A Novel Radiotracer for Evaluation of Medium Chain Fatty Acid

At the end of the reaction, the flask was placed again in an ice bath, and water (2 mL) was slowly added. The resulting two-phase mixture was stirred ...
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Bioconjugate Chem. 2004, 15, 121−127

121

8-Cyclopentadienyltricarbonyl 99mTc 8-Oxooctanoic Acid: A Novel Radiotracer for Evaluation of Medium Chain Fatty Acid Metabolism in the Liver Byung Chul Lee,†,‡ Yearn Seong Choe,†,* Dae Yoon Chi,‡ Jin-Young Paik,† Kyung-Han Lee,† Yong Choi,† and Byung-Tae Kim† Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Kangnam-ku, Seoul 135-710, Korea and Department of Chemistry, Inha University, 253 Yonghyundong, Namgu, Inchon 402-751, Korea. Received July 19, 2003

8-Cyclopentadienyltricarbonyl 99mTc 8-oxooctanoic acid (99mTc-CpTTOA; 1a) was synthesized for evaluation of medium chain fatty acid metabolism in the liver. 99mTc-CpTTOA was prepared in high radiochemical yield (50-63%) by a double ligand transfer reaction of methyl 8-ferrocenyl-8-oxooctanoate and Na99mTcO4 in the presence of CrCl3 and Cr(CO)6, followed by hydrolysis. This radiotracer was shown to be stable (>90% at 6 h) when incubated with human serum. Aqueous extraction of the radioactivity from the liver and blood samples of mice suggested that 99mTc-CpTTOA was mainly metabolized via β-oxidation in the liver, and the radioactivity was retained longer in CCl4-treated mice than in control mice, possibly due to impaired β-oxidation in the former. Planar images of rats injected with 99mTc-CpTTOA showed accumulation of the radioactivity in the liver, kidneys, and bladder with rapid hepatic clearance as a function of time. Analysis of the metabolites from the liver and urine samples of rats further supported that 99mTc-CpTTOA was metabolized to 4-cyclopentadienyltricarbonyl 99mTc 4-oxobutanoic acid (99mTc-CpTTBA; 1c) via β-oxidation. The results suggested that this radiotracer might be of valuable use in the evaluation of fatty acid metabolism in the liver.

INTRODUCTION

Fatty acids are important substrates for energy metabolism, particularly in the heart and liver. In the normal state, glucose and fatty acids are utilized as energy substrates in ATP production. On the other hand, in the fasted state, glucose is synthesized from amino acids (gluconeogenesis) and the rate of fatty acid oxidation is enhanced (1, 2). Therefore, the β-oxidation of fatty acid can be a marker for energy metabolism of the heart and liver. Long chain fatty acids are not only metabolized via β-oxidation but also stored as triglycerides, while medium chain fatty acids are predominantly metabolized via β-oxidation that degrades the fatty acid by the removal of two-carbon units (3, 4). Fatty acid is taken up in many organs, enters the mitochondria, and is metabolized. Unlike long chain fatty acid, medium chain fatty acid enters the mitochondria without the use of carnitine shuttle system (5). For past decades, fatty acids were radiolabeled with 11C, 18F, 123I, and 99mTc (3-4, 6-14) and utilized for evaluation of fatty acid metabolism using positron emission tomography (PET) and single photon emission computed tomography (SPECT). Among the radionuclides, single photon emitting 99mTc is desirable due to its ideal physical properties (141 keV, t1/2 ) 6 h) (15, 16) and ready availability. Therefore, efforts were made to develop 99mTc-labeled fatty acid analogues (4, 7-12). Early attempts included preparation of 99mTclabeled fatty acid analogues containing strong chelating groups such as DTPA and EDTA, and these radiotracers were poorly accumulated in the myocardium (7, 8). In * Corresponding author. Tel: +82-2-3410-2623, Fax: +822-3410-2639, E-mail: [email protected]. † Sungkyunkwan University School of Medicine. ‡ Inha University.

other cases, 99mTc cores such as 99mTc-N2S2 and Tc-N3S ligand were successfully coordinated to long chain fatty acid analogues, but the radiolabeled analogues showed poor uptake in the myocardium that may be partly due to relatively large size of the 99mTc cores or inappropriate lipophilicity and thus are not recognized as fatty acids. 99mTc-labeled iminodiacetic acid was rapidly taken up to the liver and the biliary system and thus applied for hepatobiliary imaging (11, 12). As a same token, radiolabeled medium chain fatty acids have been studied for evaluation of the liver function, based on their rapid accumulation in the liver (17). p-[123I]Iodophenylenanthic acid and p-[123I]iodophenylvaleric acid were studied for assessment of hepatic viability. The results showed that they were taken up to and cleared from the liver rapidly. [1-11C]Octanoate showed the similar pattern of the liver uptake and clearance to other medium chain fatty acid analogues. This radiotracer along with p-[123I]iodophenylenanthic acid were metabolized by β-oxidation in the liver with slower hepatic clearance in CCl4-treated mice than in control mice due to impaired β-oxidation (3, 6). The results demonstrated that both are promising diagnostic agents for assessment of the liver function. [99mTc]MAMA-hexanoic acid, a 99mTc-labeled medium chain fatty acid analogue, was also rapidly accumulated in the liver, and the metabolism via β-oxidation was confirmed by detection of the radiometabolite, [99mTc]MAMA-butyric acid (4). [99mTc]MAG-hexanoic acid, a polar analogue, however, showed poor accumulation in the liver and was immediately excreted from the kidneys to the bladder, because of its low lipophilicity. Recent preparation of cyclopentadienyltricarbonyl 99mTc (99mTcCpTT) extended its utilization to the preparation of the various 99mTc complexes (18-20). This small lipophilic core is attractive, because the ferrocene with various 99m

