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) 99mTc-
3-15-[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.
droxycarbonylmethyl)-2-picolylamino group can be labeled with oil in 81% yield: 1H NMR (CDCl3, 400 MHz) δ 8.52 (dd, 1H,
[99mTc(CO)3(H2O)3]+ to form a 99mTc(CO)3-[N-(acetyloxy)-2- J ) 2.0, 1.6 Hz), 7.65 (t, 1H, J ) 8.0 Hz), 7.54 (d, 1H, J ) 7.6
picolylamino] core, an ideal tridentate chelator. Hz), 7.14 (t, 1H, J ) 6.0 Hz), 3.90 (s, 2H), 3.66 (t, 3H, J ) 7.6
In this work, we prepared 99mTc(CO)3-[N-(acetyloxy)-2- Hz), 3.29 (s, 2H), 2.62 (t, 2H, J ) 7.6 Hz), 2.30 (t, 2H, J ) 7.6
picolylamino]pentadecanoic acid (1a) by the facile incorporation Hz), 1.64-1.58 (m, 2H), 1.51-1.41 (m, 9H), 1.31-1.18 (m,
of [99mTc(CO)3]+ into a N-(hydroxycarbonylmethyl)-2-picoly- 22H); 13C NMR (CDCl3, 100 MHz) δ 174.0, 170.1, 160.0,
lamino fatty acid analog and evaluated its potential as a substrate 148.8, 136.1, 122.7, 121.6, 80.4, 60.3, 55.9, 54.1, 51.1, 33.9,
for fatty acid metabolism. The β-oxidation products of 1a were 29.4; MS (FAB) m/z 477 (M+ + H); HRMS calcd for
compared with the proposed radioactive metabolite standards, C28H49N2O4 477.3693, found 477.3697.
99mTc(CO) -5-[N-(acetyloxy)-2-picolylamino]pentanoic acid (1b)
3 Synthesis of Methyl 5-[N-(tert-Butyloxycarbonylmethyl)-
and 99mTc(CO)3-3-[N-(acetyloxy)-2-picolylamino]propionic acid 2-picolylamino]pentanoate (4b). Compound 3 (250 mg, 1.12
(1c). mmol) was dissolved in CH3CN (7 mL), and to this solution
was added DIEA (980 µL, 5.63 mmol). After stirring for 30
EXPERIMENTAL SECTION min at room temperature, methyl 5-bromopentanoate (240 µL,
1.69 mmol) was added dropwise. The remainder of the
Materials and Methods. Chemicals and solvents were
procedure was as described above. Flash column chromatog-
obtained from Sigma-Aldrich (St. Louis, MO), and HPLC
raphy (2:1 hexane-ethyl acetate) gave 4b (280 mg) as a light
solvents were obtained from J. T. Baker (Phillipsburg, NJ). 1H
yellow oil in 74% yield: 1H NMR (CDCl3, 500 MHz) δ 8.55-
NMR spectra were obtained using a Varian Unity Inova 500NB
8.53 (m, 1H), 7.67 (td, 1H, J ) 8.0, 2.0 Hz), 7.55 (d, 1H, J )
(500 MHz) spectrometer (Palo Alto, CA) at the Cooperative
7.6 Hz), 7.18-7.15 (m, 1H), 3.93 (s, 2H), 3.66 (s, 3H), 3.31 (s,
Center for Research Facilities, Sungkyunkwan University (Su-
2H), 2.68 (t, 2H, J ) 7.6 Hz), 2.30 (t, 2H, J ) 7.6 Hz), 1.68-
won, Korea), and a Varian Gemini-400 (Palo Alto, CA) at Inha
1.62 (m, 2H), 1.56-1.50 (m, 2H), 1.48 (s, 9H); MS (FAB) m/z
University, and 13C NMR spectra were obtained at 100 MHz.
