Radiolabeled cannabinoid receptor 2 ligand

Abstract

To provide a compound that can be used as a radiolabeled ligand.SOLUTION: The present invention provides a compound represented by formula (I) in the figure or a pharmaceutically acceptable salt thereof. In the formula, A is CH or N; R1 and R2 are both ethyl at the same time; and R3 is fluoropropyl or hexadeuteriofluoropropyl; provided that at least one of R1, R2 and R3 comprises at least one radionuclide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to radiolabeled cannabinoid receptor 2 ligands. [Background technology]

[0002] In particular, the present invention relates to a compound of formula (I):

[0003] [ka] [In the formula, R 1 and R 2 are both simultaneously ethyl; and R 3 is 3-fluoropropyl (where R 1 , R 2 and R 3 at least one of which contains at least one radionuclide), or a pharmaceutically acceptable salt thereof.

[0004] Cannabinoid receptors are a type of cell membrane receptor that belong to the G protein-coupled receptor superfamily. Currently, there are two known subtypes, termed cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). CB1 receptors are widely expressed. They are primarily expressed in the central nervous system (i.e., amygdala, cerebellum, and hippocampus) and to a lesser extent in the periphery. CB2, encoded by the CNR2 gene, is expressed mostly peripherally on cells of the immune system such as macrophages, B cells, and T cells (Ashton, JC et al. Curr Neuropharmacol 2007, 5(2), 73-80; Miller, AM et al. Br J Pharmacol 2008, 153(2), 299-308; Centonze, D., et al. Curr Pharm Des 2008, 14(23), 2370-42), as well as in the digestive system (Wright, KL et al. Br J Pharmacol 2008, 153(2), 263-70). CB2 receptors are also present in the brain, where they are found primarily in microglia and not in neurons (Cabral, GA et al. Br J Pharmacol 2008, 153(2), 240-51).

[0005] Interest in CB2 receptor agonists has grown significantly since some of the earliest compounds were discovered to be effective in treating chronic pain (Beltramo, M. Mini Rev Med Chem 2009, 9(1), 11-25), atherosclerosis (Mach, F. et al. J Neuroendocrinol 2008, 20 Suppl 1, 53-7), bone mass regulation (Bab, I. et al. Br J Pharmacol 2008, 153(2), 182-8), neuroinflammation (Cabral, GA et al. J Leukoc Biol 2005, 78(6), 1192-7), ischemia / reperfusion injury (Pacher, P. et al. Br J Pharmacol 2008, 153(2), 252-62), and systemic fibrosis (Akhmetshina, A. et al. Arthritis Rheum 2009, 60(4), 1129-36; Garcia-Gonzalez, E. et al. Rheumatology (Oxford) 2009, 48(9), 1050-6) and liver fibrosis (Julien, B. et al. Gastroenterology 2005, 128(3), 742-55; Munoz-Luque, J. et al. J Pharmacol Exp Ther 2008, 324(2), 475-83).

[0006] Ischemia / reperfusion (I / R) injury is not only the primary cause of tissue damage in conditions such as stroke, myocardial infarction, cardiopulmonary bypass and other vascular surgeries, and organ transplantation, but also a major mechanism of end-stage organ failure, exacerbating the course of circulatory shock of various etiologies. All of these conditions are characterized by inadequate tissue oxygenation due to disruption of normal blood supply. Reoxygenation, e.g., reperfusion, is the ultimate treatment for restoring normal tissue oxygenation. However, the lack of oxygen and nutrients from the blood creates conditions in which restoration of circulation leads to further tissue damage. The damage caused by reperfusion injury is due, in part, to the inflammatory response of the injured tissue. Leukocytes transported to the area by newly returned blood release many inflammatory factors, such as interleukins, as well as free radicals, in response to tissue injury. The restored blood flow reintroduces oxygen into cells, which damages intracellular proteins, DNA, and cell membranes.