10.1021/bc0341268 CCC: $27.50 © 2004 American Chemical Society Published on Web 12/30/2003

122 Bioconjugate Chem., Vol. 15, No. 1, 2004

substituents can be readily prepared via Friedel-Crafts acylation, and thus biomolecules can be efficiently labeled with 99mTc-CpTT. Small size of the core is also expected to minimize the perturbation of biological activity of the fatty acid. In this work, we prepared 99mTc-CpTTOA (1a) by facile incorporation of 99mTc-CpTT into a medium chain fatty acid analogue. Its metabolism in the liver was evaluated and the metabolite was identified using the proposed radiometabolites, 6-cyclopentadienyltricarbonyl 99m Tc 6-oxohexanoic acid (99mTc-CpTTHA; 1b) and 99m Tc-CpTTBA (1c). EXPERIMENTAL SECTION

Materials and Methods. Solvents and reagents were purchased from the following commercial sources: SigmaAldrich Company (Milwaukee, WI) and Acros Organics (Geel, Belgium). 1H NMR were obtained on Varian Gemini-200 and 400 (Palo Alto, CA) and a JEOL JNMLA 300 spectrometer, and 13C NMR at 50 and 100 MHz. Chemical shifts were reported in parts per million (ppm, δ units). Electron impact (EI) and chemical ionization (CI) mass spectra were obtained on a GC/MS QP5050A spectrometer (Shimadzu, Kyoto, Japan). Fast atom bombardment (FAB) mass spectra were obtained on a JMS 700 (JEOL Ltd., Tokyo, Japan). Elemental analyses were performed at the Inha University (CE instrument, Italy). HPLC was carried out on a Thermo Separation Products System (Fremont, CA) with a semipreparative column (Alltech Econosil silica gel, 10 µm, 10 × 250 mm) and an analytical column (YMC C18, 5 µm, 4.6 × 250 mm). The eluant was simultaneously monitored by a UV detector (254 nm) and a NaI(Tl) radioactivity detector. TLC was performed on Merck F254 silica plates and analyzed on a Bioscan radio-TLC scanner (Washington DC). Na99mTcO4 was eluted on a daily basis from 99 Mo/99mTc generators (DuPont Pharmaceuticals Co., Wilmington, DE, and Daiichi Radioisotope Labs., Chiba, Japan). Images of rats were obtained on a gamma camera (Trionix Research Lab. Inc., Twinsburg, OH) using a pinhole collimator. Synthesis of Methyl 8-Ferrocenyl-8-oxooctanoate (3a). The flask was charged with ferrocene (150 mg, 0.806 mmol), methyl 8-chloro-8-oxooctanoate (114 µL, 0.806 mmol), and CH2Cl2 (1.5 mL). The solution was then chilled thoroughly in an ice bath. To this solution was added anhydrous aluminum chloride (113 mg, 0.846 mmol) carefully for 20 min using a spatula under N2. The reaction mixture, which turned to deep violet color, was allowed to stir for 30 min in an ice-bath and for another 2 h at room temperature. At the end of the reaction, the flask was placed again in an ice bath, and water (2 mL) was slowly added. The resulting two-phase mixture was stirred vigorously for 30 min. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed in vacuo. The residue was purified by flash column chromatography (15:85 ethyl acetate-hexane) to afford 3a (244 mg, 85%) as a red oil: 1H NMR (300 MHz, CDCl3) δ 4.78 (t, J ) 2.0 Hz, 2H, -Fe-Cp-H (R)), 4.49 (t, J ) 2.0 Hz, 2H, -Fe-Cp-H (β)), 4.19 (s, 5H, Cp-H), 3.67 (s, 3H, OCH3), 2.70 (t, J ) 7.3 Hz, 2H, CH2), 2.33 (t, J ) 7.6 Hz, 2H, CH2), 1.74-1.62 (m, 4H), 1.40-1.37 (m, 4H); 13 C NMR (50 MHz, CDCl3) δ 202.030, 171.746, 76.694, 69.669, 67.279, 66.862, 49.020, 37.132, 31.572, 26.694, 26.565, 22.355, 21.907; MS (EI) m/z 356 (M+). Anal. (C19H24O3Fe) C, H. Methyl 6-Ferrocenyl-6-oxohexanoate (3b). The flask was charged with ferrocene (500 mg, 2.688 mmol),

Lee et al.