337 (M+ + H); HRMS calcd for C18H29N2O4 337.2128, found
Chemical shifts (δ) are reported as parts per million (ppm)
337.2132.
downfield of an internal tetramethylsilane standard. Fast atom
bombardment (FAB) mass spectra were obtained using a JMS- Synthesis of Methyl 3-[N-(tert-Butyloxycarbonylmethyl)-
700 Mstation (JEOL, Ltd., Tokyo, Japan) at the Korea Basic 2-picolylamino]propionate (4c). Compound 3 (400 mg, 1.75
Science Institute (Seoul, Korea). HPLC was carried out using mmol) was dissolved in CH3CN (12 mL), and to this solution
a Thermo Separation Products System (Fremont, CA) equipped was added NaHCO3 (453.6 mg, 5.40 mmol). After stirring for
with a semipreparative column (Alltech Econosil silica gel, 10 30 min at room temperature, methyl 3-bromopropionate (257
µ, 10 × 250 mm) or an analytical column (YMC-Pack C18, 5 µL, 2.34 mmol) was added dropwise. The mixture was then
µ, 4.6 × 250 mm). Eluant was simultaneously monitored using stirred at 60 °C overnight. Solvent was removed in vacuo, and
a UV detector (254 nm) and a NaI(Tl) radioactivity detector. the residue was extracted with dichloromethane (30 mL × 3),
TLC was performed on Merck F254 silica plates, which were washed with water (30 mL × 3), and dried over anhydrous
analyzed using a Bioscan radio-TLC scanner (Washington, DC). Na2SO4. Flash column chromatography (1:1 hexane-ethyl
Na99mTcO4 was eluted on a daily basis from a 99Mo/99mTc acetate) gave 4c (320 mg) as a yellow oil in 58% yield: 1H
generator (Samyoung Unitech, Co., Ltd., DuPont Pharmaceu- NMR (CDCl3, 500 MHz) δ 8.55-8.54 (m, 1H), 7.67 (td, 1H,
ticals Co., and Daiichi Radioisotope Labs). Radioactivity was J ) 8.0, 2.0 Hz), 7.50 (d, 1H, J ) 8.0 Hz), 7.19-7.16 (m, 1H),
measured in a dose calibrator (Biodex Medical Systems, Shirley, 3.99 (s, 2H), 3.66 (s, 3H), 3.57 (s, 2H), 3.08 (t, 2H, J ) 5.0
NY) and tissue radioactivity in a Wallac automated γ counter Hz), 2.53 (t, 2H, J ) 7.0 Hz), 1.48 (s, 9H); MS (FAB) m/z 309
(Boston, MA). All animal experiments were performed in (M+ + H); HRMS calcd for C16H25N2O4 309.1815, found
compliance with the rules of the Samsung Medical Center 309.1806.
Laboratory Animal Care, which comply with NIH guidelines. Synthesis of Methyl 15-[N-(Hydroxycarbonylmethyl)-2-
Synthesis of tert-Butyl 2-Picolylamino-N-acetate (3). 2-Pi- picolylamino]pentadecanoate (5a). Trifluoroacetic acid (2 mL)
colylamine (946 µL, 9.25 mmol) was added to tert-butyl was added to 4a (250 mg, 0.52 mmol) in dichloromethane (2
bromoacetate (1.7 mL, 12.03 mmol) in CH3CN (10 mL). The mL), and the reaction mixture was stirred for 2 h at room
reaction mixture was then stirred under N2 at room temperature temperature. At the end of the reaction, solvent was removed
overnight. At the end of the reaction, solvent was removed in in vacuo, and the residue was extracted with dichloromethane
vacuo, and residue was extracted with dichloromethane (30 mL (30 mL × 3), washed with water, and dried over anhydrous
× 3), washed with water, and dried over anhydrous Na2SO4. Na2SO4. Flash column chromatography (15:1 dichloromethane-
Flash column chromatography (2:1 hexane-ethyl acetate) gave methanol) gave 5a (188 mg) as a light yellow oil in 85% yield.
3 (1.48 g) as a yellow oil in 72% yield: 1H NMR (CDCl3, 500 Compounds 5b and 5c were synthesized in 79% and 62% yields,
MHz) δ 8.57 (d, 1H, J ) 5.0 Hz), 7.68-7.65 (m, 1H), 7.35 (d, respectively, using the same procedure described for 5a.
