Bioconjugate Chem. 2007, 18, 1332−1337
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TECHNICAL NOTES 99mTc(CO)
3-15-[N-(Acetyloxy)-2-picolylamino]pentadecanoic
Acid: A Potential
Radiotracer for Evaluation of Fatty Acid Metabolism Byung Chul Lee,†,§ Dong Hyun Kim,‡ Jae Hak Lee,† Hyun Ju Sung,† Yearn Seong Choe,*,‡ Dae Yoon Chi,† Kyung-Han Lee,‡ Yong Choi,‡ and Byung-Tae Kim‡ Department of Chemistry, Inha University, 253 Yonghyundong, Namgu, Inchon 402-751, Korea, and Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Kangnam-ku, Seoul 135-710, Korea. Received September 23, 2006; Revised Manuscript Received January 20, 2007
99mTc(CO)
99mTc3-15-[N-(Acetyloxy)-2-picolylamino]pentadecanoic acid (1a) was prepared by incorporating [ (CO)3]+ into 15-[N-(hydroxycarbonylmethyl)-2-picolylamino]pentadecanoic acid (2a). The overall radiochemical yield of 1a after HPLC purification was 60-63%. Radiotracer 1a was found to be chemically stable when incubated in human plasma for 4 h at 37 °C. Tissue distribution studies showed that high radioactivity accumulated in the heart with rapid clearance. The maximum heart-to-blood uptake ratio was 1.87 at 5 min after a tail-vein injection. Radioactive metabolites were analyzed in urine samples of mice and corresponded to a 9.3:1 ratio of 99mTc(CO)3-5-[N-(acetyloxy)-2-picolylamino]pentanoic acid (1b) to 99mTc(CO)3-3-[N-(acetyloxy)-2-picolylamino]propionic acid (1c), indicating that 1a is mainly metabolized to 1b via β-oxidation in the body. These results suggest that 1a is a promising radiotracer for evaluation of fatty acid metabolism in myocardium.
INTRODUCTION Fatty acids are important substrates for adenosine triphosphate (ATP) production in myocardium. In the fasting state, fatty acid oxidation is enhanced and anaerobic glycolysis is suppressed, whereas in the nonfasting state, fatty acid oxidation decreases and glucose is utilized as an energy source (1). In myocardium, long-chain fatty acids are usually metabolized via β-oxidation, which degrades fatty acids by removing two-carbon units, and only a small fraction of the fatty acids is stored in the intracellular lipid pool as triglyceride (2-4). Disturbances in fatty acid metabolism often reflect myocardial damage, such as myocardial ischemia and cardiomyopathy; for example, fatty acid metabolism is impaired due to a reduced oxygen supply during myocardial ischemia (1, 5). Therefore, radiolabeled fatty acid analogs have been investigated for evaluating fatty acid metabolism in myocardium using single-photon emission computed tomography (SPECT) and positron emission tomography (PET). 1-[11C]Palmitate, which reflects the metabolism of its natural counterpart, showed biexponential clearance, indicating rapid washout resulting from β-oxidation and slow washout due to slow turnover of the intracellular lipid pool (5, 6). ω-[11C]Palmitate showed greater heart uptake than 1-[11C]palmitate due to its reduced metabolism (7). Several 123I-labeled fatty acid analogs have been extensively studied, i.e., 17-[123I]iodoheptadecanoic acid, 15-(p-[123I]iodophenyl)pentadecanoic acid (IPPA), 15-(p-[123I]iodophenyl)-3methylpentadecanoic acid (BMIPP), and 15-(p-[123I]iodophenyl)3,3-dimethylpentadecanoic acid (DMIPP) (8-10). [123I]BMIPP * Corresponding author. Tel: +82-2-3410-2623. Fax: +82-2-34102639. E-mail:
[email protected]. † Inha University. ‡ Sungkyunkwan University School of Medicine. § Present address: Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea.
