RADIOLABELED CANNABINOID RECEPTOR 2 LIGAND
20210115012 · 2021-04-22
Assignee
Inventors
- Simon M. Ametamey (Zuerich, CH)
- Luca Gobbi (Basel, CH)
- Uwe Grether (Basel, CH)
- Julian Kretz (Basel, CH)
- Dieter MURI (Basel, CH)
- Ahmed Haider Wahauib (Zuerich, CH)
Cpc classification
A61K51/0455
HUMAN NECESSITIES
C07B2200/05
CHEMISTRY; METALLURGY
A61K51/0459
HUMAN NECESSITIES
C07D401/04
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a compound of formula (I)
##STR00001##
wherein R.sup.1, R.sup.2, and R.sup.3 are defined as in the description and in the claims. The compound of formula (I) can be used as a radiolabeled ligand.
Claims
1. A compound of formula (I) ##STR00031## wherein A is CH or N; R.sup.1 and R.sup.2 are both ethyl at the same time; and R.sup.3 is fluoropropyl or hexadeuteriofluoropropyl; provided that at least one of R.sup.1, R.sup.2 and R.sup.3 comprises at least one radionuclide; or a pharmaceutically acceptable salt thereof.
2. A compound according to claim 1, wherein A is CH.
3. A compound according to claim 1 or 2, wherein both R.sup.1 and R.sup.2 comprise at least one radionuclide or R.sup.3 comprises a radionuclide.
4. A compound according to any one of claims 1 to 3, wherein the at least one radionuclide is independently selected from [.sup.3H], [.sup.18F] and [.sup.11C].
5. A compound according to any one of claims 1 to 4, wherein R.sup.1 and R.sup.2 are both —C.sup.3HH—C.sup.3HH.sub.2 at the same time.
6. A compound according to any one of claims 1 to 5, wherein R.sup.3 is —CH.sub.2—CH.sub.2—CH.sub.2.sup.18F or —CD.sub.2-CD.sub.2-CD.sub.2.sup.18F.
7. A compound according to any one of claims 1 to 6 selected from 3-(18F)Fluoranylpropyl 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate; (1,1,2,2,3,3-Hexadeuterio-3-(18F)fluoranyl-propyl) 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate; and 3-Fluoropropyl 2-(1,2-ditritioethyl)-2-[[6-[[(1 S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]-3,4-ditritio-butanoate; or a pharmaceutically acceptable salt thereof.
8. The use of a compound according to any one of claims 1 to 7 for localizing a CB2 receptor in a patient or sample.
9. The use of a compound according to any one of claims 1 to 7 for imaging a CB2 receptor in a patient or sample.
10. The use of a compound according to any one of claims 1 to 7 for determining whether another compound binds to a CB2 receptor.
11. The use according to claim 10 further comprising measuring the binding constant of said another compound to the CB2 receptor.
12. The use according to claim 10 or 11 in the presence of the CB receptor.
13. The use of a compound according to any one of claims 1 to 7 to determine whether a disease is characterized by a change in the expression of the CB2 receptor.
14. The use according to claim 13, wherein the disease is pain, atherosclerosis, age-related macular degeneration, diabetic retinopathy, glaucoma, diabetes mellitus, inflammation, inflammatory bowel disease, ischemia-reperfusion injury, acute liver failure, liver fibrosis, lung fibrosis, kidney fibrosis, systemic fibrosis, acute allograft rejection, chronic allograft nephropathy, diabetic nephropathy, glomerulonephropathy, cardiomyopathy, heart failure, myocardial ischemia, myocardial infarction, systemic sclerosis, thermal injury, burning, hypertrophic scars, keloids, gingivitis pyrexia, liver cirrhosis or tumors, regulation of bone mass, neurodegeneration, stroke, transient ischemic attack or uveitis.
15. A compound according to any one of claims 1 to 7 for use in the diagnosis of a disease in a patient or tissue.
16. A compound for use according to claim 15, wherein the disease is characterized by a change in the expression of the CB2 receptor in said patient or tissue compared to the expression of the CB2 receptors in a healthy subject or tissue.
17. A compound according to claim 15 or 16, wherein the diagnosis comprises the step of comparing the expression of the CB2 receptor in the patient or tissue to the expression of the CB2 receptor in a healthy subject or tissue.
18. The use of a compound according to any one of claims 1 to 7 to predict whether a patient affected with a disease is likely to respond to a treatment involving the administration of a CB2 ligand.
19. The use according to claim 18 comprising comparing the expression of the CB2 receptor in the patient to the expression of the CB2 receptor in a healthy subject or tissue.
20. The use of a compound according to any one of claims 1 to 7 to assess the efficacy of a medical therapy in a patient, comprising monitoring the CB2 receptor density in the patient before, during and/or after said medical therapy.
21. The use of a compound according to any one of claims 1 to 7 for determining the dose of a CB2 ligand that needs to be administered to a patient in need thereof.
22. A method for identifying a compound that binds to a CB2 receptor comprising the following steps: (a) contacting the compound suspected to bind to the CB2 receptor with a sample comprising a CB2 receptor and a compound according to any one of claims 1 to 7; and (b) monitoring whether the compound suspected to bind to the CB2 receptor influences the binding of the compound according to any one of claims 1 to 7 to the CB2 receptor.
23. A method according to claim 22 further comprising the step of measuring the binding strength to the CB2 receptor of the compound suspected to bind to the CB2 receptor.
24. A method according to claim 23 further comprising measuring the binding constant to the CB2 receptor of the compound suspected to bind selectively to the CB2 receptor.
25. A method for identifying a cellular receptor as a CB2 receptor comprising the following steps: (a) contacting a sample suspected to comprise a CB2 receptor with a compound according to any one of claims 1 to 7; and (b) monitoring whether the binding of the compound according to any one of claims 1 to 7 has occurred; and (c) optionally further contacting the sample with another known CB2 ligand and monitoring whether said known CB2 ligand has displaced the compound according to any one of claims 1 to 7 from its binding site.