adipoyl chloride (390 µL, 2.688 mmol), and CH2Cl2 (20 mL). The solution was then chilled thoroughly in an ice bath. To this solution was added anhydrous aluminum chloride (394 mg, 2.955 mmol) carefully for 20 min using a spatula under N2. The reaction mixture, which turned to deep violet color, was allowed to stir for 40 min in an ice-bath and for another 30 min at room temperature. Without further purification, the reaction mixture was heated at 90 °C for 1 h after slow addition of methanol (9 mL) and triethylamine (2 mL). At the end of the reaction, the solvents were removed in vacuo, and the residue was purified by flash column chromatography (20:80 ethyl acetate-hexane) to afford 3b (389 mg, 44%) as a red oil: 1H NMR (200 MHz, CDCl3) δ 4.76 (t, J ) 1.8 Hz, 2H, Fe-Cp-H (R)), 4.48 (t, J ) 1.8 Hz, 2H, FeCp-H (β)), 4.18 (s, 5H, Cp-H), 3.67 (s, 3H, OCH3), 2.72 (t, J ) 7.0 Hz, 2H, CH2), 2.37 (t, J ) 7.0 Hz, 2H, CH2) 1.751.68 (m, 4H); 13C NMR (50 MHz, CDCl3) δ 203.976, 173.918, 78.950, 72.145, 69.717, 69.247, 51.532, 39.181, 33.901, 24.729, 23.848; MS (CI) m/z 329 (M + H)+. Anal. (C17H20O3Fe) C, H. Methyl 8-Ferrocenyl-8-oxobutanoate (3c). A red oil: 1H NMR (200 MHz, CDCl3) δ 4.79 (t, J ) 1.8 Hz, 2H, -Fe-Cp-H (R)), 4.49 (t, J ) 1.8 Hz, 2H, -Fe-Cp-H (β)), 4.23 (s, 5H, Cp-H), 3.71 (s, 3H, OCH3), 3.07 (t, J ) 6.6 Hz, 2H, CH2), 2.68 (t, J ) 6.4 Hz, 2H, CH2); 13C NMR (50 MHz, CDCl3) δ 202.027, 173.516, 78.312, 72.167, 69.869, 69.118, 51.767, 34.143, 27.611; MS (CI) m/z 301 (M + H)+. Anal. (C15H16O3Fe) C, H. Synthesis of 8-Ferrocenyl-8-oxooctanoic Acid (4). A solution of the ester 3a (70 mg, 0.197 mmol) in 1.5 mL of ethanol and 1.5 mL of 20% NaOH (aq) was heated under reflux for 1 h. The reaction mixture was cooled and acidified with 5 mL of 20% HCl in an ice bath and then stirred vigorously for a few minutes to make free acid from the sodium salt. The product was collected by suction filtration at room temperature, washed with 10 mL of water, and dried. Pure 8-ferrocenyl-8-oxooctanoic acid (4) was obtained in 90% yield (60 mg) as a red solid: mp 80.2 °C; 1H NMR (300 MHz, CDCl3) δ 4.78 (t, J ) 2.0 Hz, 2H, -Fe-Cp-H (R)), 4.49 (t, J ) 2.0 Hz, 2H, -FeCp-H (β)), 4.19 (s, 5H, Cp-H), 2.70 (t, J ) 7.4 Hz, 2H, CH2), 2.38 (t, J ) 7.6 Hz, 2H, CH2), 1.74-1.62 (m, 4H), 1.30-1.22 (m, 4H); 13C NMR (50 MHz, CDCl3) δ 204.628, 179.450, 79.079, 72.145, 69.733, 69.346, 39.570, 33.942, 29.102, 28.904, 24.527, 24.360; MS(FAB) m/z 342 (M + H)+. Anal. (C18H22O3Fe) C, H. Synthesis of Methyl 8-Cyclopentadienyltricarbonylrhenium-8-oxooctanoate (5a). A mixture of the ester 3a (52.8 mg, 0.147 mmol), NH4ReO4 (13 mg, 0.047 mmol), Cr(CO)6 (58 mg, 0.265 mmol), and CrCl3 (15 mg, 0.094 mmol) was placed in a 4 mL reaction vial containing a magnetic stir bar. Dry methanol (300 µL) was added to the vial that was sealed and heated at 160 °C for 1 h. At the end of the reaction, the mixture was cooled in an ice bath for 15 min and the solvent was removed in vacuo. Flash column chromatography (1:4 ethyl acetate-hexane) of the crude mixture afforded 5a (26 mg, 35%) as a gray solid: mp 70.2 °C; 1H NMR (300 MHz, CDCl3) δ 5.99 (t, J ) 2.4 Hz, 2H, Re-Cp-H (R)), 5.40 (t, J ) 2.3 Hz, 2H, Re-Cp-H (β)), 3.67 (s, 3H, OCH3), 2.58 (t, J ) 7.3 Hz, 2H, CH2), 2.31 (t, J ) 7.5 Hz, 2H, CH2), 1.71-1.61 (m, 4H), 1.38-1.33 (m, 4H); 13C NMR (50 MHz, CDCl3) δ 194.993, 191.777, 174.063, 96.337, 87.765, 85.094, 51.367, 38.645, 33.919, 28.783, 28.631, 24.671, 24.110; MS (FAB) m/z 507 (M + H)+; HRMS calcd for C17H20O6187Re 507.0818, found 507.0812. Methyl 6-Cyclopentadienyltricarbonylrhenium6-hexanoate (5b). A pale yellow oil: 1H NMR (400 MHz,