and [123I]DMIPP showed prolonged myocardial retention versus [123I]IPPA, because methyl branching slowed their myocardial metabolism. Due to its lower liver uptake than [123I]DMIPP, [123I]BMIPP showed promise for the diagnosis of various myocardial diseases (11). 16-[18F]Fluoropalmitic acid (FPA) and methylbranched ω-[18F]fluoroheptadecanoic acids at C3 (3-MFHA) or C5 (5-MFHA) have also been investigated, and 5-MFHA showed an initial myocardial uptake and a retention intermediate between those of FPA and 3-MFHA (12). 14-[18F]Fluoro-6thiaheptadecanoic acid, which was designed for prolonged myocardial retention, was found to be similar to [123I]BMIPP in terms of myocardial uptake and retention (13, 14). 99mTc-labeled fatty acid analogs are attractive due to ready availability and the nearly ideal physical properties of 99mTc (141 keV, t1/2 ) 6 h) (15, 16). Early attempts to develop 99mTclabeled long-chain fatty acid analogs included the preparation of 99mTc-labeled fatty acid analogs containing a strong chelating group, such as DTPA or EDTA (17, 18). However, poor accumulation of these radiotracers in myocardium initiated the developments of more specific radiotracers for in vivo studies of fatty acid metabolism. During these developments, 99mTc cores were successfully coordinated to the ω-positions of fatty acid analogs, but radiolabeled fatty acid analogs showed poor myocardial uptake (18-21). Recently, the Magata group found that [99mTc]MAMA-hexadecanoic acid (HDA) shows high initial heart uptake and subsequent rapid clearance, and that it is metabolized to [99mTc]MAMA-butyric acid via β-oxidation in the body (22). Over the past decade, small 99mTc cores have been developed, including 99mTc(CO)3-(cyclopentadienyl) (2325), 99mTc(CO)3-[N,N-(diacetyloxy)amino], 99mTc(CO)3-[NR(histidinyl)], 99mTc(CO)3-[N-(acetyloxy)-2-picolylamino] cores, and others (26-28). [99mTc(CO)3(H2O)3]+ is an attractive precursor for the introduction of the small [99mTc(CO)3]+ into biomolecules (26, 27, 29). As part of a strategy to develop 99mTc(CO) -labeled long-chain fatty acid analogs, the N-(hy3
10.1021/bc060299w CCC: $37.00 © 2007 American Chemical Society Published on Web 05/16/2007
Technical Notes
droxycarbonylmethyl)-2-picolylamino group can be labeled with [99mTc(CO)3(H2O)3]+ to form a 99mTc(CO)3-[N-(acetyloxy)-2picolylamino] core, an ideal tridentate chelator. In this work, we prepared 99mTc(CO)3-[N-(acetyloxy)-2picolylamino]pentadecanoic acid (1a) by the facile incorporation of [99mTc(CO)3]+ into a N-(hydroxycarbonylmethyl)-2-picolylamino fatty acid analog and evaluated its potential as a substrate for fatty acid metabolism. The β-oxidation products of 1a were compared with the proposed radioactive metabolite standards, 99mTc(CO) -5-[N-(acetyloxy)-2-picolylamino]pentanoic acid (1b) 3 and 99mTc(CO)3-3-[N-(acetyloxy)-2-picolylamino]propionic acid (1c).