26. A method for measuring in a sample the percentage of CB2 receptors occupied by a compound suspected to bind to the CB2 receptor when said compound is put in contact with the sample, comprising the following steps: (a) contacting a sample comprising at least one CB2 receptor with a compound according to any one of claims 1 to 7 to determine a baseline signal; (b) contacting the sample with the dose of said compound suspected to bind to the CB2 receptor and a compound according to any one of claims 1 to 7; (c) monitoring the displacement of the compound according to any one of claims 1 to 7 by the compound suspected to bind to the CB2 receptor; and (d) calculating the percentage of the CB2 receptors that is occupied by the compound suspected to bind to the CB2 receptor.
27. A method for determining the dose of a CB2 ligand that needs to be administered to a vertebrate, in particular a human subject, in need thereof comprising the following steps: (a) administering to the vertebrate a compound according to any one of claims 1 to 7 and determining the baseline signal; (b) administering to the vertebrate different doses of CB2 ligand and concomitantly administering to the vertebrate a compound according to any one of claims 1 to 7; (c) monitoring the displacement of the compound according to any one of claims 1 to 7 by the different doses of CB2 ligand; and (d) calculating the percentage of CB2 receptors that is occupied by the CB2 ligand and determining the dose/occupancy relationship.
28. A method for determining whether a disease is characterized by a change in the expression of the CB2 receptor comprising the following steps: (a) contacting a sample or administering to a subject affected with said disease and a healthy sample or a healthy subject with a compound according to any one of claims 1 to 7; (b) monitoring in both samples whether the binding of the compound according to any one of claims 1 to 7 has occurred; and (c) comparing in both samples the amount of compound according to any one of claims 1 to 7 that is bound to the CB2 receptors.
29. A use, a method or a compound for use according to any one of claims 8 to 28 wherein positron emission tomography (PET), single-photon emission computed tomography (SPECT) or autoradiography is used during the monitoring.
30. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7.
31. A compound according to any one of claims 1 to 7 for use as a diagnostic agent.
32. A compound according to any one of claims 1 to 7 for use in the diagnostic of pain, atherosclerosis, age-related macular degeneration, diabetic retinopathy, glaucoma, diabetes mellitus, inflammation, inflammatory bowel disease, ischemia-reperfusion injury, acute liver failure, liver fibrosis, lung fibrosis, kidney fibrosis, systemic fibrosis, acute allograft rejection, chronic allograft nephropathy, diabetic nephropathy, glomerulonephropathy, cardiomyopathy, heart failure, myocardial ischemia, myocardial infarction, systemic sclerosis, thermal injury, burning, hypertrophic scars, keloids, gingivitis pyrexia, liver cirrhosis or tumors, regulation of bone mass, neurodegeneration, stroke, transient ischemic attack or uveitis.
33. A process for the manufacture of a compound according to any one of claims 1 to 7 comprising on of the following steps: (a) reacting a compound of formula (A) ##STR00032## a nucleophilic [.sup.18F]fluoride reagent, or (b) reacting a compound of formula (B) ##STR00033## with [.sup.3H].sub.2; wherein LG is a leaving group and wherein R.sup.1 to R.sup.3 are as defined in any one of claims 1 to 6.
34. A compound manufactured according to a process of claim 32.
35. The invention as hereinbefore described.
Description
EXAMPLES
Abbreviations
[0101] rac-BINAP=racemic 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl); CAN=chemical abstracts service number; DCM=dichloromethane; DIPEA=N-ethyl-N-isopropylpropan-2-amine; DMF=dimethylformamide; DPPA=diphenylphosphoryl azide; EI=electron impact; EtOAc=ethyl acetate; HATU=1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; LAH=Lithiumaluminiumhydrid; LC=liquid chromatography; LiTMP=lithium 2,2,6,6-tetramethylpiperidine; MS=mass spectrometry; NMR=nuclear magnetic resonance; NMR data are reported in parts per million (S) relative to internal tetramethylsilane and are referenced to the deuterium lock signal from the sample solvent (d.sub.6-DMSO unless otherwise stated); coupling constants (J) are in Hertz; PTSA=p-toluenesulfonic acid; Rt=retention time; SOR=specific optical rotation; TBTU=O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyl-uronium-tetrafluoroborate; THF=tetrahydrofuran.
Experimental
[0102] All reactions were undertaken in flame dried glassware. Analytical grade solvents were used for reactions and when required, dry solvents were used without further purification. Reagents were purchased from reputable commercial suppliers and used without further purification, unless otherwise stated. All .sup.1H NMR spectra were recorded on a Bruker Advance Ultra Shield 300 MHz spectrometer. Chemical shifts are reported relevant to the stated deuterated solvent. Mass spectra were recorded on PE Sciex API 150EX LC/MS Turbo Spray System. Flash chromatography was conducted using an Isco Combi Flash companion, using prepacked silica columns (230-400 mesh, 40-63 μm) of various sizes from various commercial suppliers. Thin layer chromatography was carried on pre-coated plates (20×20 cm, silica gel F254) purchased from Merck KgaA and was visualized using a 254 nm CAMAG UV lamp or using a basic potassium permanganate solution. All reactions were monitored using a combination of thin layer chromatography, LCMS and .sup.1H NMR
Example 1
3-[.SUP.18.F]Fluoropropyl 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate
[0103] ##STR00009##
a) Bis(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl butanedioate
[0104] ##STR00010##
[0105] A 2 L one-necked, round bottom flask was equipped with a stirrer, a Dean-Stark trap and a condenser. The flask was charged with succinic anhydride (64 g, 0.64 mol, 1 eq.). I-menthol (200 g, 1.3 mol, 2 eq.), p-toluenesulfonic acid monohydrate (1.1 g, 6.39 mmol, 0.01 eq.) and tolune (576 mL). The mixture was heated under reflux for 24 h, cooled to 25° C., diluted with hexane (640 mL) and poured into a mixture of aqueous saturated sodium bicarbonate (800 mL), methanol (320 mL) and water (320 mL). The layers were separated and the aqueous phase was extracted with hexane (2×320 mL). The organic phases were combined, washed with brine (640 mL), dried over sodium sulphate and filtered. The solvent was removed under reduced pressure and the crude product was dissolved in methanol (240 mL). The solution was cooled to +4° C. for 16 h to form colorless crystals which were collected by filtration with suction. The crystals were purified by recrystallization from methanol (240 mL) to afford pure bis(1R,2S,5R)-5-methy-2-(propan-2-yl)cyclohexyl butanedioate (212 g, 84%).