1H, J ) 5.5 Hz), 7.19-7.17 (m, 1H), 3.96 (s, 2H), 3.39 (s, 5a. 1H NMR (CD3OD, 500 MHz) δ 8.67-8.66 (m, 1H), 7.91
2H), 1.48 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ 171.2, 159.2, (td, 1H, J ) 8.0, 1.5 Hz), 7.53 (d, 1H, J ) 8.0 Hz), 7.47-7.44
149.1, 136.2, 121.8, 121.7, 80.8, 54.4, 51.0, 27.8; MS (FAB) (m, 1H), 4.56 (s, 2H), 3.76 (s, 2H), 3.66 (s, 3H), 3.27-3.23
m/z 223 (M+ + H); HRMS calcd for C12H19N2O2 223.1447, (m, 2H), 2.32 (t, 2H, J ) 7.0 Hz), 1.80-1.75 (m, 2H), 1.63-
found 223.1451. 1.60 (m, 2H), 1.30 (s, 20H); 13C NMR (CDCl3, 100 MHz) δ
Synthesis of Methyl 15-[N-(tert-Butyloxycarbonylmethyl)- 174.0, 170.1, 160.0, 148.8, 136.1, 122.7, 121.6, 80.4, 60.3, 55.9,
2-picolylamino]pentadecanoate (4a). Compound 3 (300 mg, 54.1, 51.1, 33.9, 29.4, 27.98, 27.4, 24.7; MS (FAB) m/z 421
1.35 mmol) was dissolved in CH3CN (12 mL), and to this (M+ + H); HRMS calcd for C24H41N2O4 421.3066, found
solution was added DIEA (1.18 mL, 6.75 mmol). After stirring 421.3066.
for 30 min at room temperature, methyl 15-bromopentade- 5b. 1H NMR (CD3OD, 500 MHz) δ 8.67-8.65 (m, 1H), 7.92
canoate (543 mg, 1.62 mmol) was added dropwise. The reaction (td, 1H, J ) 7.8, 2.0 Hz), 7.54 (d, 1H, J ) 8.0 Hz), 7.53-7.45
mixture was then stirred at 75 °C overnight, extracted with (m, 1H), 4.55 (s, 2H), 3.78 (s, 2H), 3.67 (s, 3H), 3.27-3.24
dichloromethane (30 mL × 3), washed with water, and dried (m, 2H), 2.38 (t, 2H, J ) 2.0 Hz), 1.83-1.79 (m, 2H), 1.69-
over anhydrous Na2SO4. Flash column chromatography (4:1 1.66 (m, 2H); MS (FAB) m/z 281 (M+ + H); HRMS calcd for
hexane-ethyl acetate) afforded 4a (521 mg) as a light yellow C14H21N2O4 281.1502, found 281.1495.
1334 Bioconjugate Chem., Vol. 18, No. 4, 2007 Lee et al.
5c. 1H NMR (CD3OD, 500 MHz) δ 8.62 (d, 1H, J ) 4.0 for 4 h at 70 °C. The solution was then cooled and neutralized
Hz), 7.92 (td, 1H, J ) 7.5, 1.5 Hz), 7.57 (d, 1H, J ) 8.0 Hz), with 1 mL of 0.1 N HCl in an ice bath. The reaction mixture
7.46-7.44 (m, 1H), 4.43 (s, 2H), 3.70 (s, 2H), 3.42-3.39 (m, was then extracted with dichloromethane, and the organic layer
2H), 3.36 (s, 3H), 2.76-2.74 (m, 2H); MS (FAB) m/z 253 (M+ was dried over anhydrous Na2SO4 and concentrated. Sold-phase
+ H); HRMS calcd for C12H17N2O4 253.1189, found 253.1192. purification (C18 Sep-Pak cartridge) gave 6c (9.3 mg) as a light
Synthesis of 15-[N-(Hydroxycarbonylmethyl)-2-picoly- yellow solid in 32% yield: mp 257-259 °C; 1H NMR (CD3-
lamino]pentadecanoic Acid (2a). Compound 5a (40 mg, 0.10 OD, 500 MHz) δ 8.83 (d, 1H, J ) 6.0 Hz), 8.10 (td, 1H, J )
mmol) was dissolved in a 1:1 mixture of 0.4 N NaOH and 8.0, 1.5 Hz), 7.72 (d, 1H, J ) 8.0 Hz), 7.56 (t, 1H, J ) 5.5
MeOH (3 mL) and stirred for 4 h at 70 °C. The solution was Hz), 4.84-4.76 (m, 2H), 4.58-4.55 (m, 2H), 3.93-3.90 (m,
then cooled and neutralized with 1.5 mL of 0.1 N HCl in an 2H), 2.92-2.87 (m, 2H); MS (FAB) m/z 509 (M+ + H); HRMS
ice bath. The reaction mixture was then extracted with dichlo- calcd for C14H14N2O7187Re 509.0359, found 509.0359.
