Compound Targeting Norepinephrine Transporter
20220096667 · 2022-03-31
Assignee
Inventors
Cpc classification
C07D251/08
CHEMISTRY; METALLURGY
C07B2200/05
CHEMISTRY; METALLURGY
C07C279/08
CHEMISTRY; METALLURGY
A61K51/0406
HUMAN NECESSITIES
International classification
C07B59/00
CHEMISTRY; METALLURGY
C07C279/08
CHEMISTRY; METALLURGY
Abstract
A compound according to following formula can be prepared,
##STR00001##
where R1 is an F or I residue.
Claims
1: A compound according to following formula ##STR00028## wherein R.sub.1 is a halogen residue.
2: A method, comprising: administering the compound according to claim 1.
3: The method according to claim 2, wherein the compound is administered to a living human or animal body suspected of having a disorder in generation, degradation, distribution, or function of norepinephrine transporter (NET).
4: The method according to claim 3, wherein after administration of the compound, the living human or animal body is scanned with Positron Emission Tomography (PET).
5: The method according to claim 4, wherein the disorder in generation, degradation, distribution, or function of NET is associated with a cardiovascular disease, a dysregulated blood pressure, a renal disorder, a neuroendocrine tumor disease, or Parkinson's disease.
6: A method for synthesizing the compound according to claim 1, comprising: a) removing the methyl group of the following compound 14 ##STR00029## to receive the following compound 15 ##STR00030## wherein R1 is a halogen residue, b) performing a chloroalkylation of compound 15 with 1-chloro-3-iodopropane, to receive the following compound 25 ##STR00031## c) performing a Wittig reaction with compound 25 with a (methoxymethyl)triphenylphosphonium halide and potassium tert-butoxide or sodium hydride, to receive the following compound 26 ##STR00032## d) reducing compound 26 in a two-step one pot reaction by addition of mercury acetate to allow formation of an intermediate followed by addition of an alkali metal borohydride, to receive the following compound 28 ##STR00033## e) performing a Mitsunobu reaction with compound 28 in the presence of triphenylphosphine, an azodicarboxylate, and the following compound 24 ##STR00034## to receive the following compound 29 ##STR00035## f) replacing the chlorine atom at the terminal of the alkyl chain of the compound resulting from any of b) to e) by iodine in a Finkelstein reaction, followed by a tosylation using silver p-toluenesulfonate in the dark, to receive the following compound 30 ##STR00036## wherein a)-f) are performed in any order to obtain the compound 30, and g) radiolabeling compound 30 by a nucleophilic substitution using an alkali metal salt of .sup.18F followed by deprotection under acidic condition at a temperature of at least 70° C. to receive the following compound ##STR00037## wherein the halogen residue is an F or an I residue.
7: The method according to claim 6, wherein the (methoxymethyl)triphenylphosphonium halide of c) is (methoxymethyl)triphenylphosphonium chloride.
8: The method according to claim 6, wherein the azodicarboxylate of e) is diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD).
9: The method according to claim 6, wherein the alkali metal borohydride of d) is sodium borohydride or potassium borohydride.
10: The method according to claim 6, wherein the alkali metal salt of .sup.18F used in g) is K.sup.18F.
11: The method according to claim 6, wherein the deprotection under acidic condition of g) is performed at a temperature of at most 100° C.
12: The method according to claim 6, wherein synthesis of compound 24 comprises: h) reacting benzyl carbamate, N,N′-dimethylurea, and water at a temperature of at least 80° C., to receive the following compound 23 ##STR00038## i) reacting compound 23 with hydrogen catalyzed by palladium on activated charcoal, to receive compound 22 ##STR00039## and j) reacting compound 22 with the following compound 13 ##STR00040## in the presence of N,N-diisopropylethylamine (DIPEA) to receive compound 24, wherein Boc is a tert-butoxycarbonyl protecting group.
13: The method according to claim 12, wherein compound 13 of j) is synthesized by reacting 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea and 5-chlorobenzotriazole in the presence of mercury (II) chloride (HgCl.sub.2).
Description
EMBODIMENTS OF THE INVENTION
[0040]
[0041]
[0042]
[0043]
[0044]
[0045]
[0046]
[0047]
[0048]
[0049]
[0050]
[0051]
[0052]
[0053]
[0054]
[0055]
[0056]
[0057]
SYNTHESIS OF THE COMPOUND ACCORDING TO THE INVENTION
[0058] The conditions of the synthesis according to
(a) 1-chloro-3-iodopropane, K.sub.2CO.sub.3, acetone, 60° C., 14 h, 87%; (b) (methoxymethyl)triphenylphosphonium chloride, potassium tert-butoxide (KOtBu), THF, 0° C.->r.t., 24 h, 50%; (c) Hg(OAc).sub.2, THF, H2O, 0° C., 15 min; (d) NaBH.sub.4, K.sub.2CO.sub.3, H.sub.2O, 0° C., 30 min, 68%; (e) compound 24, triphenylphosphine (PPh.sub.3), diisopropyl azodicarboxylate (DIAD), THF, 0° C.->r.t., 16 h, 84%; (f) Nal, acetone, 70° C., 14 h, 99%; (g) silver p-toluenesulfonate, CH3CN, 0° C.->r.t., dark, 72 h, 98%.
[0059] Conditions of the synthesis according to
(a) H.sub.2O, 100° C., 14 h, 77%; (b) CH.sub.3OH, H.sub.2O, 100° C., 14 h, 35%; (c) H2, Pd/C, r.t., 14 h, 88%; (d) compound 13, DIPEA, CH.sub.3CN, 50° C., 14 h, 58%; (e) compound 23, HgCl.sub.2, DMF, N.sub.2, 50° C., 14 h.
Detail of Synthesis of the Compounds of FIGS. 1 to 3:
[0060] Solvents were dried according to published methods and freshly distilled before use. All other reagents were commercially available compounds and were used without further purification unless noted otherwise. All reactions were carried out under nitrogen atmosphere. .sup.1H and .sup.13C NMR spectral data were obtained from a Bruker Avance II 400 MHz. .sup.1H and .sup.13C NMR chemical shifts (δ) are reported in parts per million (ppm) relative to internal standard TMS and coupling constants (J) are in Hz.
[0061] Thin layer chromatography (TLC) was performed on silica gel 60 with a 254 nm fluorescent indicator. UV light or iodine vapor were used for detection.