[0106] SOR value: [−87.64° ] at t≅25° C., 1.0132% solution in CHCl.sub.3.
b) 1,2-Bis(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (1S,2S)-cyclopropane-1,2-dicarboxylate
[0107] ##STR00011##
[0108] A 1.8 M solution of butyllithium in THF (152.2 mmol, 84 mL) was added to 225 mL of THF at 0° C. under a N atmosphere. Under stirring lithium tetramethylpiperidide (28.2 mL, 167 mmol) was added drop wise over a 20 min period. Stirring was continued at 0° C. for 1 h. Then the reaction mixture was cooled to −78° C. A solution of bis(R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl butanedioate (30 g, 76.1 mmol) in THF (60 mL) was added drop wise over a 20 min period. The yellow solution was stirred for 1 h. Bromochloromethane (4.08 mL, 60.91 mmol) was added drop wise over a 20 min period. The mixture was stirred for 3 h at −78° C. A saturated aqueous solution of NH.sub.4Cl (120 mL) was added. After stirring for 30 min at 25° C. the mixture was extracted with EtOAc (3 10×150 mL). The combined organic layers were washed with brine (200 mL), dried over Na.sub.2SO.sub.4, filtered, and concentrated in vacuo. The crude was purified by column chromatography (SiO.sub.2, 100-200 mesh, 0.5-1% of ethyl acetate and hexane) to afford the title compound (38 g, 42%) as colorless crystals. Recrystallization of this material from methanol (380 mL) provided pure 1,2-bis(R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (1S,2S)-cyclopropane-1,2-dicarboxylate (27 g, 36%).
[0109] SOR value: [+18.18° ] at t≅25° C., 1.0288% solution in CHCl.sub.3.
c) (1S,2S)-Cyclopropane-1,2-dicarboxylic acid mono-((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl) ester
[0110] ##STR00012##
[0111] To a solution of 1,2-bis(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (1S,2S)-cyclopropane-1,2-dicarboxylate (25 g, 61.58 mmol) in isopropanol (250 mL) was added a 5 M solution of NaOH (13.54 mL, 67.73 mmol) at 25° C. The mixture was stirred at 70° C. for 16 h. The organic solvent was removed under reduced pressure. Water (2(0 mL) was added and the mixture was washed with diethyl ether (2×150 mL). The aqueous layer was acidified with 2 N HCl (pH ˜2) and extracted with ethyl acetate (3×250 mL). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to get (1S,2S)-2-({[(R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]oxy}carbonyl)cyclopropane-1-carboxylic acid (11.4 g, 69%) as off white semisolid.
d) (1R,2S,5R)-5-Methyl-2-(propan-2-yl)cyclohexyl (1S,2S)-2-(hydroxymethyl) cyclopropane-1-carboxylate
[0112] ##STR00013##
[0113] To a stirred solution of (1S,2S)-cyclopropane-1,2-dicarboxylic acid mono-((R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl) ester (20 g, 74.63 mmol) in THF (200 mL) was added a 1 M solution of borane in THF (56 mL) drop wise at −78° C. The mixture was stirred for 1 h at 25° C. and quenched with aq. NH.sub.4Cl solution (150 mL). The organic solvent was removed under reduced pressure. Water was added (50 mL) and the mixture was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with brine (80 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (15-19% ethyl acetate/hexane) to get (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl(1S,2S)-2-(hydroxymethyl)cyclopropane-1-carboxylate (13.7 g, 72%) as yellowish semi solid.
e) (1R,2S,5R)-5-Methyl-2-(propan-2-yl)cyclohexyl (1S,2S)-2-[(benzyloxy) methyl]cyclopropane-1-carboxylate
[0114] ##STR00014##
[0115] To a stirred solution of (R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (S,2S)-2-(hydroxymethyl) cyclopropane-1-carboxylate (20 g, 78.74 mmol) in DMF (140 mL) was added NaH (4.72 g, 118.11 mmol) at 0° C. The mixture was stirred at 25° C. for 30 min. Benzylbromide (18.7 mL, 157.5 mmol) was added and stirring was continued at 25° C. for 30 min. Aqueous NH.sub.4C solution (150 mL) was added and the mixture was extracted with EtOAc (2×150 mL). The combined organic layers were washed with water (3×120 m), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (1.9% EtOAc/hexane) to get (R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (1S,2S)-2-[(benzyloxy)methyl]cyclopropane-1-carboxylate (22 g, 8%) as light yellow oil.
f) [(1S,2S-2-[(Benzyloxy)methyl]cyclopropyl]methanol
[0116] ##STR00015##
[0117] To a stirred solution of (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (1S,2S)-2-[(benzyloxy) methyl] cyclopropane-1-carboxylate (10 g, 29.01 mmol) in THF (200 mL) was added LAH (58.1 mL, 1 M in THF) at 0° C. The reaction mixture was stirred for 40 min at 0° C. and quenched with aq NH.sub.4Cl solution (100 mL). The organic solvent was removed under reduced pressure. The solution was extracted with ethyl acetate (3×100 mL). The combined organic layers were brought to dryness and the crude was purified using silica gel column chromatography (30-35% ethyl acetate/hexane) to get [(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methanol (5.33 g, 95%) as light yellow oil.
g) 6-{[(1S,2S)-2-[(Benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridine-2-carboxylic acid
[0118] ##STR00016##
[0119] To a solution of 5-bromo-6-chloropyridine-2-carboxylic acid (CAN 959958-25-9, 4 g, 19.80 mmol) in DMF (45 mL) was added NaH (2.77 g, 69.31 mmol) portion wise at 0° C. and stirred for 20 min at 0° C. [(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methanol (4.18 g, 21.78 mmol) in DMF (15 mL) was added drop wise at 0° C. The mixture was stirred for 15 min at 25° C., heated to 80° C. for 3 h, cooled to 25° C. and quenched with 2 N aq. HCl to pH ˜2. Water (100 mL) was added and the mixture was extracted with EtOAc (3×150 mL). The combined organic layers were washed with water (4×50 mL) and brine (50 mL), dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to get 6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridine-2-carboxylic acid (7.7 g, 99%) as off white sticky liquid.