EXPERIMENTAL SECTION Materials and Methods. Chemicals and solvents were obtained from Sigma-Aldrich (St. Louis, MO), and HPLC solvents were obtained from J. T. Baker (Phillipsburg, NJ). 1H NMR spectra were obtained using a Varian Unity Inova 500NB (500 MHz) spectrometer (Palo Alto, CA) at the Cooperative Center for Research Facilities, Sungkyunkwan University (Suwon, Korea), and a Varian Gemini-400 (Palo Alto, CA) at Inha University, and 13C NMR spectra were obtained at 100 MHz. Chemical shifts (δ) are reported as parts per million (ppm) downfield of an internal tetramethylsilane standard. Fast atom bombardment (FAB) mass spectra were obtained using a JMS700 Mstation (JEOL, Ltd., Tokyo, Japan) at the Korea Basic Science Institute (Seoul, Korea). HPLC was carried out using a Thermo Separation Products System (Fremont, CA) equipped with a semipreparative column (Alltech Econosil silica gel, 10 µ, 10 × 250 mm) or an analytical column (YMC-Pack C18, 5 µ, 4.6 × 250 mm). Eluant was simultaneously monitored using a UV detector (254 nm) and a NaI(Tl) radioactivity detector. TLC was performed on Merck F254 silica plates, which were analyzed using a Bioscan radio-TLC scanner (Washington, DC). Na99mTcO4 was eluted on a daily basis from a 99Mo/99mTc generator (Samyoung Unitech, Co., Ltd., DuPont Pharmaceuticals Co., and Daiichi Radioisotope Labs). Radioactivity was measured in a dose calibrator (Biodex Medical Systems, Shirley, NY) and tissue radioactivity in a Wallac automated γ counter (Boston, MA). All animal experiments were performed in compliance with the rules of the Samsung Medical Center Laboratory Animal Care, which comply with NIH guidelines. Synthesis of tert-Butyl 2-Picolylamino-N-acetate (3). 2-Picolylamine (946 µL, 9.25 mmol) was added to tert-butyl bromoacetate (1.7 mL, 12.03 mmol) in CH3CN (10 mL). The reaction mixture was then stirred under N2 at room temperature overnight. At the end of the reaction, solvent was removed in vacuo, and residue was extracted with dichloromethane (30 mL × 3), washed with water, and dried over anhydrous Na2SO4. Flash column chromatography (2:1 hexane-ethyl acetate) gave 3 (1.48 g) as a yellow oil in 72% yield: 1H NMR (CDCl3, 500 MHz) δ 8.57 (d, 1H, J ) 5.0 Hz), 7.68-7.65 (m, 1H), 7.35 (d, 1H, J ) 5.5 Hz), 7.19-7.17 (m, 1H), 3.96 (s, 2H), 3.39 (s, 2H), 1.48 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 171.2, 159.2, 149.1, 136.2, 121.8, 121.7, 80.8, 54.4, 51.0, 27.8; MS (FAB) m/z 223 (M+ + H); HRMS calcd for C12H19N2O2 223.1447, found 223.1451. Synthesis of Methyl 15-[N-(tert-Butyloxycarbonylmethyl)2-picolylamino]pentadecanoate (4a). Compound 3 (300 mg, 1.35 mmol) was dissolved in CH3CN (12 mL), and to this solution was added DIEA (1.18 mL, 6.75 mmol). After stirring for 30 min at room temperature, methyl 15-bromopentadecanoate (543 mg, 1.62 mmol) was added dropwise. The reaction mixture was then stirred at 75 °C overnight, extracted with dichloromethane (30 mL × 3), washed with water, and dried over anhydrous Na2SO4. Flash column chromatography (4:1 hexane-ethyl acetate) afforded 4a (521 mg) as a light yellow
Bioconjugate Chem., Vol. 18, No. 4, 2007 1333
oil in 81% yield: 1H NMR (CDCl3, 400 MHz) δ 8.52 (dd, 1H, J ) 2.0, 1.6 Hz), 7.65 (t, 1H, J ) 8.0 Hz), 7.54 (d, 1H, J ) 7.6 Hz), 7.14 (t, 1H, J ) 6.0 Hz), 3.90 (s, 2H), 3.66 (t, 3H, J ) 7.6 Hz), 3.29 (s, 2H), 2.62 (t, 2H, J ) 7.6 Hz), 2.30 (t, 2H, J ) 7.6 Hz), 1.64-1.58 (m, 2H), 1.51-1.41 (m, 9H), 1.31-1.18 (m, 22H); 13C NMR (CDCl3, 100 MHz) δ 174.0, 170.1, 160.0, 148.8, 136.1, 122.7, 121.6, 80.4, 60.3, 55.9, 54.1, 51.1, 33.9, 29.4; MS (FAB) m/z 477 (M+ + H); HRMS calcd for C28H49N2O4 477.3693, found 477.3697. Synthesis of Methyl 5-[N-(tert-Butyloxycarbonylmethyl)2-picolylamino]pentanoate (4b). Compound 3 (250 mg, 1.12 mmol) was dissolved in CH3CN (7 mL), and to this solution was added DIEA (980 µL, 5.63 mmol). After