[0062] Purity was evaluated via LCMS performed on an LCMS-system by Shimadzu Products, containing a LC20AB liquid chromatograph, an SPD-20A UV/Vis detector and a DGU-20A3R degassing unit. Stationary phase was a Synergi 4 μm fusion-RP (150×4.6 mm) column (Phenomenex, Aschaffenburg, Germany). Mass spectra were obtained by a Shimadzu LCMS-2020 (confirming purity ≥95%). For column chromatography, silica gel 60, 230-400 mesh by Merck was used. For preparative thin layer chromatography, silica gel 60 PF.sub.254 by Merck was used.
tert-Butyl (E)-((tert-butoxycarbonyl)imino)(5-chloro-1H-benzo[d][1,2,3]triazol-1-yl)methyl)carbamate (13)
[0063] 1,3-Bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea and 5-chlorobenzotriazole were dissolved in dimethylformamide and triethyl amine (0.35 g, 3.45 mol). Mercury(II) chloride (HgCl.sub.2) was added and the reaction mixture was stirred at 50° C. for 24 h. The mixture was washed with sodium hydrocarbonate and water and extracted with ethyl acetate (8×50 ml). The combined organic layers were dried over sodium sulfate. The crude product was dissolved in dichloromethane and purified by column (5:1-1:1 petroleum ether:ethyl acetate) to give the target compound 13 as a white solid (0.74 g, 18.7 mmol. 57% yield) which was used in the next step without further purification. ESI-MS (m/z): 396.05 [M+H].sup.+.
3-Fluoro-4-hydroxybenzaldehyde (15)
[0064] 3-Fluoro-4-methoxybenzaldehyde 14 (5.00 g, 32.5 mmol) was mixed with 48% HBr (30 mL), heated to 140° C. and stirred under argon atmosphere for 3 h. The mixture was diluted with water (150 mL) and extracted with dichloromethane (2×100 mL). The combined organic layers were washed with brine solution and dried over sodium sulfate. The solvent was removed in vacuo to give compound 15 as a brown solid (4.36 g, 30.2 mmol, 97% yield). NMRs are in accordance to literature. (Jiang 2014) ESI-MS (m/z): 141.00 [M+H].sup.+.
5-Benzyl-1,3-dimethyl-1,3,5-triazinan-2-one (21)
[0065] Benzylamine (9.81 g, 91.7 mmol) was dissolved in formaldehyde (14.7 g, 183 mmol, 40% aq. solution) and heated to 100° C. under argon atmosphere. N,N′-dimethylurea (8.07 g, 91.7 mmol) was added and the mixture was stirred for 14 h. The mixture was washed with water and extracted with dichloromethane (2×200 mL). The combined organic layers were washed with brine solution and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was purified by column chromatography (1:1 petroleum etherethyl acetate) to give the title compound 21 as yellow crystals (15.5 g, 70.9 mmol, 77% yield). .sup.1H NMR (CDCl.sub.3, 400 MHz) δ=2.85 (s, 6H), 3.90 (s, 2H), 4.16 (s, 4H), 7.28 (m, 5H) ppm. .sup.13C NMR (CDCl.sub.3, 101 MHz) δ=32.47, 55.36, 67.72, 127.70, 128.60, 129.11, 137.49, 156.04 ppm. ESI-MS (m/z): 220.10 [M+H].sup.+.
Benzyl 3,5-dimethyl-4-oxo-1,3,5-triazinane-1-carboxylate (23)
[0066] Benzyl carbamate (1.00 g, 6.62 mmol) was dissolved in formaldehyde (1.18 g, 14.6 mmol, 40% aq. solution) and methanol. The mixture was heated to 100° C. under argon atmosphere, N,N′-dimethylurea (0.58 g, 6.62 mmol) was added and the mixture was stirred for 14 h. The solvent was removed in vacuo and crude product was washed with water and extracted with dichloromethane (2×200 mL). The combined organic layers were washed with brine solution and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was purified by column chromatography (1:1 petroleum etherethyl acetate, then 25:1 dichloromethane:methanol) to give the title compound 23 as a colourless liquid (0.60 g, 2.29 mmol, 35% yield). .sup.1H NMR (CDCl.sub.3, 400 MHz) δ=2.79 (s, 6H), 4.62 (s, 4H), 5.09 (s, 2H), 7.26-7.27 (5H) ppm. .sup.13C NMR (CDCl.sub.3, 101 MHz) δ=32.66, 60.34, 68.15, 128.04, 128.40, 128.58, 135.56, 154.39, 156.07 ppm. ESI-MS (m/z): 264.00 [M+H].sup.+.
1,3-Dimethyl-1,3,5-triazinan-2-one (22)
[0067] Compound 23 (603 mg, 2.29 mmol) was dissolved in methanol, palladium on activated charcoal (30 mg) was added and the reaction mixture was stirred at r.t. for 14 h under hydrogen atmosphere. The mixture was filtered and the filtrate was concentrated in vacuo to give the title compound 22 as white solid (259 mg, 2.01 mmol, 88% yield). .sup.1H NMR (CDCl.sub.3, 400 MHz) δ=2.77 (s, 1H), 2.85 (s, 5H), 4.16 (s, 4H) ppm.
tert-Butyl (Z)-(((tert-butoxycarbonyl)imino)(3,5-dimethyl-4-oxo-1,3,5-triazinan-1-yl)methyl)carbamate (24)
[0068] To a solution of compound 22 (137 mg, 1.06 mmol) and compound 13 (420 mg, 1.06 mmol) in acetonitrile, DIPEA (274 mg, 2.13 mmol) was added and the mixture was stirred at 50° C. for 14 h. The solvent was removed in vacuo. The residue was diluted with water (50 mL) and extracted with ethyl acetate (2×30 ml) and dichloromethane (2×30 ml). The combined organic phases were washed with brine, dried over sodium sulfate and the solvent was removed in vacuo. The crude product was purified by column chromatography (6:1 then 2:1 petroleum ether:ethyl acetate, 1:1:0.05 petroleum etherethyl acetate:triethylamine) to give the target compound 24 as white crystals (226 mg, 0.61 mmol, 58% yield). .sup.1H NMR (CDCl.sub.3, 400 MHz) δ=1.47-1.48 (m, 18H), 2.01-2.02 (m, 1H), 2.90-2.91 (m, 6H), 4.69 (s, 6H) ppm. .sup.13C NMR (CDCl.sub.3, 101 MHz) δ=28.16, 33.18, 60.47, 62.31, 81.82, 154.01, 157.30 ppm. ESI-MS (m/z): 372.20 [M+H].sup.+.