[0120] LCMS: Column Zorbax Ext C 18 (50×4.6 mm), 5μ, (mobile phase: from 90% [10 mM NH.sub.4OAc in water] and 10% [CH.sub.3CN] to 70% [10 mM NH.sub.4OAc in water] and 30% [CH.sub.3CN] in 1.5 min, further to 10% [10 mM NH.sub.4OAc in water] and 90% [CH.sub.3CN] in 3.0 min, held this mobile phase composition to 4 min and finally back to initial condition in 5 min). Purity is 76.8%, Rt=2.60 min, MS calculate: 391, MS found: 391.8 ([M+H].sup.+).
h) Ethyl 2-[(6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridin-2-yl)formamido]-2-ethylbutanoate
[0121] ##STR00017##
[0122] To a solution of 6-{[(S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridine-2-carboxylic acid (15.5 g, 39.54 mmol) in DMF (100 mL) were added DIPEA (27.49 mL, 158.16 mmol), ethyl 2-amino-2-ethylbutanoate (CAN 189631-96-7, 7.73 g, 39.54 mmol) and TBTU (15.25 g, 47.449 mmol). The reaction mixture was stirred at 25° C. for 16 h, poured into water (170 mL) and extracted with EtOAc (3×200 mL). The combined organic layers were washed with water (4×120 mL) and brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and brought to dryness. The crude was purified via silica gel column chromatography (25% ethyl acetate/hexanes) to get ethyl 2-[(6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridin-2-yl)formamido]-2-ethylbutanoate (20.5 g, 97%) as light brown oil.
[0123] LCMS: Column Zorbax Ext C 18 (50×4.6 mm), 5μ, (mobile phase: from 90% [10 mM NH.sub.4OAc in water] and 10% [CH.sub.3CN] to 70% [10 mM NH.sub.4OAc in water] and 30% [CH.sub.3CN] in 1.5 min, further to 10% [10 mM NH.sub.4OAc in water] and 90% [CH.sub.3CN] in 3.0 min, held this mobile phase composition to 4 min and finally back to initial condition in 5 mm). Purity is 91.47%. Rt=2.58 min. MS calculate: 533, MS found: 533.0 (M+H+.sup.+).
i) Ethyl 2-[(6-{1[(1S,2S)-2-[(benzyloxy) methyl]cyclopropyl] methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]-2-ethylbutanoate
[0124] ##STR00018##
[0125] To a solution ethyl 2-[(6-{[(S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridin-2-yl)formamido]-2-ethylbutanoate (4.0 g, 7.5 mmol) in toluene (160 mL) were added 3-methoxyazetidine (1.39 g, 11.3 mmol) and cesium carbonate (7.33 g, 22.5 mmol). The mixture was degassed with argon for 10 min. Rac-BINAP (0.935 g, 1.50 mmol) and Pd(II)acetate (0.34 g, 1.50 mmol) were added. The mixture was heated to 110° C. for 3 h, diluted with EtOAc (100 mL), filtered through a celite bed and washed with EtOAc (3×100 mL). The filtrate was concentrated and the crude purified through silica gel column chromatography (42-50% ethyl acetate/hexanes) to get ethyl 2-[(6-{1[(S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]-2-ethylbutanoate (3.1 g, 76%) as light brown oil.
[0126] LCMS: Column Zorbax Ext C 18 (50×4.6 mm), 5μ, (mobile phase: from 90% [10 mM NH.sub.4OAc in water] and 10% [CH.sub.3CN] to 70% [10 mM NH.sub.4OAc in water] and 30% [CH.sub.3CN] in 1.5 min, further to 10% [10 mM NH.sub.4OAc in water] and 90% [CH.sub.3CN] in 3.0 min, held this mobile phase composition to 4 min and finally back to initial condition in 5 min). Purity is 96.7%. Rt=2.37 min, MS calculate: 539, MS found: 539.9 ([M+H].sup.+).
j) Ethyl 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoat
[0127] ##STR00019##
[0128] A stirred solution of ethyl 2-[(6-{1[(S,2S)-2-[(benzyloxy) methyl]cyclopropyl] methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]-2-ethylbutanoate (26 g, 48.24 mmol) in 735 mL EtOAc:MeOH (10:1) was degassed for 30 min. Pd/C (10%) (6.5 g) was added. The mixture was hydrogenated under a hydrogen atmosphere at 40 PSI for 28 h at 25° C., filtered through a celite bed and washed with 10% MeOH/EtOAc (4×200 mL). The filtrate was evaporated under reduced pressure to get the crude. The crude was purified applying silica gel column chromatography (10-50% EtOAc:hexanes) to get ethyl 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido] butanoate (19.3 g, 89%) as colorless sticky liquid.
[0129] SOR value: [+15.51°] at t≅20° C., 0.2514% in MeOH.
[0130] LCMS: Column Zorbax Ext C 18 (50×4.6 mm), 5μ, (mobile phase: from 90% [10 mM NH.sub.4OAc in water] and 10% [CH.sub.3CN] to 70% [10 mM NH.sub.4OAc in water] and 30% [CH.sub.3CN] in 1.5 min. further to 10%1 [10 mM NH.sub.4OAc in water] and 90% [CH.sub.3CN] in 3.0 min, held this mobile phase composition to 4 min and finally back to initial condition in 5 min). Purity is 98.9%, Rt=3.26 min, MS calculate: 449, MS found: 449.9 ([M+H].sup.+).
k) 2-Ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoic acid
[0131] ##STR00020##
[0132] In a 25 ml round-bottomed flask, ethyl 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoate (100 mg, 0.22 mmol) was combined with THF (2.0 mL), MeOH (2.2 mL) and water (2.0 mL) to give a light yellow solution. KOH pellets (62 mg, 1.11 mmol) were added. The mixture was heated to 90° C. After 18 h the organic solvent was removed under reduced pressure. The aqueous phase was diluted with water (20 mL) and extracted with diethyl ether (2×10 mL). The combined organic layers were discarded. The aqueous phase was adjusted to pH˜2 (1 M HCl) and extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine (10 mL), dried, filtered and brought to dryness under reduced pressure to get pure 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoic acid (90 mg, 96%) as colorless sticky mass.