romethane, and the organic layer was dried over anhydrous Radiochemical Synthesis of 99mTc(CO)3-15-[N-(Acetyloxy)-
Na2SO4 and concentrated. Flash column chromatography (9:1 2-picolylamino]pentadecanoic Acid (1a). Kit Preparation of
CH2Cl2-MeOH) gave 2a (22 mg) as light yellow solid in 54% [99mTc(CO)3(H2O)3]+. 99mTcO4- (740 MBq, 300 µL) was added
yield. Compound 2b was prepared in 46% yield using the to a 10 mL sealed kit vial containing a lyophilized formulation
procedure described for 2a and C-18 Sep-Pak purification. (IsoLink). The vial was then shaken vigorously and placed in
2a. 1H NMR (CD3OD, 500 MHz) δ 8.55 (d, 1H, J ) 4.5 an oil bath for 30 min at 75 °C. It was then cooled to room
Hz), 7.84 (td, 1H, J ) 7.8, 1.0 Hz), 7.61 (d, 1H, J ) 7.5 Hz), temperature, and the mixture was neutralized with 0.1 N HCl
7.37-7.34 (m, 1H), 4.10 (s, 2H), 3.36 (s, 2H), 2.79 (t, 2H, J ) (180 µL). The radio-TLC yield of [99mTc(CO)3(H2O)3]+ was
7.0 Hz), 2.17 (t, 2H, J ) 7.5 Hz), 1.62-1.59 (m, 4H), 1.28 (s, greater than 95%.
20H); MS (FAB) m/z 429 (M+ + Na); HRMS calcd for In-House Preparation of [99mTc(CO)3(H2O)3]+. A mixture of
C23H38N2O4Na 429.2732, found 429.2729. Na2CO3 (4 mg, 38 µmol), NaK tartrate tetrahydrate (2 mg), and
2b. 1H NMR (CD3OD, 500 MHz) δ 8.69-8.68 (m, 1H), NaBH4 (5 mg, 130 µmol) was placed in a 10 mL reaction vial
8.01-7.97 (m, 1H), 7.59-7.57 (m, 1H), 7.54-7.51 (m, 1H), and dissolved in water (500 µL), and the vial was then sealed.
4.59 (d, 2H, J ) 3.0 Hz), 4.06 (d, 2H, J ) 6.0 Hz), 3.67-3.66 After bubbling CO in this solution for 10 min, 99mTcO4- (370
(m, 2H), 2.40-2.34 (m, 2H), 1.89-1.77 (m, 2H), 1.69-1.64 MBq, 200 µL) was added, and the resulting solution was stirred
(m, 2H); MS (FAB) m/z 289 (M+ + Na); HRMS calcd for for 30 min at 75 °C, cooled to room temperature, and then
C13H18N2O4Na 289.1167, found 289.1169. neutralized with 0.1 N HCl (180 µL). Radio-TLC yield was
Synthesis of Re(CO)3-15-[N-(Acetyloxy)-2-picolylamino]- greater than 95%.
pentadecanoic Acid (6a). (NEt4)2[Re(CO)3Br3] was prepared
Radiochemical Synthesis of 1a. Compound 2a (1.1 mg, 2.7
as described in the literature (30). (NEt4)2[Re(CO)3Br3] (187
µmol) was dissolved in a 4:1 mixture of methanol and water
mg, 0.24 mmol) was added to 2a (60 mg, 0.16 mmol) in MeOH
(300 µL), and to this solution was added [99mTc(CO)3(H2O)3]+
(1 mL) and stirred for 2 h at 65 °C. After reaction, solvent was
(370 MBq, 250 µL). The reaction mixture was then stirred in a
removed in vacuo, and the resulting oily residue was purified
sealed vial for 30 min at 75 °C. The crude mixture was loaded
by flash column chromatography (10:1 CH2Cl2-MeOH) to give
onto a C18 Sep-Pak cartridge, which was washed with water
6a (57 mg) as a light yellow solid in 53% yield. Compound 6b
(10 mL) and then with methanol (2 mL). The methanol layer
was synthesized in 51% yield using the procedure described
was concentrated under N2 and purified by HPLC using a 8:92
for 6a.
