRIPT phase, which was increased upon pre-treatment of etomoxir (0.66 ± 0.02 %ID/g vs. 0.76 ± 0.02 %ID/g, n = 4, P = 0.0068). At the same time, the aqueous fraction decreased upon etomoxir
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treatment (0.15 ± 0.01 %ID/g vs. 0.06 ± 0.01 %ID/g, n = 4, P = 0.0002). The pellet activity did
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not change significantly upon treatment of etomoxir (0.19 ± 0.02 %ID/g vs. 0.18 ± 0.04, n = 4, P
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= 0.66).
To our knowledge, there are no reported investigations of
18
F-4 (FTO) in mice; therefore, a
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biodistribution of 18F-4 in fasted CD-1 mice was also performed for the purpose of comparison
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to 18F-6 (Table 1). The heart uptake of 18F-4 at 30 min p.i. was 1.8 ± 0.61 %ID/g, decreasing to 1.7 ± 0.27 %ID/g at 2 h p.i. Etomoxir decreased the heart uptake of 18F-4 to 0.82 ± 0.23 %ID/g at
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2 h p.i. The high bone uptake of 18F-4 (3.19 ± 2.31 %ID/g at 30 min p.i., 7.5 ± 1.82 %ID/g at 2 h p.i.) indicated significant defluorination in vivo, which was not observed for
18
F-6 (0.80 ±
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0.54 %ID/g, P = 0.0233, at 30 min p.i.; 0.60 ± 0.23 %ID/g, P < 0.0001, at 2 h p.i.). PET imaging
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with 18F-4 in a nude mouse showed clear myocardial uptake (SUV = 1.1) and high bone uptake
Discussion
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as the result of in vivo defluorination (Figure 3).
Fatty acid metabolism is the main source of energy for the myocardium. Therefore, metabolically trapped PET tracers that are sensitive to changes in FAO could determine changes in myocardial FA metabolism. Quantification of these changes in myocardial diseases with altered FA metabolism such as diabetes mellitus or myocardial ischemia could have a major impact on therapeutic regimens [23]. While numerous
18
F-FAs have been introduced for FAO
imaging with promising results, there is still a need to develop new tracers for probing the FAO
ACCEPTED MANUSCRIPT mechanisms [15, 24, 25]. To date, FA analogs with 4-thia substitutions appear to be the most sensitive analogs, because their Co-A esters are substrates for CPT-I mediated transesterification
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prior to transport into the mitochondria [24]. Additionally, radiofluorinated oleate analogs
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showed superior imaging properties in the rat heart relative to radiofluorinated palmitate-based agents [8, 16, 24]. For these reasons, we chose 4-thia-oleate to contain the resultant triazole by
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utilizing click chemistry.
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Click chemistry is an efficient way to build bioactive small molecules and could enable modular
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synthesis of a library of FA analogs to probe myocardial FAO. Therefore, we adopted the copper-catalyzed alkyne azide cycloaddition reaction to synthesize a clicked 4-thia-oleate analog
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(18F-6) in order to explore the influence of the -terminal modification of the fatty acid with the triazole group. We also synthesized
18
F-FTO for comparison, because it has been previously
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shown in rat heart to have good sensitivity to FAO and have high target tissue uptake [24].
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In the process of working out the chemistry of 14, we developed a novel and convergent
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synthesis of the precursor for FTO (18), making it amenable for further structural modifications that might improve the pharmacokinetics, sensitivity to FAO, and the resultant image quality. We improved the synthesis of 18 by reducing the synthetic steps from 9 to 3 using readily available starting materials.
18
F-FTO was employed as a reference tracer for the newly
synthesized clicked oleate analog (Scheme 2) [24].