stirring for 30 min at room temperature, methyl 5-bromopentanoate (240 µL, 1.69 mmol) was added dropwise. The remainder of the procedure was as described above. Flash column chromatography (2:1 hexane-ethyl acetate) gave 4b (280 mg) as a light yellow oil in 74% yield: 1H NMR (CDCl3, 500 MHz) δ 8.558.53 (m, 1H), 7.67 (td, 1H, J ) 8.0, 2.0 Hz), 7.55 (d, 1H, J ) 7.6 Hz), 7.18-7.15 (m, 1H), 3.93 (s, 2H), 3.66 (s, 3H), 3.31 (s, 2H), 2.68 (t, 2H, J ) 7.6 Hz), 2.30 (t, 2H, J ) 7.6 Hz), 1.681.62 (m, 2H), 1.56-1.50 (m, 2H), 1.48 (s, 9H); MS (FAB) m/z 337 (M+ + H); HRMS calcd for C18H29N2O4 337.2128, found 337.2132. Synthesis of Methyl 3-[N-(tert-Butyloxycarbonylmethyl)2-picolylamino]propionate (4c). Compound 3 (400 mg, 1.75 mmol) was dissolved in CH3CN (12 mL), and to this solution was added NaHCO3 (453.6 mg, 5.40 mmol). After stirring for 30 min at room temperature, methyl 3-bromopropionate (257 µL, 2.34 mmol) was added dropwise. The mixture was then stirred at 60 °C overnight. Solvent was removed in vacuo, and the residue was extracted with dichloromethane (30 mL × 3), washed with water (30 mL × 3), and dried over anhydrous Na2SO4. Flash column chromatography (1:1 hexane-ethyl acetate) gave 4c (320 mg) as a yellow oil in 58% yield: 1H NMR (CDCl3, 500 MHz) δ 8.55-8.54 (m, 1H), 7.67 (td, 1H, J ) 8.0, 2.0 Hz), 7.50 (d, 1H, J ) 8.0 Hz), 7.19-7.16 (m, 1H), 3.99 (s, 2H), 3.66 (s, 3H), 3.57 (s, 2H), 3.08 (t, 2H, J ) 5.0 Hz), 2.53 (t, 2H, J ) 7.0 Hz), 1.48 (s, 9H); MS (FAB) m/z 309 (M+ + H); HRMS calcd for C16H25N2O4 309.1815, found 309.1806. Synthesis of Methyl 15-[N-(Hydroxycarbonylmethyl)-2picolylamino]pentadecanoate (5a). Trifluoroacetic acid (2 mL) was added to 4a (250 mg, 0.52 mmol) in dichloromethane (2 mL), and the reaction mixture was stirred for 2 h at room temperature. At the end of the reaction, solvent was removed in vacuo, and the residue was extracted with dichloromethane (30 mL × 3), washed with water, and dried over anhydrous Na2SO4. Flash column chromatography (15:1 dichloromethanemethanol) gave 5a (188 mg) as a light yellow oil in 85% yield. Compounds 5b and 5c were synthesized in 79% and 62% yields, respectively, using the same procedure described for 5a. 5a. 1H NMR (CD3OD, 500 MHz) δ 8.67-8.66 (m, 1H), 7.91 (td, 1H, J ) 8.0, 1.5 Hz), 7.53 (d, 1H, J ) 8.0 Hz), 7.47-7.44 (m, 1H), 4.56 (s, 2H), 3.76 (s, 2H), 3.66 (s, 3H), 3.27-3.23 (m, 2H), 2.32 (t, 2H, J ) 7.0 Hz), 1.80-1.75 (m, 2H), 1.631.60 (m, 2H), 1.30 (s, 20H); 13C NMR (CDCl3, 100 MHz) δ 174.0, 170.1, 160.0, 148.8, 136.1, 122.7, 121.6, 80.4, 60.3, 55.9, 54.1, 51.1, 33.9, 29.4, 27.98, 27.4, 24.7; MS (FAB) m/z 421 (M+ + H); HRMS calcd for C24H41N2O4 421.3066, found 421.3066. 5b. 1H NMR (CD3OD, 500 MHz) δ 8.67-8.65 (m, 1H), 7.92 (td, 1H, J ) 7.8, 2.0 Hz), 7.54 (d, 1H, J ) 8.0 Hz), 7.53-7.45 (m, 1H), 4.55 (s, 2H), 3.78 (s, 2H), 3.67 (s, 3H), 3.27-3.24 (m, 2H), 2.38 (t, 2H, J ) 2.0 Hz), 1.83-1.79 (m, 2H), 1.691.66 (m, 2H); MS (FAB) m/z 281 (M+ + H); HRMS calcd for C14H21N2O4 281.1502, found 281.1495.
1334 Bioconjugate Chem., Vol. 18, No. 4, 2007