[0007] Remote ischemic preconditioning (RIPC) represents a strategy for harnessing the body's endogenous protective capacity against injury caused by ischemia and reperfusion. It explains the intriguing phenomenon that transient, nonlethal ischemia and reperfusion in one organ or tissue confers resistance to the subsequent development of "lethal" ischemia-reperfusion injury in a distant organ or tissue. Although several hypotheses have been proposed, the actual mechanism by which transient ischemia and reperfusion in an organ or tissue provides protection is currently unknown.

[0008] The humoral hypothesis proposes that endogenous substances (such as adenosine, bradykinin, opioids, CGRP, endocannabinoids, angiotensin I, or other unidentified humoral factors) produced in distant organs or tissues enter the bloodstream and activate their respective receptors in target tissues, thereby mobilizing various intracellular pathways of cardioprotection involved in ischemic preconditioning.

[0009] Recent data indicate that endocannabinoids and their receptors, especially CB2, may be involved in preconditioning and contribute to the prevention of reperfusion injury by downregulating the inflammatory response (Pacher, P. et al. Br J Pharmacol 2008, 153(2), 252-62). Specifically, recent studies using CB2 agonists have demonstrated the efficacy of this concept in reducing I / R injury in the heart (Defer, N. et al. Faseb J 2009, 23(7), 2120-30), brain (Zhang, M. et al. J Cereb Blood Flow Metab 2007, 27(7), 1387-96), liver (Batkai, S. et al. Faseb J 2007, 21(8), 1788-800) and kidney (Feizi, A. et al. Exp Toxicol Pathol 2008, 60(4-5), 405-10).

[0010] Moreover, over the past few years, a growing body of literature has shown that CB2 may also be of interest in the subchronic and chronic phases: specific upregulation of CB1 and CB2 has been shown in animal models of chronic fibrotic diseases to be associated with the appropriate expression of CB2 in myofibroblasts, cells involved in the progression of fibrosis (Garcia-Gonzalez, E. et al. Rheumatology (Oxford) 2009, 48(9), 1050-6; Yang, YY et al. Liver Int 2009, 29(5), 678-85).

[0011] Activation of CB2 receptors by selective CB2 agonists has indeed been shown to exert antifibrotic effects in diffuse systemic sclerosis (Garcia-Gonzalez, E. et al. Rheumatology (Oxford) 2009, 48(9), 1050-6), and CB2 receptors have been implicated in experimental skin fibrosis (Akhmetshina, A. et al. Arthritis Rheum 2009, 60(4), 1129-36) and in liver pathophysiology, including fibrogenesis associated with chronic liver disease (Lotersztajn, S. et al. Gastroenterol Clin Biol 2007, 31(3), 255-8; Mallat, A. et al. Expert Opin Ther Targets 2007, 11(3), 403-9; Lotersztajn, S. et al. Br J Pharmacol 2008, 153(2), 286-9) has emerged as a key target.

[0012] The need to specifically detect CB2 in tissues arose with growing interest in this receptor. Using appropriate tools to assess CB2 expression and receptor occupancy in patients or samples can validate target cells of expression and allow for dose titration of CB2 ligands in human trials or for diagnostic purposes.

[0013] To date, there is a lack of efficient tools for detecting CB2 receptor protein in tissues, which is a result of the low expression level of CB2 receptors. Another reason for the lack of specific antibodies as a detection tool for CB2 may be due to the obvious difficulty of using CB2 as an immunogen.

[0014] A number of PET tracers targeting the CB2 receptor have been reported in recent years (Caille, F et al., Mol. Pharmaceutics, 2017, 14 (11), 4064-4078 and references cited therein). All of these are short-lived radioisotopes. 11C (decay half-life 20.3 min) or lack selectivity for CB1 receptors and have the disadvantage of being highly lipophilic, resulting in unfavorable signal-to-noise ratios. 18 New PET tracers containing F-labels are highly desirable. 18 The F isotope (decay half-life of 110 minutes) will greatly expand the distribution and use of tracers after production. 18 The low positron emission energy of F makes this isotope the preferred PET radionuclide for acquiring images with higher spatial resolution.