8-Cyclopentadienyltricarbonyl

99mTc

8-Oxooctanoic Acid

CDCl3) δ 5.97 (t, J ) 2.0 Hz, 2H, Re-Cp-H (R)), 5.39 (t, J ) 2.0 Hz, 2H, Re-Cp-H (β)), 3.66 (s, 3H, OCH3), 2.61 (t, J ) 7.2 Hz, 2H, CH2), 2.35 (t, J ) 7.0 Hz, 2H, CH2), 1.73-1.64 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 194.689, 191.778, 173.766, 95.786, 87.886, 85.195, 51.590, 38.309, 33.684, 24.239, 23.557; MS (FAB) m/z 479 (M + H)+; HRMS calcd for C15H16O6187Re 479.0505, found 479.0515. Methyl 4-Cyclopentadienyltricarbonylrhenium4-oxobutanoate (5c). A pale yellow oil: 1H NMR (400 MHz, CDCl3) δ 6.03 (t, J ) 2.4 Hz, 2H, Re-Cp-H (R)), 5.40 (t, J ) 2.4 Hz, 2H, Re-Cp-H (β)), 3.69 (s, 3H, OCH3), 2.92 (t, J ) 6.8 Hz, 2H, CH2), 2.31 (t, J ) 6.4 Hz, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ 193.241, 191.710, 172.857, 95.156, 87.977, 85.217, 51.961, 33.366, 27.635; MS (FAB) m/z 451 (M + H)+; HRMS calcd for C13H12O6187Re 451.0192, found 451.0188. Synthesis of 8-Cyclopentadienyltricarbonylrhenium-8-oxooctanoic Acid (2a). A solution of 5a (100 mg, 0.197 mmol) in 3 mL of 20% NaOH (aq) and ethanol (1:1) was heated at 80 °C for 1 h. The solution was cooled and acidified with 5 mL of 0.1 N HCl in an ice bath. The reaction mixture was extracted with CH2Cl2, and the organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by flash column chromatography (60:40 ethyl acetatehexane) to give 2a (79 mg, 82%) as a pale yellow oil: 1 H NMR (200 MHz, CDCl3) δ 5.99 (t, J ) 2.4 Hz, 2H, Re-Cp-H (R)), 5.40 (t, J ) 2.4 Hz, 2H, Re-Cp-H (β)), 2.59 (t, J ) 7.2 Hz, 2H, CH2), 2.36 (t, J ) 7.3 Hz, 2H, CH2), 1.73-1.62 (m, 4H), 1.39-1.26 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 195.136, 191.816, 179.453, 96.149, 87.863, 85.142, 38.597, 33.715, 28.681, 28.590, 24.391, 24.034; MS (FAB) m/z 493 (M + H)+; HRMS calcd for C16H18O6187Re 493.0662, found 493.0616. 6-Cyclopentadienyltricarbonylrhenium-6-oxohexanoic Acid (2b). A pale yellow oil: 1H NMR (400 MHz, CDCl3) δ 5.99 (t, J ) 2.4 Hz, 2H, Re-Cp-H (R)), 5.40 (t, J ) 2.2 Hz, 2H, Re-Cp-H (β)), 2.62 (t, J ) 7.0 Hz, 2H, CH2), 2.39 (t, J ) 7.2 Hz, 2H, CH2), 1.77-1.62 (m, 4H); 13 C NMR (100 MHz, CDCl3) δ 194.765, 191.816, 179.316, 95.686, 87.939, 85.255, 38.263, 33.707, 23.966, 23.443; MS (FAB) m/z 465 (M + H)+; HRMS calcd for C14H14O6187Re 465.0348, found 465.0356. 4-Cyclopentadienyltricarbonylrhenium-4-oxobutanoic Acid (2c). A pale yellow oil: 1H NMR (400 MHz, CDCl3) δ 6.03 (t, J ) 2.4 Hz, 2H, Re-Cp-H (R)), 5.41 (t, J ) 2.4 Hz, 2H, Re-Cp-H (β)), 2.90 (t, J ) 6.4 Hz, 2H, CH2), 2.74 (t, J ) 6.4 Hz, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ 195.580, 191.717, 179.305, 94.966, 88.053, 85.263, 33.123, 27.635; MS (FAB) m/z 437 (M + H)+; HRMS calcd for C12H10O6187Re 437.0035, found 437.0055. Radiochemical Synthesis of 99mTc-CpTTOA (1a). A solution of Na99mTcO4 eluted from a generator was placed in a glass tube and was concentrated to dryness under a stream of N2 at 75-80 °C. The resulting residue was treated with methanol, and the fraction soluble in methanol was collected in a pressure tube. To this were added the ester 3a (3 mg, 8.42 µmol), Cr(CO)6 (3.3 mg, 15.16 µmol), and CrCl3 (0.8 mg, 5.39 µmol). The reaction mixture was heated at 160 °C for 1 h. At the end of the reaction, the mixture was cooled in an ice bath and methanol was removed under a stream of N2. The residue was dissolved in CH2Cl2, passed through a short silica plug, and the solvent was removed. The residue was redissolved in methanol (150 µL) and treated with 0.4 N NaOH (50 µL) at 80 °C for 10 min. The solution was cooled, acidified with 50 µL of 0.5 N HCl, and extracted with CH2Cl2. 99mTc-CpTTOA (1a) was purified by HPLC