5c. 1H NMR (CD3OD, 500 MHz) δ 8.62 (d, 1H, J ) 4.0 Hz), 7.92 (td, 1H, J ) 7.5, 1.5 Hz), 7.57 (d, 1H, J ) 8.0 Hz), 7.46-7.44 (m, 1H), 4.43 (s, 2H), 3.70 (s, 2H), 3.42-3.39 (m, 2H), 3.36 (s, 3H), 2.76-2.74 (m, 2H); MS (FAB) m/z 253 (M+ + H); HRMS calcd for C12H17N2O4 253.1189, found 253.1192. Synthesis of 15-[N-(Hydroxycarbonylmethyl)-2-picolylamino]pentadecanoic Acid (2a). Compound 5a (40 mg, 0.10 mmol) was dissolved in a 1:1 mixture of 0.4 N NaOH and MeOH (3 mL) and stirred for 4 h at 70 °C. The solution was then cooled and neutralized with 1.5 mL of 0.1 N HCl in an ice bath. The reaction mixture was then extracted with dichloromethane, and the organic layer was dried over anhydrous Na2SO4 and concentrated. Flash column chromatography (9:1 CH2Cl2-MeOH) gave 2a (22 mg) as light yellow solid in 54% yield. Compound 2b was prepared in 46% yield using the procedure described for 2a and C-18 Sep-Pak purification. 2a. 1H NMR (CD3OD, 500 MHz) δ 8.55 (d, 1H, J ) 4.5 Hz), 7.84 (td, 1H, J ) 7.8, 1.0 Hz), 7.61 (d, 1H, J ) 7.5 Hz), 7.37-7.34 (m, 1H), 4.10 (s, 2H), 3.36 (s, 2H), 2.79 (t, 2H, J ) 7.0 Hz), 2.17 (t, 2H, J ) 7.5 Hz), 1.62-1.59 (m, 4H), 1.28 (s, 20H); MS (FAB) m/z 429 (M+ + Na); HRMS calcd for C23H38N2O4Na 429.2732, found 429.2729. 2b. 1H NMR (CD3OD, 500 MHz) δ 8.69-8.68 (m, 1H), 8.01-7.97 (m, 1H), 7.59-7.57 (m, 1H), 7.54-7.51 (m, 1H), 4.59 (d, 2H, J ) 3.0 Hz), 4.06 (d, 2H, J ) 6.0 Hz), 3.67-3.66 (m, 2H), 2.40-2.34 (m, 2H), 1.89-1.77 (m, 2H), 1.69-1.64 (m, 2H); MS (FAB) m/z 289 (M+ + Na); HRMS calcd for C13H18N2O4Na 289.1167, found 289.1169. Synthesis of Re(CO)3-15-[N-(Acetyloxy)-2-picolylamino]pentadecanoic Acid (6a). (NEt4)2[Re(CO)3Br3] was prepared as described in the literature (30). (NEt4)2[Re(CO)3Br3] (187 mg, 0.24 mmol) was added to 2a (60 mg, 0.16 mmol) in MeOH (1 mL) and stirred for 2 h at 65 °C. After reaction, solvent was removed in vacuo, and the resulting oily residue was purified by flash column chromatography (10:1 CH2Cl2-MeOH) to give 6a (57 mg) as a light yellow solid in 53% yield. Compound 6b was synthesized in 51% yield using the procedure described for 6a. 6a. mp 221-225 °C; 1H NMR (CDCl3, 500 MHz) δ 8.85 (d, 1H, J ) 5.5 Hz), 7.95 (td, 1H, J ) 8.0, 1.5 Hz), 7.51 (d, 1H, J ) 7.5 Hz), 7.44 (t, 1H, J ) 6.0 Hz), 4.51-4.48 (m, 2H), 4.37-4.34 (m, 2H), 3.74-3.70 (m, 2H), 3.58-3.54 (m, 2H), 2.36-2.33 (m, 2H), 1.61-1.59 (m, 2H), 1.27 (s, 20H); MS (FAB) m/z 677 (M+ + H); HRMS calcd for C26H38N2O7187Re 677.2237, found 677.2245. 6b. mp 237-240 °C; 1H NMR (CD3OD, 500 MHz) δ 8.83 (d, 1H, J ) 5.5 Hz), 8.10 (td, 1H, J ) 8.2, 1.0 Hz), 7.72 (d, 1H, J ) 8.0 Hz), 7.55 (t, 1H, J ) 6.0 Hz), 4.75-4.72 (m, 2H), 4.57-4.54 (m, 2H), 3.95-3.91 (m, 2H), 3.64-3.62 (m, 2H), 2.42 (t, 2H, J ) 7.0 Hz), 1.71-1.69 (m, 2H); MS (FAB) m/z 537 (M+ + H); HRMS calcd for C16H18N2O7187Re 537.0672, found 537.0668. Synthesis of Re(CO)3-3-[N-(Acetyloxy)-2-picolylamino]propionic Acid (6c). (NEt4)2[Re(CO)3Br3] (187 mg, 0.24 mmol) was added to 5c (50 mg, 0.16 mmol) in MeOH (3 mL) and stirred for 2 h at 65 °C. After reaction, solvent was removed in vacuo, and the resulting oily residue was purified by flash column chromatography (20:1 CH2Cl2-MeOH) to give Re coordinated methyl ester (38 mg) as a light yellow solid in 46% yield: mp 187-192 °C; 1H NMR (CDCl3, 500 MHz) δ 8.86 (d, 1H, J ) 5.0 Hz), 7.96 (td, 1H, J ) 8.0, 1.5 Hz), 7.51 (d, 1H, J ) 8.0 Hz), 7.46 (t, 1H, J ) 6.5 Hz), 4.44-4.36 (m, 2H), 3.94-3.91 (m, 2H), 3.80 (s, 3H), 3.39-3.36 (m, 2H), 2.87 (t, 2H, J ) 7.0 Hz); MS (FAB) m/z 523 (M+ + H); HRMS calcd for C15H16N2O7187Re 523.0516, found 523.0522. The methyl ester (30 mg, 0.057 mmol) was dissolved in a 1:1 mixture of 0.4 N NaOH and MeOH (1.5 mL) and stirred
Lee et al.