4-(3-Chloropropoxy)-3-fluorobenzaldehyde (25)
[0069] To a solution of compound 15 (2.50 g, 17.9 mmol) and potassium carbonate (2.71 g, 19.6 mmol) in acetone, 1-chloro-3-iodopropane (3.64 g, 17.9 mmol) was added and the reaction mixture was stirred at 60° C. for 14 h. The solvent was evaporated and the residue was diluted with water (100 mL) and extracted with ethyl acetate (2×50 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was purified by column chromatography (5:1 petroleum ether:ethyl acetate). The solvent was removed in vacuo to give compound 25 as a pale-yellow liquid (3.36 g, 15.6 mmol, 87% yield). .sup.1H NMR (CDCl.sub.3, 400 MHz) δ=2.23-2.32 (m, 2H), 3.74-3.78 (m, 2H), 4.22-4.28 (m, 2H), 7.58-7.80 (m, 3H), 9.85 (s, 1H) ppm. .sup.13C NMR (CDCl.sub.3, 101 MHz) δ=31.98, 41.14, 65.69, 113.62, 113.81, 115.94, 116.15, 123.30, 123.37, 130.20, 130.30, 147.54, 148.66, 150.66-153.12 (J(C-F)=254.4 Hz), 189.96 ppm. ESI-MS (m/z): 217.10 [M+H].sup.+.
(E)-1-(3-Chloropropoxy)-2-fluoro-4-(2-methoxyvinyl)benzene (26)
[0070] (Methoxymethyl)triphenylphosphonium chloride (538 mg, 1.57 mmol) was dissolved in dry tetrahydrofuran and cooled to 0° C. Potassium tert-butoxide (228 mg, 2.04 mmol) was added portionwise and the mixture was stirred for 1 h. Compound 25 (200 mg, 1.00 mmol) in dry tetrahydrofuran was added dropwise under cooling and the reaction was warmed to room temperature over night. After mixture was quenched with water, the solvent was removed in vacuo and the residue was diluted with water (100 mL) and extracted with ethyl acetate (2×50 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was purified by column chromatography (15:1 petroleum ether:ethyl acetate) to give the target compound 26 as a colorless oil (192 mg, 0.78 mmol, 50% yield). .sup.1H NMR (CDCl.sub.3, 400 MHz) δ=2.25 (m, 2H), 3.37 (s, 2H), 3.76-3.77 (m, 3H), 4.16 (m, 2H), 5.13-5.14 (d, J=6.9 Hz, 1H), 5.70-5.73 (d, J=13.0 Hz, 0.5H), 6.08-6.10 (d, J=7.0 Hz, 0.5H), 6.87-6.89 (m, 3H) ppm. .sup.13C NMR (CDCl.sub.3, 101 MHz) δ=32.47, 41.52, 41.55, 56.70, 60.78, 66.15, 66.29, 112.69, 112.88, 115.08, 115.10, 115.84, 115.86, 116.02, 116.21, 121.13, 121.17, 147.57, 148.78, 151.91-154.35 (J(C—F)=245.1 Hz), 153.80 ppm. 2-(4-(3-Chloropropoxy)-3-fluorophenyl)ethan-1-ol (28) Compound 26 (90 mg, 0.37 mmol) was dissolved in tetrahydrofuran and a solution of mercury(II) acetate (129 mg, 0.41 mmol) in water was added under cooling in an ice/water bath. The reaction mixture was stirred for 15 mins and sodium borohydride (56 mg, 1.48 mmol) in a saturated potassium carbonate solution was added dropwise. The reaction mixture was stirred for 30 min, diluted with water and extracted with ethyl acetate (4×10 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was purified by column chromatography (4:1 petroleum ether:ethyl acetate) to give the target compound 28 as colorless oil (58.5 mg, 0.25 mmol, 68% yield). .sup.1H NMR (CDCl.sub.3, 400 MHz) δ=2.22-2.28 (m, 2H), 2.77-2.80 (t, J=6.5 Hz, 2H), 3.75-3.83 (m, 4H), 4.15-4.18 (t, J=5.8 Hz, 2H), 6.91-6.98 (m, 3H) ppm. .sup.13C NMR (CDCl.sub.3, 101 MHz) δ=31.27, 38.82, 41.32, 41.43, 62.64, 65.98, 113.39, 113.46, 116.63, 116.84, 125.48, 125.56, 133.36, 133.39, 144.45, 144.85, 153.22-156.17 (J(C-F)=251.5 Hz) ppm.
tert-butyl (E)-((tert-butoxycarbonyl)imino)(3,5-dimethyl-4-oxo-1,3,5-triazinan-1-yl)methyl)(4-(3-chloropropoxy)-3-fluorophenethyl)carbamate (29)
[0071] Compound 28 (72 mg, 0.31 mmol), compound 24 (172 mg, 0.46 mmol) and triphenyl phosphine (122 mg, 0.46 mmol) were dissolved in dry tetrahydrofuran. A solution of DIAD (94 mg, 0.46 mmol) in dry tetrahydrofuran was added dropwise at 0° C. and the reaction mixture was slow warmed up to room temperature and stirred for 16 h. The mixture was diluted with water (40 mL) and extracted with diethyl ether (4×10 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was purified by column chromatography (2.5:1.5:0.5 petroleum ether:acetone:ethyl acetate) to give the target compound 29 as a colorless liquid (152 mg, 0.26 mmol, 84% yield). .sup.1H NMR (CDCl.sub.3, 400 MHz) δ=1.47 (s, 18H), 2.22-2.25 (m, 2H), 2.84 (m, 6H), 3.57 (bs, 4H), 3.74-3.77 (m, 2H), 4.13-4.16 (m, 2H), 4.52 (bs, 4H), 6.89-6.95 (m, 3H) ppm. .sup.13C NMR (CDCl.sub.3, 101 MHz) δ=28.14, 28.25, 41.43, 48.87, 60.49, 61.02, 66.02, 80.84, 83.00, 115.48, 116.55, 116.73, 124.40, 124.43, 131.50, 131.56, 145.60, 145.71, 150.98, 151.52-153.97 (J(C-F)=246.8 Hz), 152.31, 156.98, 158.39 ppm. ESI-MS (m/z): 586.25, 588.25 [M+H].sup.+.
tert-butyl (E)-((tert-butoxycarbonyl)imino)(3,5-dimethyl-4-oxo-1,3,5-triazinan-1-yl)methyl)(3-fluoro-4-(3-odopropoxy)phenethyl)carbamate (30a)
[0072] Compound 29 (72 mg, 0.12 mmol) and sodium iodine (37 mg, 0.25 mmol) were dissolved in acetone and the reaction mixture was stirred at 70° C. for 14 h. The mixture diluted with water (40 mL) and extracted with diethyl ether (4×10 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to give the target compound 30a as a yellow liquid (83 mg, 0.12 mmol, 99% yield) and was used directly in the next step without further purification. ESI-MS (m/z): 678.20 [M+H].sup.+, 700.20 [M+Na].sup.+.