[0133] LCMS: Column Zorbax Ext C 18 (50×4.6 mm), 5μ, (mobile phase: from 90% [10 mM NH.sub.4OAc in water] and 10% [CH.sub.3CN] to 70% [10 mM NH.sub.4OAc in water] and 30% [CH.sub.3CN] in 1.5 min, further to 10% [10 mM N.sub.4OAc in water] and 90% [CH.sub.3CN] in 3.0 min, held this mobile phase composition to 4 min and finally back to initial condition in 5 min). Purity is 95.5%, Rt=2.00 min, MS calculate: 419, MS found: 420.4 ([M+H].sup.+).
l) 3-{[(4-Methylbenzene)sulfonyl]oxy}propyl 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoate
[0134] ##STR00021##
[0135] To a solution of 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoic acid (260 mg, 0.62 mmol) in DMF (5 mL) were added K.sub.2CO.sub.3 (256 mg, 1.85 mmol) and 3-{1[(4-methylbenzene)sulfonyl]oxy}propyl 4-methylbenzene-1-sulfonate (711 mg, 1.85 mmol). The reaction mixture was stirred for 16 h at 25° C., poured into water, quenched with aq. 1 (N) HCl and extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (30 mL), dried, filtered and concentrated in vacuo to get crude product which was purified by combiflash using silica column and 20-80% EtOAc in hexane to get pure 3-{[(4-methylbenzene)sulfonyl]oxy}propyl 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoate (255 mg, 65%) as colorless sticky mass.
[0136] LCMS: Column Zorbax Ext C 18 (50×4.6 mm), 5μ, (mobile phase: from 90% [10 mM NH.sub.4OAc in water] and 10% [CH.sub.3CN] to 70% [10 mM NH.sub.4OAc in water] and 30% [CH.sub.3CN] in 1.5 min, further to 10% [10 mM NH.sub.4OAc in water] and 90% [CH.sub.3CN] in 3.0 min, held this mobile phase composition to 4 min and finally back to initial condition in 5 min). Purity is 90.7%, Rt=3.48 min, MS calculate: 633, MS found: 634.4 ([M+H].sup.+).
m) 3-[.SUP.18.F]Fluoropropyl 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate
[0137] [.sup.18F]Fluoride ions were obtained from bombardment of 98% enriched .sup.18O-water via the e .sup.18O(p,n).sup.18F nuclear reaction in a Cyclone 18/9 cyclotron (18-MeV; IBA Belgium), trapped on an anion exchange cartridge (Waters SepPak Accell QMA cartridge carbonate) and subsequently eluted with a solution of K.sub.2CO.sub.3 (1 mg/mL) and Krypofix.sub.222 (2.5 mg/mL) in water/MeCN 1:3. Volatiles were removed at 110° C. under reduced pressure and with a gentle flow of nitrogen. Azeotropic drying was carried out using MeCN (3×1 mL). Upon addition of 3-{[(4-methylbenzene)sulfonyl]oxy}propyl 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoate (1 mg in 0.5 mL MeCN), the reaction mixture was stirred at 90° C. for 10 minutes. The reaction mixture was subsequently diluted with water (2.5 mL) and the crude was purified by semi-preparative HPLC (Merck-Hitachi L2130 system), equipped with a radiation detector VRM 202 (Comecer, Netherlands) in combination with an ACE 5 C-18-300 (250×10.0 mm, 5μm) column and a gradient solvent system: 0.1% H3PO4 in H2O (solvent A), MeCN (solvent B); 0.0-8.0 min, 20% B; 8.1-30.0 min, 20-90% B; 30.1-35.0 min, 90% B; 35.1-37.0 min, 90-20% B; 37.1-43.0 min, 20% B. A flow rate of was 4 mL/min was used and the UV signal detection was conducted at 230 nm. Product fraction of the semipreparative HPLC was collected in 35 mL of water and passed through a C18 cartridge (Waters, pre-conditioned with 5 mL EtOH and 5 mL water). The cartridge was washed with water (5 mL) and the title compound subsequently eluted using 0.5 mL EtOH. The radioligand 3-(18F)fluoranylpropyl 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate was formulated with 5% EtOH in water for injection (WFI). 3-(18F)Fluoranylpropyl 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate was obtained with molar activities ranging from 52-65 GBq/μmol and excellent radiochemical purity (>99%). The decay-corrected radiochemical yield was 9.0±0.4%. Analytical quality control was performed to determine radiochemical and chemical purity, specific activity, and chemical identity on an Agilent 1100 series HPLC system, equipped with UV detector and a GabiStar radiodetector (Raytest). A reverse phase ACE C18-AR column (50×4.6 mm, 3 mm) was used in combination with the following separation conditions: 0.1% TFA in H2O (solvent A), MeCN (solvent B); 0.0-2.0 min, 20% B; 2.1-12.0 min, 20-90% B; 12.1-14.0 min, 90% B; 14.1-15.0 min, 90-20% B; 15.1-20.0 min, 20% B. The flow rate of 1 mL/min was used and the UV signal was recorded at 230 nm. Molar activities were calculated by comparing the UV intensity of the formulated products with calibration curves of the corresponding non-radioactive standards.
Example 2
(1,1,2,2,3,3-Hexadeuterio-3-[.SUP.18.F]fluoranyl-propyl) 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate
[0138] ##STR00022##
a) [1,1,2,2,3,3-Hexadeuterio-3-(p-tolysulfonyloxy)propyl] 4-methylbenzenesulfonate
[0139] ##STR00023##
[0140] To a solution of 1,1,2,2,3,3-hexadeuteriopropane-1,3-diol (73 mg, 0.87 mmol) in DCM (1 mL) were added 2,6-lutidine (0.5 mL, 4.34 mmol) and tosyl chloride (496 mg, 2.60 mmol, 3 eq.). The reaction mixture was stirred for 17 h at 25° C., diluted with DCM (20 mL), washed with aq. 1 N HCl (10 mL), dried, filtered and concentrated in vacuo. The crude was purified by column chromatography on silica gel (5-30% EtOAc in hexane) to get the title compound (205 mg, 61%) as white solid.