[0015] The incorporation of fluorine atoms into the chemical structure of a small molecule has a significant effect on its physicochemical and biological properties (K. Muller et al, 2007, 317 (5846), 1881-1886). Therefore, when searching for candidate structures for radiofluorinable PET tracers, it is not easy to identify suitable compounds that maintain and combine all the desired properties within a single molecule.

[0016] Compounds of formula (I) as defined above have surprisingly been identified to have desirable properties and have been found to exhibit greatly reduced non-specific binding.

[0017] Compounds of formula (I) were found to bind specifically and selectively to membranes prepared from cells recombinantly expressing CB2 receptors. Furthermore, compounds of formula (I) were found to specifically label CB2 receptors in spleen tissue, an organ with high expression of both CB1 and CB2 receptors. Furthermore, there was no binding by compounds of formula (I) in spleen tissue isolated from CB2 receptor-deficient mice. In this specific case, the total binding signal was significantly greater than that of unlabeled (R 1 =CH3) compound.

[0018] Thus, compounds of formula (I) can be used for dose-finding of CB2 ligands in human trials or for diagnostic purposes, e.g., in tissue autoradiography and PET imaging, for assessing receptor expression and receptor occupancy.

[0019] In this description, the term "radionuclide" defines an isotope of an atom that has an unstable nucleus and undergoes radioactive decay. Specific radionuclides of the present invention are 3 H], [ 18 F], [ 11 C] and [ 14 C], and for more details [ 3 H] and [ 18 F].

[0020] The term "binding constant" refers to the equilibrium constant associated with the binding reaction of a ligand to a receptor.

[0021] The term "selective binding" characterizes the binding of a ligand to a very specific type of receptor.

[0022] The present invention thus relates to: Compounds of formula (I) wherein A is CH; R 1 and R 2 Both contain at least one radionuclide, or R 3 a compound of formula (I) containing one radionuclide; At least one radionuclide is 3 H], [ 18 F] and [ 11 C], a compound of formula (I); R 1 and R 2 But both at the same time -C 3 HH-C 3 HH2, a compound of formula (I); R 3 But -CH2-CH2-CH2 18 F or -CD2-CD2-CD2 18 F; below: 2-Ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid 3-[ 18 F]fluoropropyl; 2-Ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid (1,1,2,2,3,3-hexadeuterio-3-[ 18 F]fluoro-propyl); and 2-(1,2-ditrithioethyl)-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]-3,4-ditrithiobutanoate 3-fluoropropyl or a pharmaceutically acceptable salt thereof; Use of a compound of formula (I) for identifying CB2 receptors in a patient, animal or sample; Use of a compound of formula (I) for imaging CB2 receptors in a patient, animal or sample; Use of a compound of formula (I) to determine whether another compound binds to the CB2 receptor; use of a compound of formula (I) for determining whether another compound binds to the CB2 receptor, the use further comprising measuring the binding constant of said another compound to the CB2 receptor; Use of a compound of formula (I) to determine whether another compound binds to the CB2 receptor in vivo by receptor occupancy testing using PET; Use as above in the presence of CB1 receptors; Use of a compound of formula (I) to determine whether a disease is characterized by altered expression of CB2 receptors; Use of a compound of formula (I) for determining whether a disease is characterized by altered expression of CB2 receptors, wherein the disease is pain, atherosclerosis, age-related macular degeneration, diabetic retinopathy, glaucoma, diabetes, inflammation, inflammatory bowel disease, ischemia-reperfusion injury, acute liver failure, liver fibrosis, pulmonary fibrosis, renal fibrosis, systemic fibrosis, acute allograft rejection, chronic allograft nephropathy, diabetic nephropathy, glomerular nephropathy, cardiomyopathy, heart failure, myocardial ischemia, myocardial infarction, systemic sclerosis, thermal injury, scald, hypertrophic scar, keloid, gingivitis, fever, cirrhosis or liver tumor, bone mass regulation, neurodegeneration, stroke, transient ischemic attack or uveitis; A compound of formula (I) for use in the diagnosis in a patient or tissue of a disease; a compound of formula (I) for use in the diagnosis in a patient or tissue of a disease as defined above, wherein the disease is characterized by an altered expression of the CB2 receptor in said patient or tissue compared to the expression of the CB2 receptor in healthy subjects or tissue; A compound as defined above for use wherein the diagnosis comprises comparing the expression of CB2 receptors in a patient or tissue with the expression of CB2 receptors in a healthy subject or tissue; the use of a compound of formula (I) to predict whether an affected patient is likely to respond to a treatment comprising the administration of a CB2 ligand; Use of a compound of formula (I) for predicting whether an affected patient is likely to respond to treatment comprising the administration of a CB2 ligand, comprising comparing the expression of CB2 receptors in the patient with the expression of CB2 receptors in healthy subjects or tissues; Use of a compound of formula (I) for assessing the efficacy of drug therapy in a patient, comprising monitoring CB2 receptor density (i.e., CB2 receptor expression) in the patient before, during and / or after said drug therapy; and Use of a compound of formula (I) to determine the required dose of a CB2 ligand to be administered to a patient in need thereof.