Bioconjugate Chem., Vol. 15, No. 1, 2004 123

at a flow 4 mL/min using a 91:9:0.09 mixture of hexane: methanol:acetic acid. 99mTc-CpTTOA collected from HPLC was concentrated, redissolved in ethanol, and diluted in saline to give a final solution of 10% ethanol in saline. A portion of the sample was treated with bovine serum albumin to give 5% of the total volume. An aliquot was analyzed by reversed phase HPLC at a flow 1 mL/min using a 70:30 mixture of methanol-0.1% acetic acid (aq). 99m Tc-CpTTHA (1b) and 99mTc-CpTTBA (1c) were prepared as in the synthesis of 1a. In Vitro Stability Studies. An aliquot (18.5 MBq) of 99m Tc-CpTTOA in 10% ethanol-saline was added to human serum (1 mL) and incubated at 37 °C, and the solution was analyzed at 30, 60, 240, and 360 min by radio-TLC using ethyl acetate-hexane (7:3) as the developing solvents. Animals. A group of mice (ICR, male, 28 g) was orally administered 30% CCl4 (1 mL CCl4/kg) in olive oil after 6 h-long fast, whereas control mice were administered the same volume of olive oil under the same conditions (3, 21). These two groups were fasted for another 2 h and then fed ad libitum for 15 h. Biological Studies. Control and CCl4-treated mice were fasted for another 3 h before administration of 99m Tc-CpTTOA (1.5 MBq/mouse) via tail vein. At 2, 30, 60, and 120 min postinjection, both groups of mice (n ) 3/time point) were sacrificed by cervical dislocation, and samples of blood and liver were isolated, weighed, and counted. The injected dose was calculated from standards prepared from the injection solution. The data were expressed as percent injected dose per gram of tissue (%ID/g). The 30 min samples of the serum and the liver were homogenized, extracted with a 2:1:1 mixture of CHCl3/MeOH/0.001 N NaOH, and centrifuged (3000 rpm, 30 min) (22). The resulting chloroform and aqueous layers (1 mL each) were counted, respectively. 99m Tc-CpTTOA was injected to 6-h-long fasted SD rats and planar images were obtained at 5, 30, and 60 min postinjection. Two sets of experiment were carried out, one with the radiotracer in 10% ethanol-saline and the other with additional 5% bovine serum albumin. In another set of experiment, SD rats (n ) 2/time point) were injected with 99mTc-CpTTOA, and samples of the liver and the bladder were collected at 30 and 60 min postinjection. The liver samples were homogenized and centrifuged as previously, whereas urine samples were filtered through a membrane (MW cutoff 10 000). The samples were analyzed by both HPLC at a flow rate of 1 mL/min using a 70:30 mixture of methanol and 0.1% acetic acid (aq) as the eluants and radio-TLC using a 50:50 mixture of methanol and 0.2% acetic acid (aq) as the developing solvents. RESULTS AND DISCUSSION

Synthesis of 8-Cyclopentadienyltricarbonylrhenium-8-oxooctanoic Acid (2a). The ester 3a was prepared by Friedel-Crafts acylation from ferrocene and methyl 8-chloro-8-oxooctanoate in the presence of AlCl3 (23). A double ligand transfer reaction of the ester 3a and NH4ReO4 using CrCl3 and Cr(CO)6 was carried out to provide Re compound 5a. This reaction was well described by Spradau and Katzenellenbogen (18), in that ReO4- is reduced and carbonylated, which undergoes a Cp ligand transfer reaction with the ferrocenyl precursor. The optimal reaction solvent was methanol among various solvents studied. Hydrolysis of the resulting compound in 0.4 N NaOH (aq) and methanol (1:3) afforded 8-cyclopentadienyltricarbonylrhenium-8-oxooctanoic acid

124 Bioconjugate Chem., Vol. 15, No. 1, 2004

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Table 1. Comparison of Radiochemical Yields Depending on Precursors and Solvents in the Double Ligand Transfer Reaction

Table 2. Comparison of Hydrolysis Conditions of the Methyl Ester of 99mTc-CpTTOA (1a)

precursor

solventa

yieldb (no. of reacion)

reagent

NaOH/ methanola

ratio of unknown peak to 1ab

3 4 4

methanol THF 2-propanol

58-82 (8) NRc (3) 12-15 (2)

20% NaOH/ethanol 0.1 N NaOH/methanol 0.2 N NaOH/methanol 0.4 N NaOH/methanol 0.4 N NaOH/methanol 0.5 N NaOH/methanol 0.7 N KOH/methanol

1:1 1:1 1:1 1:1 1:3 1:1 1:1

NRc 4/1 3/1 3.3/1 1/3 9/1 8/1

a A total volume was 200 µL. b Yield was based on radio-TLC data. c NR: no reaction.

(2a). A double ligand transfer reaction of the carboxylic acid 4 in methanol gave the 5a instead of the 2a (2-5% yield), possibly due to methyl ester formation of the carboxylic acid by methanol. The ester 3b was prepared from ferrocene and methyl 6-chloro-6-oxohexanoyl chloride in the presence of AlCl3, followed by methyl ester formation in methanol in the presence of triethylamine. Re complexes 2b and 2c were prepared as the synthesis of 2a. Radiochemical Synthesis of 99mTc-CpTTOA (1a). 99m Tc-CpTTOA was synthesized via the same route as the preparation of Re-fatty acid 2a (18-20). The ester 3a was reacted with 99mTcO4- in the presence of CrCl3 and Cr(CO)6 at 160 °C for 1 h. When either the acid 4 was used as the precursor or THF (or 2-propanol) as the solvent, the radiochemical yield of 1a was low as shown in Table 1 and some unknown radioactive peaks appeared on radio-TLC. Therefore, the ester 3a and methanol were employed to produce 1a as shown in Scheme 1. Hydrolysis of the 99mTc-labeled compound was attempted in 20% NaOH (aq) and ethanol (24) as described for the hydrolysis of the 5a, but unknown radioactive compounds were detected upon analysis by radio-TLC. Although the unknown peaks were not identified, they were likely to be derived from cleavage of the 99mTc core. Therefore, hydrolysis conditions were varied to give a maximal yield of the desired product and to minimize the production of

a The reaction was carried out in a total volume of 200 µL (80 °C, 10 min). b Measured by radio-TLC. c NR: no reaction.