[0141] LCMS: Column Zorbax Ext C 18 (50×4.6 mm), 5μ, (mobile phase: from 90% [10 mM NH4OAc in water] and 10% [CH3CN] to 70% [10 mM NH4OAc in water] and 30% [CH3CN] in 1.5 min, further to 10% [10 mM NH4OAc in water] and 90% [CH3CN] in 3.0 min, held this mobile phase composition to 4 min and finally back to initial condition in 5 min). Purity is 99.84%, Rt=3.48 min, MS calculate: 390. MS found: 408.1 ([M+NH.sub.4].sup.+).
b) [1,1,2,2,3,3-Hexadeuterio-3-(p-tolylsulfonyloxy)propyl] 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate
[0142] ##STR00024##
[0143] To a solution of 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoic acid (example 1 k, 50 mg, 0.12 mmol) in DMF (5.0 mL) were added K.sub.2CO.sub.3 (49 mg, 0.35 mmol) and [1,1,2,2,3,3-hexadeuterio-3-(p-tolylsulfonyloxy)propyl] 4-methylbenzenesulfonate (93 mg, 0.24 mmol). The reaction mixture was stirred for 17 h, quenched with water (30 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine (20 mL), dried, filtered and concentrated in vacuo. The crude was purified by column chromatography on silica gel (30-80% EtOAc in hexane) to obtain the title compound (50 mg, 67%) as colorless liquid.
[0144] LCMS: Column Zorbax Ext C 18 (50×4.6 mm), 5μ, (mobile phase: from 90% [10 mM NH.sub.4OAc in water] and 10% [CH.sub.3CN] to 70% [10 mM NH.sub.4OAc in water] and 30% [CH.sub.3CN] in 1.5 min, further to 10% [10 mM NH.sub.4OAc in water] and 90% [CH.sub.3CN] in 3.0 min, held this mobile phase composition to 4 min and finally back to initial condition in 5 min). Purity is 95.5%, Rt=3.47 min, MS calculate: 639, MS found: 640.3 ([M+H].sup.+).
c) (1,1,2,2,3,3-Hexadeuterio-3-[.SUP.18.F]fluoranyl-propyl) 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate
[0145] [.sup.18F] Fluoride ions were obtained from bombardment of 98% enriched .sup.18O-water via the e .sup.18O(p,n).sup.18F nuclear reaction in a Cyclone 18/9 cyclotron (18-MeV; IBA Belgium). [.sup.18F]Fluoride was trapped on an anion exchange cartridge (Waters SepPak Accell QMA cartridge carbonate) and subsequently eluted with a solution of K.sub.2CO.sub.3 (mg/mL) and Krypofix.sub.222 (2.5 mg/mL) in water/MeCN 1:3. Volatiles were removed at 110° C. under reduced pressure and with a gentle flow of nitrogen. Azeotropic drying was carried out using MeCN (3×1 mL). Upon addition of [1,1,2,2,3,3-hexadeuterio-3-(p-tolylsulfonyloxy)propyl 2-ethyl-2-[6-1[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxyl-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate (1 mg in 0.5 mL MeCN), the reaction mixture was stirred at 90° C. for 10 minutes. The reaction mixture was subsequently diluted with water (2.5 mL) and the crude was purified by semi-preparative HPLC (Merck-Hitachi L2130 system), equipped with a radiation detector VRM 202 (Comecer, Netherlands) in combination with an ACE 5 C-18-300 (250×10.0 mm, 5μm) column and a gradient solvent system: 0.1% H3PO4 in H2O (solvent A), MeCN (solvent B); 0.0-8.0 min, 20% B; 8.1-30.0 min, 20-90% B; 30.1-35.0 min, 90% B; 35.1-37.0 min, 90-20% B; 37.1-43.0 min, 20% B. A flow rate of was 4 mL/min was used and the UV signal detection was conducted at 230 nm. Product fraction of the semipreparative HPLC was collected in 35 mL of water and passed through a C18 cartridge (Waters, pre-conditioned with 5 mL EtOH and 5 mL water). The cartridge was washed with water (5 mL) and the title compound subsequently eluted using 0.5 mL EtOH. The radioligand (1,1,2,2,3,3-hexadeuterio-3-(18F)fluoranyl-propyl) 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate was formulated with 5% EtOH in water for injection (WFI), radioligand (1,1,2,2,3,3-hexadeuterio-3-(18F)fluoranyl-propyl) 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate was obtained with molar activities ranging from 27-44 GBq/μmol and excellent radiochemical purity (>99%). The decay-corrected radiochemical yield was 5.0±1.1%. Analytical quality control was performed to determine radiochemical and chemical purity, specific activity, and chemical identity on an Agilent 1100 series HPLC system, equipped with UV detector and a GabiStar radiodetector (Raytest). A reverse phase ACE C18-AR column (50×4.6 mm, 3 mm) was used in combination with the following separation conditions: 0.1% TFA in H2O (solvent A), MeCN (solvent B); 0.0-2.0 min, 20% B; 2.1-12.0 min, 20-90% B; 12.1-14.0 min, 90% B; 14.1-15.0 min, 90-20% B; 15.1-20.0 min, 20% B. The flow rate of 1 mL/min was used and the UV signal was recorded at 230 nm. Molar activities were calculated by comparing the UV intensity of the formulated products with calibration curves of the corresponding non-radioactive standards.
Example 3
3-Fluoropropyl 2-(1,2-ditritioethyl)-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]-3,4-ditritio-butanoate
[0146] ##STR00025##
a) 2-Ethyl-2-(6-(((1S,2S)-2-(hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinamido)butanoyl azide
[0147] ##STR00026##
[0148] In a 30 mL round bottom flask 2-ethyl-2-(6-(((S,2S)-2-(hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolmamido)butanoic acid (example 1k, 338 mg, 802 μmol, 1 eq.) was dissolved in toluene (14 ML). Triethylamine (81 mg, 116 μL, 802 μmol, 1 eq.) and DPPA (221 mg, 173 μL, 802 μmol, 1 eq.) were added. The reaction mixture was stirred for 24 h at ambient temperature, poured onto water (20 mL) and extracted with AcOEt (3×30 mL). The combined organic layers were dried over sodium sulfate and concentrated in vacuo. The crude material was purified by flash chromatography (SiO.sub.2, 120 g, 10-70% AcOEt in heptane) to give the title compound (177 mg, 0.396 mmol, 48%) as off-white solid.