(E)-3-(4-(2-(N,N′-bis(tert-butoxycarbonyl)-3,5-dimethyl-4-oxo-1,3,5-triazinane-1-carboximidamido)ethyl)-2-fluorophenoxy)propyl 4-methylbenzenesulfonate (30)
[0073] Compound 30a (72 mg, 0.11 mmol) was dissolved in acetonitrile and silver p-toluenesulfonate (148 mg, 0.53 mmol) in darkness at 0° C. The reaction mixture was stirred at room temperature for 72 h. The crude product was diluted with water (40 mL) and extracted with ethyl acetate (4×10 ml). The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to give the target compound 30 as a yellow liquid (75 mg, 0.10 mmol, 98% yield). .sup.1H NMR (CDCl.sub.3, 400 MHz) δ=1.48 (s, 18H), 2.11-2.14 (m, 2H), 2.14 (s, 3H), 2.85 (s, 6H), 3.85 (bs, 4H), 3.89-4.01 (m, 2H), 4.22-4.25 (m, 2H), 4.55 (bs, 4H), 6.77-6.93 (m, 3H), 7.26-7.28 (m, 2H), 7.75-7.77 (d, J=8.3 Hz) ppm. .sup.13C NMR (CDCl.sub.3, 101 MHz) δ=28.15, 28.26, 29.05, 29.80, 33.22, 33.66, 60.50, 61.20, 64.87, 66.98, 80.93, 83.06, 115.32, 116.49, 116.67, 124.42, 127.98, 129.95, 131.55, 132.92, 144.92, 145.36, 145.47, 151.37, 152.28, 153.82, 156.95 ppm. ESI-MS (m/z): 722.35 [M+H].sup.+, 744.30 [M+Na].sup.+.
Radiochemistry
[0074] .sup.18F-Fluoride trapped on a Sep-Pak QMA cartridge was first washed with distilled water (3 mL) to remove .sup.18O-water. .sup.18F-Fluoride was then eluted from the cartridge with K.sub.2CO.sub.3 solution (0.3 mL, 23 μmol/mL) into a vial that contains Kryptofix.sub.222 (22.5 mg, 59.7 μmol). Acetonitrile (0.5 mL×4) was added to the vial, and the solution was dried azeotropically under argon at 120° C. Labeling is carried out by adding the solution of the precursor (4.8 mg) in dry acetonitrile (0.3 mL), followed by heating and stirring at 110° C. for 10 min. Hydrochloric acid (6N, 0.3 mL) was added to the reaction mixture, and the reaction continued for 20 min at 100° C. The mixture was cooled, diluted with 1 mL of a mixture solution of water and acetonitrile (1:1), and applied to the semi-preparative HPLC for purification. HPLC condition: Column: 9.4×250 mm 5-micron ZORBAX Eclipse XDB-C18 (P.N.: 990967-202). Mobile phase: Phase A: Millipore water with 0.1% formic acid; Phase B: Methanol with 0.1% formic acid. Condition: 0-20 min, 30%-60% B, 20-22 min 60-100% B, 22-50 min 100% B; Flow rate 3 mL/min. Condition of autoradiography: TLC with normal phase silica gel 60. Solvent system: 3 mL methanol+20 μL formic acid, Rf.: 0.6. .sup.131I-MIBG was purchase from GE Healthcare (Freiburg im Breigau, Germany) and used within 2 h after calibration time. .sup.131I-MIBG was chosen instead of .sup.123I-MIBG due to its relative longer half-life, which is convenient for research purposes and financial reasons.
Detail of Synthesis of the Compounds of FIGS. 4 to 6:
(E/Z)-2-odo-1-methoxy-4-(2-methoxyvinyl)benzene C.SUB.10.H.SUB.11.O.SUB.2.I (107)
[0075] ##STR00018##
[0076] (Methoxymethyl)triphenylphosphonium chloride (3.94 g, 11.50 mmol, 1.50 equiv.) was dissolved in dry tetrahydrofuran (60 mL) under inert gas atmosphere and potassium tert-butoxide (1.47 g, 13.13 mmol, 1.71 equiv.) was added at 0° C. The reaction mixture was stirred for 2 min at 0° C. before commercially available 3-iodo-4-methoxybenzaldehyde 106 (2.01 g, 7.66 mmol, 1.00 equiv.) was added. The solution was stirred at room temperature for 18 h and evaporated to dryness. The residue was partitioned between in ethyl acetate (50 mL) and water (50 mL) followed by layer separation. The aqueous layer was extracted with ethyl acetate (2×50 mL) and the combined organic layers were washed with brine (1×100 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure and the crude material was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate=12:1, R.sub.f=0.37) affording the desired product 107 as a pale yellow oil (1.79 g, 81% yield). .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ=8.02 (1H, d, J=2.13 Hz, H.sup.h′), 7.66 (1H, d, J=2.18 Hz, H.sup.h), 7.50 (1H, dd, J=2.13 Hz, 8.54 Hz, H.sup.d′), 7.15 (1H, dd, J=2.25 Hz, 8.46 Hz, H.sup.d), 6.92 (1H, d, J=12.92 Hz, H.sup.a), 6.78-6.71 (2H, m, H.sup.e/e′), 6.07 (1H, d, J=6.94 Hz, H.sup.a′), 5.70 (1H, d, J=13.00 Hz, H.sup.b), 5.09 (1H, d, J=6.98 Hz, H.sup.b′), 3.86/3.85 (6H, s, H.sup.i/i′), 3.77 (3H, s, H.sup.j′), 3.66 (3H, s, H.sup.j) ppm. .sup.13C-NMR (CDCl.sub.3, 101 MHz) δ=156.4 (C.sup.f), 156.1 (C.sup.f′), 148.5 (C.sup.a), 147.3 (C.sup.a′), 139.1 (C.sup.h′), 136.1 (C.sup.h), 131.3 (C.sup.c), 131.0 (C.sup.c′), 129.4 (C.sup.d′), 126.3 (C.sup.d), 111.2/110.7 (C.sup.e/e′), 103.9 (C.sup.b′), 103.4 (C.sup.b), 86.5 (C.sup.g), 86.0 (C.sup.g′), 60.8 (C.sup.j′), 56.7/56.6/56.5 (C.sup.j/i′/i″) ppm. ESI-MS (m/z): 290.80 [M+H].sup.+.