the unknown radiochemical impurities. As shown in Table 2, hydrolysis of the methyl ester of 99mTc-CpTTOA in a 1:3 mixture of 0.4 N NaOH and methanol gave the highest yield (72-77%). The reaction mixture was then purified by HPLC, and the retention time of 99mTcCpTTOA was 12.5 min using a 91:9:0.09 mixture of hexane:methanol:acetic acid as the eluants. An aliquot was analyzed by reversed phase HPLC, and 99mTcCpTTOA and Re-CpTTOA were eluted at 16.9 and 14.4 min, respectively. Although they were not coeluted on HPLC, 99mTc-CpTTOA was identified based on the similar chemical properties between Tc and Re complexes. The different retention times of Tc and Re complexes on HPLC have been also reported in other cases (20). Synthesis of 99mTc-CpTTOA gave higher yield than the corresponding Re complex, due to higher reactivity of 99mTc than Re. Overall radiochemical yield of 99mTc-CpTTOA was 50-63% (decay-corrected) and radiochemical purity was higher than 99% as determined by HPLC and radio-TLC. 99mTc-CpTTHA (1b) and 99m Tc-CpTTBA (1c) were prepared as in the synthesis of 1a and used as the radiometabolite standards of 1a. HPLC retention times of 1b and 1c were 11.1 and 8.2

Scheme 1 a

a Reaction conditions: (i) AlCl , CH Cl , 0 °C, 3 h; (ii) MeOH, Et N, 90 °C, 1 h; (iii) 20% NaOH (aq)-ethanol (1:1), 80 °C, 1 h; (iv) 3 2 2 3 NH4ReO4, CrCl3, Cr(CO)6, CH3OH, 160 °C, 1 h; (v) Na99mTcO4, CrCl3, Cr(CO)6, CH3OH, 160 °C, 1 h; (vi) 0.4 N NaOH (aq)-CH3OH (1:3), 80 °C, 10 min.

8-Cyclopentadienyltricarbonyl

99mTc

Table 3. In Vitro Stability of Human Serum

8-Oxooctanoic Acid 99mTc-CpTTOA

Bioconjugate Chem., Vol. 15, No. 1, 2004 125 (1a) in

time (min)

% of 99mTc-CpTTOAa

30 60 240 360

98 98 94 92

Table 4. Solubility of the Radioactivity in Liver and Blood Samples of Control and CCl4-Treated Mice at 30 min after Administration of 99mTc-CpTTOA (1a) control CCl4-treated

a 99mTc-CpTTOA

was incubated with human serum at 37 °C for 6 h and the percentage of the remaining radiotracer was analyzed by radio-TLC.

a

tissue

organica

aqueousa

liver serum liver serum

1.7 14.5 1.6 8.9

98.3 85.5 98.4 91.1

Mean average of three mice.

Figure 1. Measurement of the radioactivity in liver and blood samples of control and CCl4-treated mice. Both groups of mice were injected with 99mTc-CpTTOA, and the liver and blood were collected at 2, 30, 60, and 120 min postinjection and counted. The injected dose was calculated from standards prepared from the injection solution. The data were expressed as percent injected dose per gram of tissue (%ID/g). Closed bar: liver sample from control mice; open bar: liver sample from CCl4treated mice; right-handed striped bar: blood sample from control mice; left-handed striped bar: blood sample from CCl4treated mice.

min, respectively, when analyzed in a 70:30 mixture of methanol-0.1% acetic acid (aq) at a flow rate of 1 mL/min. In Vitro Stability Studies. Percentage of the remaining 99mTc-CpTTOA was 98% after 1 h and over 90% even after 6 h when the radiotracer was incubated with human serum at 37 °C and analyzed by radio-TLC, indicating high in vitro stability of the radiotracer (Table 3). Measurement of the Radioactivity in the Liver and Blood Samples of Control and CCl4-Treated Mice. Control and CCl4-treated mice were fasted for 3 h before injected with 99mTc-CpTTOA. Initial uptake of the radioactivity in CCl4-treated mice was similar to that in control mice at 2 min postinjection of 99mTc-CpTTOA, as shown in Figure 1. In contrast, the amounts of radioactivity in the liver of CCl4-treated mice were higher than those of control mice at 30, 60, and 120 min postinjection, reaching a maximum at 60 min. The radioactivity in the samples of blood was slightly higher in CCl4-treated mice. This was also the case when [1-11C]octanoate was evaluated in the control and CCl4-treated mice (3), although the difference in the liver uptake of the radioactivity between the two groups of mice was more profound in [1-11C]octanoate than in 99mTc-CpTTOA. This result demonstrated that the 99mTc-CpTTOA underwent slower hepatic clearance in CCl4-treated mice compared to the control mice, probably due to impaired β-oxidation caused by CCl4 that is known to induce the hepatotoxicity (21). Solubility of the Radioactivity in the Liver and Blood Samples of Control and CCl4-Treated Mice. Most of the radioactivities obtained from the blood and liver at 30 min postinjection in control and CCl4-treated mice were extracted into the aqueous layer rather than into the organic layer, indicating that the metabolites are water-soluble (Table 4). The radioactivities produced by