for 4 h at 70 °C. The solution was then cooled and neutralized with 1 mL of 0.1 N HCl in an ice bath. The reaction mixture was then extracted with dichloromethane, and the organic layer was dried over anhydrous Na2SO4 and concentrated. Sold-phase purification (C18 Sep-Pak cartridge) gave 6c (9.3 mg) as a light yellow solid in 32% yield: mp 257-259 °C; 1H NMR (CD3OD, 500 MHz) δ 8.83 (d, 1H, J ) 6.0 Hz), 8.10 (td, 1H, J ) 8.0, 1.5 Hz), 7.72 (d, 1H, J ) 8.0 Hz), 7.56 (t, 1H, J ) 5.5 Hz), 4.84-4.76 (m, 2H), 4.58-4.55 (m, 2H), 3.93-3.90 (m, 2H), 2.92-2.87 (m, 2H); MS (FAB) m/z 509 (M+ + H); HRMS calcd for C14H14N2O7187Re 509.0359, found 509.0359. Radiochemical Synthesis of 99mTc(CO)3-15-[N-(Acetyloxy)2-picolylamino]pentadecanoic Acid (1a). Kit Preparation of [99mTc(CO)3(H2O)3]+. 99mTcO4- (740 MBq, 300 µL) was added to a 10 mL sealed kit vial containing a lyophilized formulation (IsoLink). The vial was then shaken vigorously and placed in an oil bath for 30 min at 75 °C. It was then cooled to room temperature, and the mixture was neutralized with 0.1 N HCl (180 µL). The radio-TLC yield of [99mTc(CO)3(H2O)3]+ was greater than 95%. In-House Preparation of [99mTc(CO)3(H2O)3]+. A mixture of Na2CO3 (4 mg, 38 µmol), NaK tartrate tetrahydrate (2 mg), and NaBH4 (5 mg, 130 µmol) was placed in a 10 mL reaction vial and dissolved in water (500 µL), and the vial was then sealed. After bubbling CO in this solution for 10 min, 99mTcO4- (370 MBq, 200 µL) was added, and the resulting solution was stirred for 30 min at 75 °C, cooled to room temperature, and then neutralized with 0.1 N HCl (180 µL). Radio-TLC yield was greater than 95%. Radiochemical Synthesis of 1a. Compound 2a (1.1 mg, 2.7 µmol) was dissolved in a 4:1 mixture of methanol and water (300 µL), and to this solution was added [99mTc(CO)3(H2O)3]+ (370 MBq, 250 µL). The reaction mixture was then stirred in a sealed vial for 30 min at 75 °C. The crude mixture was loaded onto a C18 Sep-Pak cartridge, which was washed with water (10 mL) and then with methanol (2 mL). The methanol layer was concentrated under N2 and purified by HPLC using a 8:92 mixture of hexane and dichloromethane-2-propanol-acetic acid (88:12:0.3) at 4 mL/min. The desired fraction (tR ) 21.5-23.5 min) was collected, and solvents were removed under N2. The residue was then passed through a C18 Sep-Pak cartridge that was washed with water (10 mL) and then with MeOH (2 mL). Methanol removal gave 1a in 60-63% yield. Radiotracer 1b was prepared in an identical manner and purified using a C18 Sep-Pak cartridge at yields of 48%. In preparation of 1c, methyl ester 5c (1.3 mg, 4.64 µmol) was labeled with [99mTc(CO)3(H2O)3]+ (37 MBq, 25 µL) for 30 min at 75 °C. After solid-phase purification, the residue was redissolved in methanol (300 µL) and treated with 0.4 N NaOH (aq, 100 µL) for 10 min at 65 °C. The reaction mixture was then cooled, neutralized with 1 N HCl (40 µL), and purified using a C18 Sep-Pak cartridge and methanol as the eluant, which gave 1c in 34% yield. In Vitro Stability. An aliquot (9.7 MBq) of 1a in saline was added to human plasma (1 mL) and incubated for 4 h at 37 °C, and the resulting solution was analyzed by radio-TLC using dichloromethane-methanol (9:1). Tissue Distribution in Normal Mice. Radiotracer 1a was dissolved in saline containing 1% bovine serum albumin (BSA) and 10% ethanol at 40 °C. ICR mice (male, 25-30 g, four mice per time point) were fasted for 12 h before injecting 1a (1.11 MBq/mouse) via a tail vein. At the indicated time points (0.5, 1, 5, and 30 min), mice were sacrificed by cervical dislocation, and samples of blood were immediately obtained from the myocardial tissue via a syringe, and samples of whole organ (heart) and tissue (lung, liver, kidney, and
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Technical Notes Scheme 1
a
a Reagents and conditions: (a) CH3CN, room temperature, overnight, 72%; (b) methyl 15-bromopentadecanoate (n ) 14), methyl 5-bromopentanoate (n ) 4), or methyl 3-bromopropionate (n ) 2), DIEA or NaHCO3, CH3CN, 60-75 °C, overnight, 58-81%; (c) CF3COOH, CH2Cl2, room temperature, 2 h, 62-85%; (d) 0.4 N NaOH-MeOH, 70 °C, 4 h, 46-54%.
Scheme 2
a
Table 1. Tissue Distribution of 1a in Normal Micea % ID/g
a Reagents and conditions: (a) [99mTc(CO)3(H2O)3]+, MeOH, 75 °C, 30 min, 48-63%; (b) [99mTc(CO)3(H2O)3]+, MeOH, 75 °C, 30 min, 0.4 N NaOH-MeOH, 65 °C, 10 min, 34%; (c) (NH4)2[Re(CO)3Br3], MeOH, 65 °C, 2 h, 51-53%; (d) (NH4)2[Re(CO)3Br3], MeOH, 65 °C, 2 h, 46%; (e) 0.4 N NaOH-MeOH, 70 °C, 4 h, 32%.