2-(3-lodo-4-methoxyphenyl)ethan-1-ol C.SUB.9.H.SUB.11.O.SUB.2.I (108)
[0077] ##STR00019##
[0078] (E/Z)-2-lodo-1-methoxy-4-(2-methoxyvinyl)benzene 107 (1.79 g, 6.17 mmol, 1.00 equiv.) was suspended in tetrahydrofuran (46 mL) and water (74 mL) before mercury acetate (2.18 g, 6.84 mmol, 1.10 equiv.) was added at 0° C. The reaction mixture was stirred at 0° C. for 30 min followed by the addition of a 50% aqueous potassium carbonate solution (30 mL) and sodium borohydride (942 mg, 24.90 mmol, 4.00 equiv.). After the reaction mixture was stirred at 0° C. for 2.5 h, the precipitate was filtered off and the filtrate was extracted with ethyl acetate (3×80 mL). The combined organic layers were dried over sodium sulfate and the solvent was removed under reduced pressure. The crude material was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate=1:1, R.sub.f=0.45) to obtain alcohol 108 as a yellow oil (1.36 g, 79% yield). .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ=7.65 (1H, d, J=2.13 Hz, H.sup.h), 7.16 (1H, dd, J=2.14 Hz, 8.35 Hz, H.sup.d), 6.76 (1H, d, J=8.35 Hz, H.sup.e), 3.85 (3H, s, H.sup.l), 3.81 (2H, t, J=6.55 Hz, H.sup.a), 2.76 (2H, t, J=6.53 Hz, H.sup.b), 1.69 (1H, s, H.sup.j) ppm. .sup.13C-NMR (CDCl.sub.3, 101 MHz) δ=157.0 (C.sup.f), 139.9 (C.sup.h), 132.9 (C.sup.c), 130.2 (C.sup.d), 111.1 (C.sup.e), 86.2 (C.sup.g), 63.7 (C.sup.a), 56.5 (C.sup.i), 37.8 (C.sup.b) ppm. ESI-MS (m/z): 278.80 [M+H].sup.+.
3-lodo-4-methoxyphenethyl acetate C.SUB.11.H.SUB.13.O.SUB.31 .(113)
[0079] ##STR00020##
[0080] 2-(3-lodo-4-methoxyphenyl)ethan-1-ol 108 (1.04 g, 3.74 mmol, 1.00 equiv.) was dissolved in acetone (20 mL) and acetyl chloride (800 μL, 11.21 mmol, 3.00 equiv.) was added at 0° C. The reaction mixture was stirred at room temperature for 18 h before the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (20 mL) and washed with saturated aqueous sodium bicarbonate solution (1×20 mL) and brine (1×20 mL). The organic layer was dried over sodium sulfate and the solvent was removed under reduced pressure. The crude material was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate=3:1, R.sub.f=0.54) to obtain acetate 113 as a yellow oil (1.07 g, 90% yield). .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ=7.63 (1H, d, J=2.15 Hz, H.sup.h), 7.15 (1H, dd, J=2.16 Hz, 8.39 Hz, H.sup.d), 6.75 (1H, d, J=8.38 Hz, H.sup.e), 4.22 (2H, t, J=7.01 Hz, H.sup.a), 3.85 (3H, s, H.sup.i), 2.83 (2H, t, J=7.01 Hz, H.sup.b), 2.03 (3H, s, H.sup.k) ppm. .sup.13C-NMR (CDCl.sub.3, 101 MHz) δ=171.1 (C.sup.j), 157.1 (C.sup.f), 139.9 (C.sup.h), 132.2 (C.sup.c), 130.0 (C.sup.d), 111.0 (C.sup.e), 86.1 (C.sup.g), 64.9 (C.sup.a), 56.5 (C.sup.i), 33.8 (C.sup.b), 21.1 (C.sup.k) ppm. ESI-MS (m/z): 342.85 [M+Na].sup.+.
4-Hydroxy-3-iodophenethyl acetate C.SUB.10.H.SUB.11.O.SUB.3.I (114)
[0081] ##STR00021##
[0082] 3-lodo-4-methoxyphenethyl acetate 113 (588 mg, 1.87 mmol, 1.00 equiv.) was dissolved in dry dichloromethane (12 mL) under inert gas atmosphere and a solution of boron tribromide (445 μL, 4.69 mmol, 2.50 equiv.) in dry dichloromethane (12 mL) was added at −20° C. The reaction mixture was stirred for 2 h and partitioned between ethyl acetate (60 mL) and a 50% aqueous sodium bicarbonate solution (60 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×60 mL). The combined organic layers were washed with brine (1×180 mL) and dried over sodium sulfate. Evaporation of the solvent under reduced pressure and purification by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate=3:1, R.sub.f=0.36) afforded phenol 114 as a yellow oil (602 mg, 92% yield). .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ=7.51 (1H, d, J=2.08 Hz, H.sup.h), 7.08 (1H, dd, J=2.05 Hz, 8.28 Hz, H.sup.d), 6.91 (1H, d, J=8.29 Hz, H.sup.e), 5.47 (1H, bs, H.sup.i), 4.22 (2H, t, J=7.02 Hz, H.sup.a), 2.83 (2H, t, J=6.99 Hz, H.sup.b), 2.04 (3H, s, H.sup.k) ppm. .sup.13C-NMR (CDCl.sub.3, 101 MHz) δ=171.2 (C.sup.j), 153.8 (C.sup.e), 138.5 (C.sup.h), 132.0 (C.sup.c), 130.8 (C.sup.d), 115.1 (C.sup.e), 85.6 (C.sup.g), 65.0 (C.sup.a), 33.8 (C.sup.b), 21.1 (C.sup.k) ppm. ESI-MS (m/z): 328.85 [M+Na].sup.+.
4-(3-Chloropropoxy)-3-iodophenethyl acetate C.SUB.13.H.SUB.16.O.SUB.3.ICl
[0083] ##STR00022##
[0084] 4-Hydroxy-3-iodophenethyl acetate 114 (330 mg, 1.08 mmol, 1.00 equiv.) was dissolved in acetone (17 mL) followed by the addition of 3-chloro-1-iodopropane (177 μL, 1.65 mmol, 1.53 equiv.) and cesium carbonate (702 mg, 2.15 mmol, 2.00 equiv.). The reaction mixture was stirred at 70° C. for 18 h before water (85 mL) was added. After an extraction with ethyl acetate (3×85 mL), the combined organic layers were washed with brine (1×215 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate=5:1, R.sub.f=0.42) to give the title compound as a yellow oil (372 mg, 90% yield). .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ=7.63 (1H, d, J=2.16 Hz, H.sup.h), 7.13 (1H, dd, J=2.15 Hz, 8.33 Hz, H.sup.d), 6.76 (1H, d, J=8.35 Hz, H.sup.e), 4.22 (2H, t, J=6.99 Hz, H.sup.a), 4.14 (2H, t, J=5.68 Hz, H.sup.k), 3.84 (2H, t, J=6.33 Hz, H.sup.m), 2.83 (2H, t, J=6.99 Hz, H.sup.b, 2.23 (2H, quint, J=6.00 Hz, H.sup.l), 2.03 (3H, s, H.sup.j) ppm. .sup.13C-NMR (CDCl.sub.3, 101 MHz) δ=171.1 (C.sup.j), 156.1 (C.sup.f), 139.8 (C.sup.h), 132.5 (C.sup.c), 130.0 (C.sup.d), 112.2 (C.sup.e), 86.8 (C.sup.g), 65.6 (C.sup.k), 64.9 (C.sup.a), 41.8 (C.sup.m), 33.8 (C.sup.b), 32.3 (C.sup.l), 21.1 (C.sup.j) ppm. ESI-MS (m/z): 404.80 [M+Na].sup.+.