mixture of hexane and dichloromethane-2-propanol-acetic acid
6a. mp 221-225 °C; 1H NMR (CDCl3, 500 MHz) δ 8.85 (88:12:0.3) at 4 mL/min. The desired fraction (tR ) 21.5-23.5
(d, 1H, J ) 5.5 Hz), 7.95 (td, 1H, J ) 8.0, 1.5 Hz), 7.51 (d, min) was collected, and solvents were removed under N2. The
1H, J ) 7.5 Hz), 7.44 (t, 1H, J ) 6.0 Hz), 4.51-4.48 (m, 2H), residue was then passed through a C18 Sep-Pak cartridge that
4.37-4.34 (m, 2H), 3.74-3.70 (m, 2H), 3.58-3.54 (m, 2H), was washed with water (10 mL) and then with MeOH (2 mL).
2.36-2.33 (m, 2H), 1.61-1.59 (m, 2H), 1.27 (s, 20H); MS Methanol removal gave 1a in 60-63% yield. Radiotracer 1b
(FAB) m/z 677 (M+ + H); HRMS calcd for C26H38N2O7187Re was prepared in an identical manner and purified using a C18
677.2237, found 677.2245. Sep-Pak cartridge at yields of 48%.
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, In preparation of 1c, methyl ester 5c (1.3 mg, 4.64 µmol)
1H, J ) 8.0 Hz), 7.55 (t, 1H, J ) 6.0 Hz), 4.75-4.72 (m, 2H), was labeled with [99mTc(CO)3(H2O)3]+ (37 MBq, 25 µL) for
4.57-4.54 (m, 2H), 3.95-3.91 (m, 2H), 3.64-3.62 (m, 2H), 30 min at 75 °C. After solid-phase purification, the residue was
2.42 (t, 2H, J ) 7.0 Hz), 1.71-1.69 (m, 2H); MS (FAB) m/z redissolved in methanol (300 µL) and treated with 0.4 N NaOH
537 (M+ + H); HRMS calcd for C16H18N2O7187Re 537.0672, (aq, 100 µL) for 10 min at 65 °C. The reaction mixture was
found 537.0668. then cooled, neutralized with 1 N HCl (40 µL), and purified
using a C18 Sep-Pak cartridge and methanol as the eluant, which
Synthesis of Re(CO)3-3-[N-(Acetyloxy)-2-picolylamino]-
gave 1c in 34% yield.
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 In Vitro Stability. An aliquot (9.7 MBq) of 1a in saline was
stirred for 2 h at 65 °C. After reaction, solvent was removed in added to human plasma (1 mL) and incubated for 4 h at 37 °C,
vacuo, and the resulting oily residue was purified by flash and the resulting solution was analyzed by radio-TLC using
column chromatography (20:1 CH2Cl2-MeOH) to give Re dichloromethane-methanol (9:1).
coordinated methyl ester (38 mg) as a light yellow solid in 46% Tissue Distribution in Normal Mice. Radiotracer 1a was
yield: mp 187-192 °C; 1H NMR (CDCl3, 500 MHz) δ 8.86 dissolved in saline containing 1% bovine serum albumin
(d, 1H, J ) 5.0 Hz), 7.96 (td, 1H, J ) 8.0, 1.5 Hz), 7.51 (d, (BSA) and 10% ethanol at 40 °C. ICR mice (male, 25-30 g,
1H, J ) 8.0 Hz), 7.46 (t, 1H, J ) 6.5 Hz), 4.44-4.36 (m, 2H), four mice per time point) were fasted for 12 h before injec-
3.94-3.91 (m, 2H), 3.80 (s, 3H), 3.39-3.36 (m, 2H), 2.87 (t, ting 1a (1.11 MBq/mouse) via a tail vein. At the indicated
2H, J ) 7.0 Hz); MS (FAB) m/z 523 (M+ + H); HRMS calcd time points (0.5, 1, 5, and 30 min), mice were sacrificed by
for C15H16N2O7187Re 523.0516, found 523.0522. cervical dislocation, and samples of blood were immediately
The methyl ester (30 mg, 0.057 mmol) was dissolved in a obtained from the myocardial tissue via a syringe, and samples
1:1 mixture of 0.4 N NaOH and MeOH (1.5 mL) and stirred of whole organ (heart) and tissue (lung, liver, kidney, and
Technical Notes Bioconjugate Chem., Vol. 18, No. 4, 2007 1335
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%.
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 distribu-
tion 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 LITERATURE CITED
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