stomach) were also removed, weighed, and counted. Data are expressed as the percent injected dose per gram of tissue (% ID/g). Analysis of Metabolites. Radiotracer 1a (18.5 MBq/mouse) was injected via a tail vein into 12 h-fasted ICR mice. At 30 min after injection, mice were sacrificed by cervical dislocation, and samples of the urine were collected, filtered through a membrane filter, and analyzed by HPLC at a flow rate of 1 mL/min using a combination of solution A (0.1% TFA-water) and solution B (methanol) and a linear gradient program from 60:40 A-B to 40:60 A-B over 20 min, followed by 40:60 A-B to 0:100 A-B over 20 min. A major radioactive metabolite was recovered from the HPLC column at a recovery efficiency of 71%. Radiotracers 1a, 1b, and 1c were analyzed by HPLC using the same conditions.
RESULTS AND DISCUSSION Chemistry. The precursor 2a for [99mTc(CO)3]+ labeling was synthesized by reacting tert-butyl bromoacetate with 2-picolylamine, reacting the resulting compound 3 with methyl 15bromopentadecanoate, and the subsequent removal of the tertbutyl group and the methyl ester group (Scheme 1). Reaction conditions for the synthesis of 4a-4c depended on carbon chain length. Compound 2b was synthesized using the procedure described for 2a. Methyl ester 5c was prepared from deprotection of tert-butyl group of 4c and used directly for [99mTc(CO)3]+ labeling. Radiotracer 1a was prepared by labeling the precursor 2a with [99mTc(CO)3(H2O)3]+ for 30 min at 75 °C (Scheme 2). Hydrolysis can be carried out after [99mTc(CO)3]+ labeling of methyl ester precursors 5a-5c. This was the case for [99mTc(CO)3]+ labeling of methyl ester 5c, due to limited isolation of its polar hydrolyzed product, 3-[N-(hydroxycarbonylmethyl)2-picolylamino]propionic acid. No significant difference in the yields of [99mTc(CO)3(H2O)3]+ was apparent for kit and in-house preparations. The predicted β-oxidation metabolites, 1b and 1c, were prepared as shown in Scheme 2 and purified using a C-18 Sep-Pak cartridge.
tissue
0.5 min
1 min
5 min
30 min
blood heart lung liver kidney stomach
18.28 ( 3.32 12.67 ( 1.48 23.58 ( 1.16 27.60 ( 5.31 16.06 ( 1.82 1.24 ( 0.36
7.54 ( 1.46 6.38 ( 0.69 15.98 ( 3.32 31.88 ( 2.51 13.86 ( 2.24 0.78 ( 0.16
0.39 ( 0.14 0.73 ( 0.37 6.82 ( 1.77 30.33 ( 6.05 6.63 ( 0.98 2.27 ( 1.45
0.17 ( 0.03 0.28 ( 0.08 1.36 ( 0.21 13.06 ( 3.24 6.53 ( 0.90 1.57 ( 0.44
a
Values are given as mean ( SD for groups of four mice.
Re standards were also prepared using (NH4)2[Re(CO)3Br3] as described for the synthesis of 99mTc-labeled fatty acids. 99mTcfatty acid analogs had retention times that were similar to the Re standards on HPLC: 35.9 min vs 35.2 min, 16.7 min vs 15.7, and 11.8 min vs 11.3 min for 1a-1c vs 6a-6c. Although 99mTc and Re-fatty acid analogs were not coeluted on HPLC, the formers were identified on the basis of the similar chemical properties of 99mTc and Re complexes. The different HPLC retention times of 99mTc and Re complexes have also been reported for other cases (24, 25). In Vitro Stability. Radiotracer 1a was incubated in human plasma for 4 h at 37 °C and analyzed by radio-TLC. The results showed that 97% of 1a remained intact after 2 h and over 87% remained after 4 h, demonstrating its high chemical stability. Tissue Distribution Studies. Since fatty acids are predominantly metabolized in the fasting state, mice were fasted for 12 h prior to experiments. High radioactivity accumulated in blood, heart, lung, liver, and kidneys, and the radioactivity washed out rapidly with time from all tissues except for liver and kidneys (Table 1). Heart uptake of 1a (6.38% ID/g at 1 min and 0.73% ID/g at 5 min postinjection) was comparable to those of 99mTcMAMA-HDA (5.46% ID/g at 2 min and 2.40% ID/g at 5 min postinjection) (22). 99mTc-BAT-pentadecanoic acid and HDA were evaluated in rats, and the results were expressed as % ID/ organ (0.31-0.46% ID/organ at 5 min postinjection) (19). The maximum heart-to-blood uptake ratio of 1a was 1.87 at 5 min postinjection, which appears higher than those obtained for other 99mTc-labeled fatty acid analogs (Table 2). Heart-to-liver uptake ratios of 1a were similar to those of 99mTc-MAMA-HDA. 99mTc-[9,10-bis[N-(2′-methyl-2′-mercapto)propyl]aminooctadecanoic acid and 99mTc-[7,10-bis(2-mercapto-2-methyl)propyl]7,10-diazapentadecanoic acid showed high heart-to-blood and heart-to-liver uptake ratios in rats (Table 2), probably due to the branching effect of the fatty acid analogs (20, 21). Although direct comparisons in % ID/g of radiotracers may not be appropriate, due to different experiment conditions and animal species used for the studies, 1a may be superior to other 99mTc-labeled fatty acid analogs in terms of its facile conjugation of [99mTc(CO)3]+ to a fatty acid analog, high heart uptake, and high heart-to-blood ratios. No significant uptake was observed
1336 Bioconjugate Chem., Vol. 18, No. 4, 2007 Table 2. Comparison of in Vivo Data of
99mTc-Labeled
radiotracer 1a
99mTc-MAMA-HDA
Lee et al.