2-(4-(3-Chloropropoxy)-3-iodophenyl)ethan-1-ol C.SUB.11.H.SUB.14.O.SUB.2.ICl (117)
[0085] ##STR00023##
[0086] 4-(3-Chloropropoxy)-3-iodophenethyl acetate (189 mg, 0.49 mmol, 1.00 equiv.) was dissolved in methanol (9 mL) before potassium carbonate (650 mg, 4.70 mmol, 9.60 equiv.) was added. The reaction mixture was stirred at room temperature for 3 h and was partitioned between water (60 mL) and dichloromethane (60 mL) followed by layer separation. The aqueous layer was extracted with dichloromethane (2×60 mL) and the combined organic layers were washed with brine (1×180 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure to obtain alcohol 117 as a yellow oil (175 mg, quant.) which was used directly in the next step. .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ=7.65 (1H, d, J=2.15 Hz, H.sup.h), 7.16 (1H, dd, J=2.12 Hz, 8.32 Hz, H.sup.d), 6.77 (1H, d, J=8.36 Hz, H.sup.e), 4.14 (2H, t, J=5.72 Hz, H.sup.j), 3.87-3.78 (4H, m, H.sup.a,k), 2.77 (2H, t, J=6.48 Hz, H.sup.b), 2.27 (2H, quint, J=6.00 Hz, H.sup.j) ppm. .sup.13C-NMR (CDCl.sub.3, 101 MHz) δ=156.1 (C.sup.f), 139.9 (C.sup.h), 133.3 (C.sup.c), 130.2 (C.sup.d), 112.3 (C.sup.e), 87.0 (C.sup.g), 65.7 (C.sup.j), 63.7 (C.sup.a), 41.8 (C.sup.k), 37.9 (C.sup.b), 32.3 (C.sup.j) ppm. ESI-MS (m/z): 362.80 [M+Na].sup.+.
tert-Butyl (((tert-butoxycarbonyl)imino)(3,5-dimethyl-4-oxo-1,3,5-triazinan-1-yl)methyl)(4-(3-chloropropoxy)-3-Iodophenethyl)carbamate C.SUB.27.H.SUB.41.N.SUB.5.O.SUB.6.ICl (121)
[0087] ##STR00024##
[0088] 2-(4-(3-Chloropropoxy)-3-iodophenyl)ethan-1-ol 117 (160 mg, 0.47 mmol, 1.00 equiv.), triphenylphosphine (185 mg, 0.71 mmol, 1.51 equiv.) and 24 (175 mg, 0.47 mmol, 1.00 equiv.) were dissolved in dry tetrahydrofuran (23 mL) under inert gas atmosphere. DIAD (140 μL, 0.71 mmol, 1.51 equiv.) was added at 0° C. and the reaction mixture was stirred at room temperature for 18 h. The solvent was removed under reduced pressure and the residue was partitioned between water (20 mL) and ethyl acetate (20 mL) followed by layer separation. The aqueous layer was extracted with ethyl acetate (2×20 mL) and the combined organic layers were washed with brine (1×60 mL) and dried over sodium sulfate. Evaporation of the solvent under reduced pressure and purification by column chromatography (SiO.sub.2, petroleum ether/acetone/ethyl acetate=5:3:1, R.sub.f=0.38) gave the title compound 121 as a pale green oil (272 mg, 83% yield). .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ=7.61 (1H, d, J=1.81 Hz, H.sup.h), 7.12 (1H, dd, J=1.71 Hz, 8.25 Hz, H.sup.d), 6.74 (1H, d, J=8.31 Hz, H.sup.e), 4.50 (4H, bs, H.sup.p), 4.16-4.07 (3H, m, H.sup.i), 3.82 (2H, t, J=6.25 Hz, H.sup.k), 3.57 (2H, bs, H.sup.a), 2.88-2.80 (8H, m, H.sup.b,q), 2.26 (2H, quint, J=5.96 Hz, H.sup.j), 1.48 (9H, s, H.sup.o), 1.47 (9H, s, H.sup.o) ppm. .sup.13C-NMR (CDCl.sub.3, 101 MHz) δ=156.3 (C.sup.f,r), 152.4/151.3 (C.sup.l,m), 139.6 (C.sup.h), 132.5 (C.sup.c), 129.9 (C.sup.d), 112.3 (C.sup.e), 87.0 (C.sup.g), 83.1/80.9 (C.sup.n), 65.7 (C.sup.i), 61.2 (C.sup.p), 49.0 (C.sup.a), 41.7 (C.sup.k), 33.2 (C.sup.q), 33.1 (C.sup.b), 32.2 (C.sup.j), 28.3/28.2 (C.sup.o) ppm. ESI-MS (m/z): 694.20 [M+H].sup.+, 716.05 [M+Na].sup.+.
tert-Butyl (((tert-butoxycarbonyl)imino)(3,5-dimethyl-4-oxo-1,3,5-triazinan-1-yl)methyl)(3-odo-4-(3-odopropoxy)phenethyl)carbamate C.SUB.27.H.SUB.41.N.SUB.5.O.SUB.6.I.SUB.2 .(122)
[0089] ##STR00025##
[0090] 121 (236 mg, 0.34 mmol, 1.00 equiv.) was dissolved in acetone (30 mL) and sodium iodide (510 mg, 3.40 mmol, 10.00 equiv.) was added. The reaction mixture was stirred at 70° C. for 18 h and the solvent was removed under reduced pressure. Since LC-MS indicated incomplete conversion of the starting material, an additional amount of sodium iodide (816 mg, 5.44 mmol, 16.00 equiv.) was added. The reaction mixture was stirred at 70° C. for another day. After adding another amount of sodium iodide (816 mg, 5.44 mmol, 16.00 equiv.), the reaction mixture was stirred at 70° C. for additional 18 h before partitioning between water (15 mL) and diethyl ether (15 mL) followed by layer separation. The aqueous layer was extracted with diethyl ether (2×15 mL) and the combined organic layers were washed with brine (1×45 mL) and dried over sodium sulfate. The solvent was removed under reduced pressure to obtain iodide 122 as a yellow oil (221 mg, 83% yield) which was used directly in the next step. .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ=7.62 (1H, d, J=2.16 Hz, H.sup.h), 7.13 (1H, dd, J=2.22 Hz, 8.31 Hz, H.sup.d), 6.75 (1H, d, J=8.33 Hz, H.sup.e), 4.51 (4H, bs, H.sup.p), 4.05 (3H, t, J=5.69 Hz, H.sup.i), 3.58 (2H, bs, H.sup.a), 3.46 (3H, t, J=6.60 Hz, H.sup.k), 2.90-2.75 (8H, m, H.sup.b,q), 2.32-2.24 (2H, m, H.sup.j), 1.48 (9H, s, H.sup.o), 1.47 (10H, s, H.sup.o) ppm. .sup.13C-NMR (CDCl.sub.3, 101 MHz) δ=157.0 (C.sup.r), 156.2 (C.sup.f), 152.3/151.1 (C.sup.l,m), 139.6 (C.sup.h), 132.6 (C.sup.c), 129.8 (C.sup.d), 112.4 (C.sup.e), 87.0 (C.sup.g), 83.1/81.0 (C.sup.n), 68.7 (C.sup.i), 61.2 (C.sup.p), 49.0 (C.sup.a), 33.3 (C.sup.q), 33.1 (C.sup.b), 32.9 (C.sup.j), 28.3/28.2 (C.sup.o), 3.1 (C.sup.k) ppm. ESI-MS (m/z): 786.00 [M+H].sup.+, 807.90 [M+Na].sup.+.