Figure 2. Planar images of 99mTc-CpTTOA (1a) in rats. The radiotracer (11 MBq) was administered to SD rats via tail vein and planar images were obtained at 5, 30, and 60 min postinjection, respectively (A). Another sets of planar images were obtained at 5 and 30 min after administration of the radiotracer containing 5% bovine serum albumin (B).

esterification should be extracted into chloroform layer. The results therefore indicated that the 99mTc-CpTTOA was predominately metabolized by β-oxidation in the liver and not esterified as triglycerides, which was consistent with the results of other medium chain fatty acid analogues such as p-[123I]iodophenylenanthic acid, p-[123I]iodophenylvaleric acid, and [1-11C]octanoate in the liver (3, 6). Imaging Studies. Since fatty acids are predominantly metabolized in the fasted state, the animals were fasted for 6 h prior to the experiments. Planar images of the rats injected with 99mTc-CpTTOA showed rapid uptake of the radioactivity in the liver, kidneys and bladder at 5 min postinjection with rapid elimination of the radioactivity from the liver as a function of time (Figure 2A). Within 60 min, most of the radioactivity was observed in the kidneys and bladder. This rapid hepatic clearance indirectly suggested that this fatty acid analogue was not esterified, unlike long chain fatty acid. Uptake pattern of the 99mTc-CpTTOA was not significantly affected by the presence of 5% bovine serum albumin (Figure 2B), even though bovine serum albumin is known to facilitate the penetration of the fatty acid through the cell membranes (25). There was no significant uptake in the thyroid or salivary gland, indicating that 99mTc pertechnetate was not regenerated during the time of the study.

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tabolite appeared at the retention time of 8.2 min on HPLC and was identified as 99mTc-CpTTBA on both radio-TLC and HPLC (Figure 3). 99mTc-CpTTHA, the metabolite after one cycle of β-oxidation of 99mTcCpTTOA, was not detected (Figure 3B-D). Ratios of peak area under 99mTc-CpTTBA to 99mTc-CpTTOA were 77: 23 and 90:10 in 30 min samples of the liver and urine, respectively (Figure 3B and C). The 60 min samples of the liver were not analyzed because of the insufficient radioactivity. The parent was completely converted to the metabolite in the 60 min sample of the urine (Figure 3D). This indicated that 99mTc-CpTTOA was metabolized to 99m Tc-CpTTBA via β-oxidation in the liver and excreted to the kidneys and bladder, which was similar to the metabolism of [99mTc]MAMA-hexanoic acid in the liver (4). CONCLUSION

The labeling method using 99mTc-CpTT was advantageous, because the precursor was readily prepared by Friedel-Crafts acylation from ferrocene and fatty acid analogue, and 99mTc labeling via a double ligand transfer reaction was easily achievable. A novel radiotracer, 99m Tc-CpTTOA, was metabolized via β-oxidation in the liver, which supported that the β-oxidation was the main metabolic pathway of this medium chain fatty acid analogue. The results suggest that 99mTc-CpTTOA may be useful for evaluation of fatty acid metabolism in the liver. This is the first example of the medium chain fatty acid analogue containing 99mTc-CpTT. ACKNOWLEDGMENT

This work was supported in part by a grant of Nuclear R & D Program, Korea Ministry of Science and Technology (M20204-190027-02A0701-00150). We thank Mr. S. Y. Kim and S. W. Choi for their technical assistance. Supporting Information Available: Combustion analysis data. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED

Figure 3. HPLC profile of the standards. (A); tR ) 8.2 min (1c), 11.1 min (1b), 16.9 min (1a). HPLC analysis of the liver and urine samples of the rats injected with 99mTc-CpTTOA (1a); (B): liver, 30 min, (C): urine 30 min, and (D): urine 60 min.

It is likely that the lipophilic properties of 99mTc-CpTT contributed to rapid uptake of this fatty acid analogue to the liver because medium chain fatty acid does not use carnitine shuttle step when enters the mitochondria, whereas a polar [99mTc]MAG-hexanoic acid was hardly accumulated in the liver (4). Although 99mTc-CpTT is smaller than 99mTc-N2S2 core, difference in the size of the cores did not significantly affect on the biological activity of the corresponding medium chain fatty acid analogue. Analysis of Metabolites. When the samples of the liver and urine of rats were analyzed, the sole radiome-