[0149] .sup.1H NMR (600 MHz, CDCl.sub.3): δ ppm 8.32 (s, 2H, NH), 7.54-7.59 (d, .sup.3J=7.9 Hz, 1H, N.sub.Py—C.sub.q—CH—CH), 6.46-6.53 (d, .sup.3J=7.9 Hz, 1H, N.sub.Py—C.sub.q—CH), 4.09-4.30 (m, 8H, O—CH.sub.2, CH.sub.2—N—CH.sub.2, PO.sub.3—O—CH.sub.2, O—CH), 3.72-3.84 (m, 2H, CH—N—CH.sub.2), 3.23 (s, 3H, O—CH.sub.3), 2.35-2.51 (m, 2H, N.sub.3CO—C.sub.q—CH.sub.2), 1.68-1.89 (m, 2H, N.sub.3CO—C.sub.q—CH.sub.2), 1.24-1.34 (m, 2H, CH—CH.sub.2—CH), 0.74 (t, .sup.3J=7.5 Hz, 6H, N.sub.3CO—C.sub.q—CH.sub.2—CH.sub.3), 0.63-0.72 (m, 2H, CH—CH.sub.2—CH)
[0150] HRMS (ESI): C.sub.21H.sub.30N.sub.6O.sub.5 [M+H].sup.+ calculated=447.2304; found=447.2296.
b) 6-(((1S,2S)-2-(Hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinamide
[0151] ##STR00027##
[0152] In a 25 mL round bottom flask 2-ethyl-2-(6-(((S,2S)-2-(hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinamido)butanoyl azide (177 mg, 0.396 mmol, 1 eq.) was dissolved in toluene (10.0 mL). The reaction mixture was heated to 110° C. upon stirring for 3 h and then concentrated in vacuo. THF (3 mL) and 3N NaOH (7 mL) were added. The reaction mixture was heated to 90° C. for 1 h upon stirring, poured onto water (10 mL) and extracted with AcOEt (3×40 mL). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give the title compound (85 mg, 0.277 mmol, 70%) as light orange oil. The crude material was used in the next step without further purification.
[0153] .sup.1H NMR (600 MHz, CDCl.sub.3): δ ppm 8.18 (CO—NH.sub.2), 7.74 (d, .sup.3J=8.0 Hz, 1H, N.sub.Py—C.sub.q—CH—CH), 6.56 (d, .sup.3J=8.0 Hz, 1H, N.sub.Py—C.sub.q-C.sub.H), 3.99-4.41 (m, 7H, O—CH.sub.2, CH.sub.2—N—CH.sub.2, O—CH, HO—CH.sub.2), 3.95-4.00 (m, 2H. CH—N—CH.sub.2), 3.29 (m, 3H, O—CH.sub.3), 1.20-1.36 (CH—CH.sub.2—CH), 0.54-0.79 (m, 2H, CH—CH.sub.2—CH)
[0154] MS (ESI): C.sub.15H.sub.21N.sub.2O.sub.4 [M+H].sup.+ calculated=308.14; found=308.20.
c) 6-(((1S,2S)-2-(Hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinic acid
[0155] ##STR00028##
[0156] In a 25 mL round bottom flask 6-(((1S,2S)-2-(hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinamide (85 mg, 0.277 mmol, 1 eq.) was dissolved in methanol (3 mL) and water (5 mL). Sodium hydroxide (55 mg, 1.38 mmol, 5 eq.) was added. The reaction mixture was heated to 85° C. for 12 h upon stirring, poured onto water (10 mL) and 1N HCl (3 mL) and extracted with AcOEt (3×20 mL). The combined organic layers were dried over sodium sulfate and concentrated in vacuo. The crude material was purified by flash chromatography (SiO.sub.2, 12 g, 40-100% AcOEt in heptane) to give the title compound (64 mg, 0.207 mmol, 75%) as a light orange solid.
[0157] .sup.1H NMR (600 MHz, CDCl.sub.3): δ ppm 7.72 (dd, .sup.3J=7.9 Hz, .sup.4J=2.9 Hz, 1H, N.sub.Py—C.sub.q—CH—CH), 6.56 (d, .sup.3J=7.9 Hz, 1H, N.sub.Py—C.sub.q—CH), 3.99-4.41 (m, 7H, O—CH.sub.2, CH.sub.2—N—CH.sub.2, O—CH, HO—CH.sub.2), 3.97-3.99 (m, 2H, CH.sub.2—N—CH.sub.2), 3.28 (m, 3H, O—CH) 1.18-1.32 (CH—CH.sub.2—CH), 0.56-0.81 (m, 2H, CH—CH.sub.2—CH)
[0158] HRMS (ESI): C.sub.15H.sub.20N.sub.2O.sub.5 [M+H].sup.+ calculated=309.1379; found=309.1451.
d) 3-Fluoropropyl 2-amino-2-vinylbut-3-enoate
[0159] ##STR00029##
[0160] 3-Fluoropropan-1-ol (1.55 g, 1.61 mL, 19.8 mmol, Eq.: 18) and 2-amino-2-vinylbut-3-enoic acid hydrochloride (CAN 1865695-91-5, 180 mg, 1.1 mmol, Eq.: 1) were added to a round bottom flask. Sulfurous dichloride (1.31 g, 798 μL, 11 mmol, Eq.: 10) was added. The reaction mixture was stirred for 1 h at 80° C., poured onto water (10 mL) and extracted with CH.sub.2Cl.sub.2 (2×20 mL). The organic layers were combined, dried over sodium sulfate, filtered and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 12 g, 20% to 70% AcOEt in heptane) to give the title compound as colorless oil, LC-MS (UV peak area/ESI) 94%, 187.1083 [MH.sup.+].
e) 3-Fluoropropyl 2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]-2-vinyl-but-3-enoate
[0161] ##STR00030##
[0162] 6-(((1S,2S)-2-(Hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxazetidin-1-yl)picolinic acid (19.8 mg, 64.1 μmol, Eq.: 0.8) and 3-fluoropropyl 2-amino-2-vinylbut-3-enoate (15 mg, 80.1 μmol, Eq.: 1) were dissolved in CH.sub.2C2 (1.34 mL). N-Ethyl-N-isopropylpropan-2-amine (41.4 mg, 55.2 μL, 320 μmol, Eq.: 4) followed by 1-(bis(dimethylamino)methylene)-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium 3-oxide hexafluorophosphate(V) (36.6 mg, 96.1 μmol, Eq. 1.2) were added. The reaction mixture was stirred for 1 h at ambient temperature, poured onto water (10 mL) and extracted with CH.sub.2Cl.sub.2 (4×20 mL). The organic layers were combined, dried over sodium sulfate, filtered and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 12 g, 20% to 70% AcOEt in heptane) to give the title compound as colorless oil, LC-MS (UV peak area/ESI) 98%, 478.2399 [MH.sup.+].