3-(4-(2-(N,N′-Bis(tert-butoxycarbonyl)-3,5-dimethyl-4-oxo-1,3,5-triazinane-1-carboximidamido)ethyl)-2-iodophenoxy)propyl 4-methylbenzenesulfonate C.SUB.34.H.SUB.48.N.SUB.5.O.SUB.9.SI (123)
[0091] ##STR00026##
[0092] 122 (191 mg, 0.24 mmol, 1.00 equiv.) was dissolved in acetonitrile (10 mL) and silver p-toluenesulfonate (340 mg, 1.22 mmol, 5.00 equiv.) was added. The reaction mixture was stirred in darkness at room temperature for 72 h before the solvent was removed under reduced pressure. The residue was partitioned between water (15 mL) and ethyl acetate (15 mL) followed by layer separation. The aqueous layer was extracted with ethyl acetate (2×15 mL) and the combined organic layers were washed with brine (1×40 mL) and dried over sodium sulfate. Evaporation of the solvent under reduced pressure and purification by column chromatography by column chromatography (SiO.sub.2, petroleum ether/acetone/ethyl acetate=6:3:1, R.sub.f=0.33) afforded the target compound 123 as a colourless foam (113 mg, 56% yield). .sup.1H-NMR (CDCl.sub.3, 400 MHz) δ=7.75 (2H, d, J=8.33 Hz, H.sup.t), 7.58 (1H, d, J=2.06 Hz, H.sup.h), 7.23 (2H, d, J=8.32 Hz, H.sup.u), 7.10 (1H, dd, J=2.04 Hz, 8.33 Hz, H.sup.d), 6.62 (1H, d, J=8.22 Hz, H.sup.e), 4.52 (3H, bs, H.sup.p), 4.31 (2H, t, J=6.02 Hz, H.sup.k), 3.96 (2H, t, J=5.78 Hz, H.sup.i), 3.56 (2H, bs, H.sup.a), 2.88-2.79 (8H, m, H.sup.b,q), 2.36 (3H, s, H.sup.w), 2.19-2.10 (2H, m, H.sup.j), 1.48 (9H, s, H.sup.o), 1.47 (9H, s, H.sup.o) ppm. .sup.13C-NMR (CDCl.sub.3, 101 MHz) δ=157.0 (C.sup.r), 156.0 (C.sup.f), 152.3/151.0 (C.sup.l,m), 144.9 (C.sup.v), 139.6 (C.sup.h), 132.9 (C.sup.s), 132.5 (C.sup.c), 130.0 (C.sup.u), 129.7 (C.sup.d), 127.9 (C.sup.t), 112.1 (C.sup.e), 86.8 (C.sup.g), 83.0/81.0 (C.sup.n), 67.2 (C.sup.k), 64.4 (C.sup.l), 61.2 (C.sup.p), 49.1 (C.sup.a), 33.3 (C.sup.d), 33.2 (C.sup.b), 29.0 (C.sup.j), 28.3/28.2 (C.sup.o), 21.8 (C.sup.w) ppm. ESI-MS (m/z): 830.10 [M+H].sup.+, 852.05 [M+Na].sup.+.
Radiochemistry
[0093] Radiofluorination was performed using K.sup.18F to replace the tosylate group of precursor 123 with .sup.18F. By the addition of 6N HCl, cleavage of the boc groups and the triazinanone moiety was achieved (
Competitive Binding/Cell Uptake Studies
[0094] SK-N-SH cells expressing NET were cultivated according to the instructions from the supplier (Sigma-Aldrich Chemie GmbH, Munich, Germany). The cells were transferred to a 12 well plate and incubated in 1 mL EMEM while reaching 2×10.sup.5 cells/well density the day for testing. The medium was removed, and the cells were washed with 1 mL EMEM. A solution of .sup.18F-AF78 (3.7 kBq) in EMEM (700 μL) was added to each well with or without inhibitors (NE, MHPG, cold AF78 each with a final concentration 100 nM, 1 μM, 10 μM and 100 μM, respectively; Desipramine 10 μM). The plate was incubated for 60 min at 37° C. The cells were washed with ice-cold PBS buffer (1 mL×2) in order to remove unbound tracer. NaOH solution (0.1 N, 500 μL) was added to each well followed by the collection of cell pellet and the measurement in γ-counter (FH412, Frieseke & Höpfner, Erlangen, Germany) using differential energy windows (±20%) for .sup.18F and .sup.131I.
[0095] Cold, i. e. non-radioactive, reference compound AF78 of the compound according to the invention and the following compound 1
##STR00027##
were synthesized. The compounds were tested in SK-N-SH cells expressing NET in order to evaluate their competitive binding affinity against .sup.131I-MIBG.
[0096] NE and cold MIBG were used as references. As a result, cold reference 2 (AF78) could inhibit the cell uptake of .sup.131I-MIBG in a concentration-dependent manner. In contrast, cold reference 1 was not able to block the uptake of .sup.131I-MIBG even at the highest concentration tested. Inhibition was only 40% at 100 μM. This result provides a hint for a structure-activity relationship. The introduction of long alkyl chain at 3-position on the benzene ring seems not to be tolerated, whereas introduction at the 4-position seems to be well tolerated.
[0097] After successful radiolabeling, .sup.18F-AF78 was also tested in SK-N-SH cells in order to check cell uptake. Antidepressant desipramine (DMI) was used as the inhibitor of NET. Results are shown in
[0098] To further confirm the tracer's binding affinity, .sup.18F-labeled AF78 was used in competitive inhibition assay, to which different concentrations of NE, MHPG, and cold reference 2 (AF78) were added.