18
F-FTO shows promise as a myocardial
imaging tracer in spite of severe enzymatic defluorination in rodents; however, defluorination of -terminal FAs is not as severe in swine as in rodents [7]. When
18
F is incorporated at -n
carbons, the enzymatic defluorination is diminished, but it generates a stereocenter, resulting in an inseparable racemic mixture as the product [28]. For example, relative to FTO (2 h bone, ~7 %ID/g), FTHA, which is fluorinated at the -4 position, showed much lower bone uptake in
ACCEPTED MANUSCRIPT mice (2 h bone, 3.8 %ID/g) [15]. Previous work has shown that incorporation of a triazole into the aliphatic chain apparently reduces hepatic enzyme-mediated defluorination for the clicked
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FTHA (1 h bone, 1.2 %ID/g); however, there was no direct comparison to -fluorinated FAs
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[18]. When compared to 18F-FTO, 18F-6 showed relatively low bone uptake (7.5 ± 1.8 vs 0.60 ±
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0.23 %ID/g), confirming that the triazoles do inhibit hepatic enzymatic defluorination and that the inhibition is not dependent of the position of the triazole in the aliphatic chain.
18
F-4 and
18
F-6 in mouse heart in comparison to that of
F-FA analogs in mouse as well as in rat heart. For example, the uptake of
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other
18
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There are major differences between
18
F-6 was
sufficient to visualize the mouse myocardial tissue with PET, and was about half that of FTO 18
F-6 or
18
F-4
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(18F-4). In contrast, clicked-FHA (18F-3) demonstrated more uptake than either
(%ID/g = 3.16 ± 0.36, 0.5 h) [18]. Clicked-FHA (3) may be considered an analog of 14(R,S)-
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[18F]fluoro-6-thia-hepatadecanoic acid (FTHA), which showed extremely high uptake in mouse
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heart (%ID/g = 29.2 ± 0.3, 1 h) [15]; however, in rat heart, the uptake of FTHA dropped significantly (0.96 ± 0.18 %ID/g) [29]. The first step towards metabolism and intracellular
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trafficking of FAs is molecular transport across the across the sarcolemma into the cytoplasm of the myocardial cell. Therefore, these differences in myocardial uptake between species are likely attributable to the expression levels of FA transport proteins and transport kinetics of these FAs. FA transport proteins are a class of integral membrane proteins that mediate transport of FAs across the plasma membrane. When FATP6 was transfected in HEK293 cells, which do not express any FATPs or other transport proteins, FATP6 mediated transport of palmitate was 4fold that of oleate [30]. FATP6 is highly expressed in the mouse myocardial tissue compared to low expression of FATP1 [30]. However, in rat heart, FATP1 is expressed at higher levels than FATP6 [31]. The incorporation of fluorine and sulfur atoms, while being reasonable isoteres of
ACCEPTED MANUSCRIPT hydrogen and carbon respectively, could conceivably impair binding efficiency and transport. Moreover, the incorporation of a triazole in 5 could be deleterious to the molecular recognition 18
F-6 may
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by the FATPs leading to diminished transport rates. Thus, the structural features of
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account for the observed diminished myocardial uptake relative to FTO and clicked-FHA [32]. Taken together, the aforementioned uptakes of FTHA (18F-5), FTO (18F-4), clicked-FHA (18F-3),
18
F-6 revealed high uptake in the liver, kidney, and heart, consistent with the
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PET imaging of
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and 18F-6 could easily be influenced by the transporter levels and rates of transport.
than that of FTO
18
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biodistribution data. While the SUV of 18F-6 in the heart is 0.26 ± 0.12 (n = 4), which is lower F-4, it is still sufficient for visualization of the heart, albeit with increased
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background. Based on the biodistribution data, the heart-to-muscle ratio of p.i.) is lower than that of clicked-FHA
18
18
F-6 (2.05 at 0.5 h
F-3 (4.79 at 0.5 h p.i.), which may be due to the
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preferential expression of FATPs favoring the transportation of this heptanoic acid analog rather
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than oleate in mouse heart. The higher heart-to-muscle ratio of FTO relative to that the terminal triazole group interfered with the transportation of
18
F-6 suggests
F-6 into the myocardial
cells. Of note, the relatively high heart-to-muscle and heart-to-blood ratios of
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18
18
F-4 in rat were
not reproduced in mouse, which suggests distinct molecular processes are responsible for uptake between species and they are not confined to those processes related to -oxidation alone [24]. To determine whether the observed myocardial uptake of 18F-6 was mediated by -oxidation, the CPT-I inhibitor etomoxir was pre-administered with 18F-6. CPT-I is an enzyme that catalyzes the trans-esterification of palmitate and other fatty acid CoA esters with carnitine for subsequent transport into the mitochondria [33]. Myocardial uptake of 18F-6 was partially blocked by CPT-I inhibition, suggesting that the uptake of the tracer was due in part to its oxidization in the mitochondria (Table 1). Etomoxir was reported to reduce the CPT-I mediated -oxidation of
ACCEPTED MANUSCRIPT FTO by 50%, while we observed only a 29% reduction of 5 (Table 1) [24]. The remaining fraction of 18F-6 is presumably esterified into cellular lipids, consistent with the observed Folch
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extraction results showing the largest fraction of activity in the lipid fraction with low amounts
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of oxidized FA incorporated into cellular proteins (Figure 2). Etomoxir did not reduce the amount of radioactivity associated with the protein fraction [34]. Based on these data,
18
F-6
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would not be sensitive to reduced metabolism in myocardial tissues.