f) 3-Fluoropropyl 2-(1,2-ditritioethyl)-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxazetidin-1-yl)pyridine-2-carbonyl]amino]-3,4-ditritio-butanoate
[0163] In a 2 ml titration flask, 3-Fluoropropyl 2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]-2-vinyl-but-3-enoate (2.0 mg, 4.2 μmol, 1.0 eq.) and Pd/C (10%) (0.89 mg, 0.84 μmol, 0.2 eq.) were suspended in dimethylformamide (0.4 ml). The flask was attached to the tritium manifold (RC-TRITEC) and degassed by freeze-pump-thaw. Tritium gas was introduced, and the black suspension was vigorously stirred for 3 hours under an atmosphere of tritium at 560 mbar. The solution was cooled by liquid nitrogen and the excess tritium gas in the reaction vessel was reabsorbed on a uranium-trap for waste-tritium. The solvent was lyophilized off and labile tritium was removed by lyophilization with a methanol (3×1 ml). The remaining black residue was suspended in methanol (10 ml) and filtered over a 17 mm Titan HPLC filter (0.45μm, PTFE) to provide 8.21 GBq (222 mCi) of a crude product in a purity of >90%. The crude product was concentrated and purified by preparative HPLC (SunFire C18, 5μm, 4.6×250 mm) using acetonitrile [A] and 5% acetonitrile in water [B] as eluent (gradient: 10% [A], 90%[B] to 99%[A], 1%[B] in 12 min, hold for 3 min, then back to initial conditions for 5 min). 4.59 GBq (124 mCi) were obtained of the title compound with a radiochemical purity of 98.7% and a specific activity of 4.18 TBq/mmol (113 Ci/mmol), determined by MS spectrometry. The compound was stored as an ethanolic solution. MS m/z: 482.3 [M+H].sup.+ (1%), 484.3 [M(.sup.3H)+H].sup.+ (5%), 486.3 [M(.sup.3H.sub.2)+H].sup.+ (13%), 488.3 [M(.sup.3H.sub.3)+H].sup.+ (21%), 490.3[M(.sup.3H.sub.4)+H].sup.+ (12%), 492.3[M(.sup.3H.sub.5)+H].sup.+ (20%), 494.3 [M(.sup.3H.sub.6)+H].sup.+ (12%), 496.3 [M(.sup.3H.sub.7)+H].sup.+ (4%).
Example 4
Radioligand Binding Assay and Micro PET Studies
[0164] Stably transfected cells or spleen tissue were homogenized in 15 mmol L-1 Hepes, 0.3 mmol L-1 EDTA, 1 mmol L-1 EGTA 2 mmol L-1 MgCl2, complete EDTA-free protease inhibitor (Roche Applied Science, Rotkreuz, Switzerland), pH 7.4 using a glass potter and centrifugation at 47,800 g at 4° C. for 30 min. The pellet was then rehomogenized twice in the same buffer and centrifuged (47,800 g, 4° C., 30 min). The final pellet was then resuspended in 75 mmol L-1 Tris, 0.3 mmol L-1 EDTA, 1 mmol L-1 EGTA, 12.5 mmol L-1 MgCl2, 250 mmol L-1 sucrose, pH 7.4 at a protein concentration of 1 to 3 mg mL-1, aliquoted, frozen on dry ice and stored at −80° C.
[0165] Saturation binding was performed with 0.05 to 2.4 nM compound of formula (I) and 40 μg of membrane protein. CP55940 (10 μM) was used to define nonspecific binding. Assay buffer consisted of 50 mmol L-1 Tris-HCL 5 mmol L-1 MgCl2, 2.5 mmol L-1 EGTA, and 0.1% fatty acid-free BSA, pH 7.4. Assays were initiated by addition of membranes in a final volume of 250 μl/well. Assays were incubated for 2 h at room temperature and then vacuum filtered and rinsed with wash buffer (50 mmol L-1 Tris-HCl, 5 mmol L-MgCl2, 2.5 mmol L-1 EGTA, and 0.5% fatty acid-free BSA, pH 7.4) on a Filtermate cell harvester through Packard GF/B filters presoaked in 0.3% polyethylenimine.
[0166] For competition binding, membrane preparations were incubated either with 0.3 nM of [.sup.3H]-CP55940 in the presence or absence of increasing concentrations of unlabeled (R.sup.1═R.sup.2═CH.sub.2CH3, R.sup.3═CH.sub.2CH.sub.2CH.sub.2F) compound of formula (I) or with 1.5 nM compound of formula (I) ((R.sup.1═R.sup.2=CHTCH.sub.2T, R.sup.3═CH.sub.2CH.sub.2CH.sub.2F)) and increasing amounts of membranes (2.5-80 μg) in the presence or absence of CP55940 (10 μM) for 60 min at 30° C. in a final volume of 0.2 mL of 50 mmol L-1 Tris-HCl, 5 mmol L-1 MgCl2, 2.5 mmol L-1 EGTA, 0.1% fatty acid-free BSA and 1% DMSO. pH 7.4, buffer, gently shaking. All binding reactions were terminated by vacuum filtration onto 0.5% polyethylenimine presoaked GF/B filter plates (Packard) followed by seven brief washes with 2 mL of ice-cold binding buffer containing 0.5% fatty acid-free BSA. Plates were dried at 50° C. for 1 h and liquid scintillation counting was used to determine bound radiolabel. IC50 values and Hill slopes were determined by a four parameter logistic model using ActivityBase (ID Business Solution, Ltd.).
[0167] The results are shown in Table 1 and
[0168]
TABLE-US-00001 TABLE 1 Radioligand competition binding of [.sup.3H]CP55940 using CHO-K1 cell expressing human CB2 receptors Human CB1 Human CB2 Ki [nM] >10′000 0.7
[0169] Table 1 demonstrates high binding selectivity of unlabeled compound of formula (I) for human CB2 receptors (Ki 0.7 nM) vs human CB1 receptors (Ki>10′000 nM) in cells that recombinantly express these receptors and using the non-selective CB1/CB2 radioligand [.sup.3H]CP55940.