Ex Vivo Autoradiography and Tissue Counting Studies
[0099] Standard protocols and data analysis methods for non-invasive PET imaging of small animals have been established in the working group of the inventors (Rischpler C. et al., Eur J Nucl Med Mol Imaging. 2013; 40(7): 1077-83). Healthy male Wistar rats (Japan SLC, Inc., Japan) each weighing 200-250 g were used. The rats are anaesthetized with 2% isoflurane. 10-20 MBq of the tracer was administered via tail vein injection. Ten minutes after the tracer administration, the animals were immediately euthanized. Hearts, livers and blood were obtained for ex vivo analysis with autoradiography (Typhoon FLA 7000) and tissue counts with γ-counter (1480 WIZARD™ 3″). Following weight and decay correction of tissue counts, the heart to blood (H/B) and heart to liver (H/L) count ratio were calculated, respectively. For autoradiography, the rats were first injected via tail vein either with or without (control) NET blocker phenoxynezamine 50 mg/kg after anaesthesia. After 10 min, the tracer (10-20 MBq) was administered. The hearts were harvested 10 min later, frozen, and cut into 20 μm short axis slices using a cryostat (Cryotome FSE, Thermo Fisher Scientific). To obtain the distribution of the tracer, autoradiography plates (Fuji SR-type image plate, Fujifilm Corporation, Tokyo, Japan) were exposed to the short axis slices immediately for 18 h and thereafter imaged using a digital autoradiographic system (Typhoon FLA 7000).
Cardiac Imaging Studies in Rats and Monkeys
[0100] In an autoradiography study of left ventricular short axis (LVSA) slices from rats, .sup.18F-AF78 showed homogeneous tracer uptake throughout the left ventricular walls in the healthy myocardium. The result is shown in
[0101] An ex vivo tissue counting study showed that the tracer has a reasonable uptake in the heart as demonstrated in both Heart-to-Blood (H/B) and Heart-to-Liver (H/L) ratio counting 12.54 and 6.14, respectively. This uptake could be specifically blocked by PhB (50 mg/kg) and led to approximately 6 fold decline in the H/B ratio and 5 fold decline in the H/L ratio, counting 2.41 and 1.30, respectively. Results of the tissue distribution study are shown in
Cardiac PET Imaging in Animals
[0102] Rats were maintained anaesthetized during the whole experiment by 2% isoflurane. All scans were obtained using a dedicated small-animal PET system (microPET FOCUS 120, SIEMENS, Germany). The PET imaging protocol was designed to assess the systemic and myocardial tracer distribution of .sup.18F-AF78. Shortly before the injection of 10-20 MBq of .sup.18F-AF78 via the tail vein, a dynamic PET scan was initiated. In order to evaluate the cardiac uptake mechanism of the tracer, rats were pretreated with phenoxybenzamine (50 mg/kg intravenously, Sigma-Aldrich, Tokyo, Japan). Ten minutes after the pretreatment, 10-20 MBq of .sup.18F-AF78 was administered via the tail vein. A 10 min dynamic PET session was started shortly before the tracer injection. PET image was acquired in list-mode format. The data were sorted into 3-dimensional sonograms, which were then rebind with a Fourier algorithm to reconstruct dynamic images using a 2-dimensional ordered-subset expectation maximization (OSEM) algorithm. All images were corrected for .sup.18F decay, random, and dead time; correction for attenuation was not performed (Higuchi JNM 2013). The obtained PET images were analyzed with the public domain tool AMIDE imaging software (A Medical Imaging Data Examiner, version 1.01).
[0103] Systemic distribution of .sup.18F-AF78 .sup.18F-37 in the healthy rat myocardium by in vivo PET with (NET Blockade) or without (No pretreatment) pretreatment with NET blocker PhB (50 mg/kg iv injection) 10 min before the tracer injection is shown in
[0104] For control, Cynomolgus monkey (male, 5.3 kg) was maintained anaesthetized during the imaging study using 3.3% seboflurane. Shortly before receiving an intravenous injection of the tracer (44 MBq), a 120 min dynamic PET session was started. In addition, to investigate the inhibition of cardiac neuronal uptake-1, DMI (1 mg/kg, iv.) was injected to Cynomolgus monkey (female, 5.6 kg) 10 min before the tracer (18.2 MBq) injection and PET assessment started. PET was conducted using a PCA-2000A positron scanner (Toshiba Medical Systems Corporation, Otawara, Japan). Sinograms were collected with the following pattern: 10 sec×12, 30 sec×6, 300 sec×23. The tissue uptake was determined as arbitrary unit (A.U.), from which a time-activity curves of different organs were generated.
[0105] A tissue bio-distribution consistent with that observed in rat could be observed with clear long term and stable cardiac uptake during the total 120 min scan whereas uptake in adjacent liver and lungs was low. Representative time activity curves derived from .sup.18F-AF78 dynamic images in control and sympathetic nerve blocked monkey were generated from the scanning data. The time activity curves expressed in arbitrary unit, A.U. versus time in minutes are shown
[0106] Further biodistribution studies were performed in rats (n=4) 10 min after injection of the tracer .sup.18F-37 demonstrating favourable heart-to-blood (H/B, approx. 6:1) and heart-to-liver (H/L, approx. 2:1) ratios (
[0107] Static PET images of cardiac uptake of .sup.18F-AF78 in healthy rats, rabbits, pigs and monkeys (
[0108] A further biodistribution study with .sup.18F-AF78 in monkey has been conducted. Results calculated as SUV (standardized uptake value) are shown in
[0109] A kinetic study has been performed with .sup.18F-AF78 in monkeys' hearts. The results confirmed the specific uptake of .sup.18F-AF78 via NET and stable vesicular storage mechanism in the sympathetic nerve terminals. When injected 10 min before the tracer administration, NET blocker desipramine (DMI) specifically blocked the tracer uptake with significant difference (p<0.0001) compared to control (
Statistical Analysis
[0110] All results are displayed as mean±SD. The two-tailed paired Student t-test was used to compare differences between two dependent groups, and the two-tailed independent Student t-test for differences between independent groups. Multiple group comparisons were performed using analysis of variance (ANOVA). A p value of less than 0.05 was assumed to be statistically significant. Statistical analysis was performed with StatMate III (ATMS Co., Ltd.).
The Meaning of Abbreviations Used in this Specification is as Follows: DIAD, diisopropyl azodicarboxylate; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DMI, desipramine; .sup.11C-HED, .sup.11C-(−)-m-hydroxyephedrine; iv, intravenously; .sup.18F-MHPG, .sup.18F-4-fluoro-3-hydroxyphenethylguanidine; .sup.123/131I-MIBG, .sup.123/131I-meta-iodobenzylguanidine; NE, norepinephrine; NET, norepinephrine transporter; PET, positron emission tomography; PhB, phenoxybenzamine; .sup.18F-PHPG, .sup.18F-3-fluoro-4-hydroxyphenethylguanidine; SPECT, single photon emission computed tomography; THF, tetrahydrofuran