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The incorporation of structural features in FAs to block -oxidation and trap the metabolite(s) in
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the myocardial cells must be balanced against the need to maintain affinity and trafficking of the FA by proteins involved in the metabolic process. Our results suggest that molecular recognition
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of FAs by membrane associated and cytosolic proteins is an important determinant of uptake. Therefore, future work will entail moving the triazole group away from the -terminus toward
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the acyl terminus in an attempt to increase the myocardial uptake of the clicked oleate analogs.
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Previous work demonstrated that moving polar modifications away from the -terminus increased myocardial uptake of 99mTc-labeled fatty acid analogs [35]. Since small changes in the
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structure of the fatty acid backbone could have significant impact on the myocardial uptake, it is foreseeable that an instructive structure-activity relationship could be obtained with this new synthetic route that allows one to generate a library of clicked fatty acid analogs. In addition to their utility as potential myocardial PET imaging agents, the generation of such a library of 18Flabeled fatty acids that retain affinity for their transport proteins and are substrates for oxidation could be used to probe a variety of other disease conditions and organ functionalities including hepatic functions [36, 37], diabetes [38, 39], neuroinflammation, and a range of tumor types that utilize fatty acids as a major energy source [41, 42].
ACCEPTED MANUSCRIPT Conclusion Here we report a novel clicked oleic acid analog (14) that was synthesized via click chemistry
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convergently in good yield (16% overall), and was efficiently labeled with 18F-fluoride (90 min; 17% non-decay corrected radiochemical yield). We also optimized the synthetic route to FTO
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requiring only three steps from commercially available materials, which is a significant improvement over the former synthetic route (nine steps) that required extensive protecting
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group manipulations. The incorporation of a triazole group (18F-6) significantly reduced the in
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vivo defluorination compared with FTO (18F-4). While the uptake of analog 18F-6 was sufficient for visualizing the myocardium in mice, the preliminary metabolism data suggest only a fraction
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of the uptake (~29%) was due to fatty acid beta-oxidation. Studies are underway to explore the
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uptake/oxidation mechanism and kinetics. Acknowledgements
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The authors thank Joseph Latoche, Kathryn Day Elizabeth, Nicole DeBlasio, and Jalpa Modi for
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the excellent technique assistance. The small animal PET/CT imaging at UPCI was supported in part by P30CA047904 (UPCI CCSG).
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Table 1. Tissue biodistribution of 18F-4 and 18F-6 in fasted CD-1 mice Mean ± SD %ID/g of 18F-4
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Mean ± SD %ID/g of 18F-6 30 min
2h
2 h, etomoxir a
30 min
2h
2 h, etomoxir a
blood
0.91 ± 0.22
0.43 ± 0.09
0.24 ± 0.13
0.64 ± 0.19
0.24 ± 0.03
0.33 ± 0.17
lung
1.45 ± 0.51
0.57 ± 0.09
0.52 ± 0.08
0.78 ± 0.28
0.40 ± 0.09
0.37 ± 0.11
liver
18.37 ± 6.19
9.87 ± 3.22
6.45 ± 0.58
2.96 ± 1.38
1.59 ± 0.19
1.36 ± 0.15
kidney
7.34 ± 2.33
5.92 ± 2.73
4.27 ± 1.31
3.96 ± 1.92
2.38 ± 0.23
0.89 ± 0.29
muscle 0.46 ± 0.14
0.27 ± 0.06
0.17 ± 0.06
0.37 ± 0.22
0.26 ± 0.07
0.23 ± 0.09
heart
0.94 ± 0.28
0.48 ± 0.05b
0.34 ± 0.07b
1.8 ± 0.61
1.69 ± 0.27b
0.82 ± 0.23b
brain
0.34 ± 0.12
0.18 ± 0.04
0.11 ± 0.03
0.22 ± 0.14
0.2 ± 0.04
0.18 ± 0.03
Bone
0.80 ± 0.54
0.60 ± 0.23
0.82 ± 0.33
3.19 ± 2.31
7.5 ± 1.82
8.52 ± 2.14
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tissue
Mice were pretreated with etomoxir 3 h before injecting 18F-4 or 18F-6, n = 4.