(1) Burt, A. D., and James, O. F. W. (1994) Pathophysiology of the Liver. Pathology of the Liver (MacSween, R. N. M., Anthony, P. P., Scheuer, P. J., Burt, A. D., and Portmann, B. C., Eds.) pp 51-81, Churchill Livingstone, New York. (2) Zakim, D. (1996) Metabolism of Glucose and Fatty Acids by the Liver. Hepatology. A Textbook of Liver Disease (Zakim, D., and Boyer, T. D., Eds.) pp 58-92, Saunders, Philadelphia. (3) Yamamura, N., Magata, Y., Kitano, H., Konishi, J., and Saji, H. (1998) Evaluation of [1-11C]octanoate as a new radiopharmaceutical for assessing liver function using positron emission tomography. Nucl. Med. Biol. 25, 467-472. (4) Yamamura, N., Magata, Y., Arano, Y., Kawaguchi, T., Ogawa, K., Konishi, J., and Saji, H. (1999) Technetium-99mlabeled medium-chain fatty acid analogues metabolized by β-oxidation: radiopharmaceutical for assessing liver function. Bioconjugate Chem. 10, 489-495. (5) Bennett, M. J. (1994) The enzymes of mitochondrial fatty acid oxidation. Clin. Chim. Acta 226, 211-224. (6) Yamamura, N., Magata, Y., Konishi, J., and Saji, H. (1999) Evaluation of radioiodinated medium chain fatty acids as new diagnostic agents for the determination of hepatic viability. Eur. J. Nucl. Med. 26, 1597-1605. (7) Eckelman, W. C., Karesh, S. M., and Reba, R. C. (1975) New compounds: fatty acid and long chain hydrocarbon derivatives containing a strong chelating agent. J. Pharma. Sci. 64, 704-706. (8) Karesh, S. M., Eckelman, W. C., and Reba, R. C. (1977) Biological distribution of chemical analogues of fatty acids

8-Cyclopentadienyltricarbonyl

99mTc

8-Oxooctanoic Acid

and long chain hydrocarbons containing a strong chelating agent. J. Pharm. Sci. 86, 225-228. (9) Mach, R. H., Kung, H. F., Jungwiwattanaporn, P., and Guo, Y. Z. (1991) Synthesis and biodistribution of a new class of 99mTc-labeled fatty acid analogues for myocardial imaging. Nucl. Med. Biol. 18, 215-226. (10) Jones, G. S., Elmaleh D. R., Strauss, H. W., and Fischman, A. J. (1994) Synthesis and biodistribution of a new 99mtechnetium fatty acid. Nucl. Med. Biol. 21, 117-123. (11) Loberg, M. D., Cooper, M., Harvey, E., Callery, P., and Faith, W. (1976) Development of new radiopharmaceuticals based on N-substituted iminodiacetic acid. J. Nucl. Med. 17, 633-638. (12) Ryan, J., Cooper, M., Loberg, M., Harvey, E., and Sikorski, S. (1977) Technetium-99m-labeled N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid (Tc-99m HIDA): a new radiopharmaceutcial for hepatobiliary imaging studies. J. Nucl. Med. 18, 997-1004. (13) Eisenhut, M. (1982) Simple labeling of omega-phenylfatty acids by iodine isotope exchange. Int. J. Appl. Radiat. Isot. 33, 499-504. (14) Ambrose, K. R., Owen, B. A., Goodman, M. M., and Knapp, F. F., Jr. (1987) Evaluation of the metabolism in rat hearts of two new radioiodinated 3-methyl-branched fatty acid myocardial imaging agents. Eur. J. Nucl. Med. 12, 486-491. (15) Schwochau, K. (1994) Technetium radiopharmaceuticals. Fundamentals, synthesis, structure, and development. Angew. Chem., Int. Ed. 33, 2258-2267. (16) Jurisson, S., Berning, D., Jia, W., and Ma, D. S. (1993) Coordination compounds in nuclear medicine. Chem. Rev. 93, 1137-1156. (17) Gholson, C. F., Provenza, J. M., and Bacon, B. R. (1990) Hepatological considerations in patients with parenchymal liver disease undergoing surgery. Am. J. Gastroenterol. 85, 487-496.

Bioconjugate Chem., Vol. 15, No. 1, 2004 127 (18) Spradau, T. W. and Katzenellenbogen, J. A. (1998) Preparation of cyclopentadienyltricarbonylrhenium complexes using a double ligand transfer reaction. Organometallics 17, 2009-2017. (19) Cesati, R. R., III, Tamagnan, G., Baldwin, R. H., Zoghbi, S. S., Innis, R. B., and Katzenellenbogen, J. A. (1999) Synthesis of cyclopentadienyl tricarbonyl technetium phenyl-tropane derivatives by direct double ligand transfer with ferrocene precursors. J. Labelled Compd. Radiopharm. 42, S150-152. (20) Skaddan, M. B., Wust, F. R., Jonson, S., Syhre, R., Welch, M. J., Spies, H., and Katzenellenbogen, J. A. (2000) Radiochemical synthesis and tissue distribution of Tc-99m-labeled 7R-substituted estradiol complex. Nucl. Med. Biol. 27, 269278. (21) Hjelle, J. J., Grubbs, J. H., Beer, D. G., and Petersen, D. R. (1983) Time course of the carbon tetrachloride-induced decrease in mitochondrial aldehyde dehydrogenase activity. Toxicol. Appl. Pharmacol. 67, 159-165. (22) Forch, J., Less, M., and Sloane-Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509. (23) Reeves, P. C. (1963) Carboxylation of Aromatic Compound: Ferrocenecarboxylic Acid. Organic Syntheses (Mrowca, J. J., Borecki, M. M., and Sheppard, W. A., Eds.) Coll. Vol. VI, pp 625-627, John Wiley & Sons, New York. (24) Burness, D. M. (1963) 3-Methyl-2-Furoic Acid and 3-Methylfuran. Organic Syntheses (Cason, J., and Hutchinson, R. B., Eds.) Coll. Vol. IV, pp 628-630, John Wiley & Sons, New York. (25) Hamilton, J. A. (1989) Medium-chain fatty acid binding to albumin and transfer to phospholipid bilayers. Proc. Natl. Acad. Sci. U.S.A. 86, 2663-2667.

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