(22)
Fatty Acid Analogsd heart/blooda
heart/livera
min)b
12.67 (0.5 6.38 (1 min) 0.73 (5 min) 0.28 (30 min)
0.69 0.85 1.87 1.65
0.46 0.20 0.02 0.02
ICR mice
mainly 1b (ICR mice)
11.22 (0.5 min)b 5.46 (2 min) 2.40 (5 min) 0.60 (20 min) 0.32 (60 min)
1.14 1.51 0.82 0.77 0.86
0.53 0.21 0.09 0.03 0.02
ddY mice
99mTc-MAMA-BA (Wistar rats)
heart
animal
99mTc-BAT-PDA
(19)
0.31 (5 min)c 0.13 (15 min) 0.05 (30 min)
1.39 0.98 0.65
SD rats
99mTc-BAT-HDA
(19)
0.46 (5 min)c 0.16 (15 min) 0.10 (30 min)
0.98 0.59 0.46
SD rats
1.30 (5 min)b 0.50 (30 min)
1.78 1.67
0.29 0.11
Fischer CD rats
propyl]amino-ODA (20) 99mTc-[7,10-bis(2-
0.56 (15 min)b
0.71
0.21
Fischer CD rats
99mTc-[9,10-bis[N-(2′-methyl-2′-mercapto)
mercapto-2-methyl)propyl] -7,10-diaza-PDA (21) a
metabolites
% ID/g ratio. b % ID/g. c % ID/organ. d HDA, hexadecanoic acid; PDA, pentadecanoic acid; ODA, octadecanoic acid; BA, butyric acid.
the kidneys. Although the metabolites were not analyzed in a time-dependent manner, it is believed that 1b would be further metabolized to 1c in time on the basis of the presence of a small portion of 1c at 30 min postinjection on HPLC (Figure 1A). The metabolism of 1a is similar to that of 99mTc-MAMA-HDA, which is metabolized to 99mTc-MAMA-butyric acid (BA) after six cycles of β-oxidation in rats (Table 2) (22). Although the metabolism of 1a appears to be slower than 99mTc-MAMAHDA, it is probable that metabolic rates are species-dependent (mice vs rats). Metabolite analysis has not been carried out for other 99mTc-labeled fatty acid analogs (Table 2).
CONCLUSION A 99mTc-labeled fatty acid analog, 1a, was prepared in high yield by incorporating [99mTc(CO)3]+ into a fatty acid analog, 2a. An in vivo evaluation of 1a demonstrated that high radioactivity accumulated in the heart and that the radiotracer is mainly metabolized to 1b via β-oxidation in the body. Taken together, 1a may be useful for evaluating the β-oxidation of fatty acids in myocardium.
ACKNOWLEDGMENT We thank Hyun Jung Jeon for performing the tissue distribution studies and Iljung Lee and Byung Ho Ahn for their technical assistance. IsoLink kits were generous gifts from Mallinckrodt Medical B.V. (Petten, The Netherlands). This work was supported by the Korea Research Foundation Grant (MOEHRD) (KRF-2006-521-E00092). Figure 1. HPLC analysis of 30 min urine samples from mice injected via a tail vein with 1a (A). HPLC profiles of radioactive metabolite standards: tR ) 11.8 min (1c) (B), tR ) 16.7 min (1b) (C), and tR ) 35.9 min (1a) (D). HPLC analysis was conducted using an analytical column (YMC-Pack C18, 5 µ, 4.6 × 250 mm) and a NaI(T1) radioactivity detector.
by the stomach, indicating that 99mTc pertechnetate was not regenerated during the study. Analysis of Metabolites. When samples of urine were analyzed by HPLC, a 9.3:1 ratio of the radioactive metabolites at the retentions times of 16.7 and 11.8 min was observed, which were identified as 1b and 1c, respectively (Figure 1). Radioactive metabolite 1b, which is produced by five cycles of 1a β-oxidation, was the major metabolite (Figure 1A,C), which indicated that 1a was metabolized to 1b via β-oxidation and excreted to
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