b
P = 0.0027.
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a
ACCEPTED MANUSCRIPT Legends for figures
F-FTO (18F-4); 18F-FTHA (18F-5); 18F-clicked FTO (18F-6).
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18
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Figure 1. Structures of 123I-BMIPP (123I-1); 11C-Palmitate (11C-2); and 18F-Clicked FHA (18F-3);
Figure 2. Representative midventricular trans-axial PET images of 18
F-4 in fasted nude mouse
F-6 in fasted CD-1 mouse at 30 minutes p.i. with different scale bars due to significantly
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and
18
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different SUV in the heart. SUV of 18F-4 in the heart is 1.1 (n = 1). SUV of 18F-6 in the heart is 0.26 ± 0.12 (n = 4). The location of spine and fat is identified based on the co-registration of CT
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images. The radiotracers (3.7 MBq) were injected via tail vein and the mice were imaged at 2 h p.i. Static imaging was performed on an Inveon PET/CT scanner with 10 min PET scanning
18
F-6 and its metabolites in the hearts of fasted mice by Folch-
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Figure 3. Total distribution of
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followed by 5 min CT. The mice were anesthetized with isoflurane during the time of scanning.
type extraction, with and without etomoxir pre-treatment. (Statistical significant difference: *P <
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0.05, n = 4, 7 for etomoxir pre-treated group and untreated group respectively).
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Legends for schemes
Scheme 1. Synthesis of 18F-clicked-FTO (18F-6) and the cold standard, 19F-clicked-FTO (6). Scheme 2. Improved Synthesis of 18F-FTO (18F-4).
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ACCEPTED MANUSCRIPT
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F-FTO (18F-4); 18F-FTHA (18F-5); 18F-clicked FTO (18F-6).
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18
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Figure 1. Structures of 123I-BMIPP (123I-1); 11C-Palmitate (11C-2); and 18F-Clicked FHA (18F-3);
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ACCEPTED MANUSCRIPT
18
F-4
F-6
Heart
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3E-1 SUV
18
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Heart
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Spine
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Fat
and
18
1E-1 SUV
18
F-4 in fasted nude mouse
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Figure 2. Representative midventricular trans-axial PET images of
Fat
F-6 in fasted CD-1 mouse at 30 minutes p.i. with different scale bars due to significantly
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different SUV in the heart. SUV of 18F-4 in the heart is 1.1 (n = 1). SUV of 18F-6 in the heart is
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0.26 ± 0.12 (n = 4). The location of spine and fat is identified based on the co-registration of CT images. The radiotracers (3.7 MBq) were injected via tail vein and the mice were imaged at 2 h
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p.i. Static imaging was performed on an Inveon PET/CT scanner with 10 min PET scanning followed by 5 min CT. The mice were anesthetized with isoflurane during the time of scanning.
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0.6 0.4
*
0.2 0.0
Pellets
Aqueous
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Organic
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0.5 h 0.5 h+etomoxir
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0.8
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fraction of total ctivity
1.0
18
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Figure 3. Total distribution of
F-6 and its metabolites in the hearts of fasted mice by Folch-
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type extraction, with and without etomoxir pre-treatment. (Statistical significant difference: *P <
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0.05, n = 4, 7 for etomoxir pre-treated group and untreated group respectively).
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RI P
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Scheme 1. Synthesis of 18F-clicked-FTO (18F-6) and the cold standard, 19F-clicked-FTO (6).
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Scheme 2. Improved synthesis of 18F-FTO.