[0023] The present invention further provides a method for identifying a compound that binds to a CB2 receptor, comprising the steps of: (a) contacting a compound suspected of binding to the CB2 receptor with a sample containing the CB2 receptor and a compound of formula (I); and (b) monitoring whether the compound suspected of binding to the CB2 receptor affects the binding of the compound of formula (I) to the CB2 receptor. The present invention relates to a method comprising:

[0024] The present invention also relates to a method as defined above, which further comprises the step of measuring the binding strength to the CB2 receptor of a compound suspected of binding to the CB2 receptor.

[0025] The present invention also provides a method for identifying a cellular receptor as a CB2 receptor, comprising the steps of: (a) contacting a sample suspected of containing CB2 receptors with a compound of formula (I); and (b) monitoring whether binding of the compound of formula (I) has occurred; and (c) optionally further contacting the sample with another known CB2 ligand and monitoring whether the known CB2 ligand displaces the compound of formula (I) from its binding site. The present invention relates to a method comprising:

[0026] The present invention also provides a method for determining in a sample the proportion of CB2 receptors occupied by a compound suspected of binding to CB2 receptors when said compound is contacted with said sample, the method comprising the steps of: (a) contacting a sample containing at least one CB2 receptor with a compound of formula (I) and determining a baseline signal; (b) contacting the sample with a dose of the compound suspected of binding to the CB2 receptor and a compound of formula (I); (c) monitoring the displacement of the compound of formula (I) by a compound suspected of binding to the CB2 receptor; and (d) calculating the percentage of CB2 receptors occupied by the compound suspected of binding to the CB2 receptors. The present invention relates to a method comprising:

[0027] The present invention also provides a method for determining the percentage of CB2 receptors occupied by a compound suspected of binding to CB2 receptors in a living vertebrate, including a human subject, when a dose of said compound is administered to the vertebrate, the method comprising the steps of: (a) administering a compound of formula (I) to a vertebrate and determining a baseline signal; (b) co-administering to a vertebrate a dose of the compound suspected of binding to the CB2 receptor and a compound of formula (I); (c) monitoring the displacement of the compound of formula (I) by a compound suspected of binding to the CB2 receptor; and (d) calculating the percentage of CB2 receptors occupied by the compound suspected of binding to the CB2 receptors. The present invention relates to a method comprising:

[0028] The present invention also provides a method for determining the required dose of a CB2 ligand to be administered to a vertebrate, including a human subject, in need thereof, comprising the steps of: (a) administering a compound of formula (I) to a vertebrate and determining a baseline signal; (b) administering to the vertebrate various doses of a CB2 ligand and simultaneously administering to the vertebrate a compound of formula (I); (c) monitoring the displacement of the compound of formula (I) by various doses of the CB2 ligand; and (d) calculating the percentage of CB2 receptors occupied by the CB2 ligand to determine a dose / occupancy relationship; The present invention relates to a method comprising:

[0029] In step (a) above, the baseline signal is considered to be 100%.

[0030] The present invention also provides a method for determining whether a disease is characterized by an altered expression of the CB2 receptor, comprising the steps of: (a) contacting a sample with a compound of formula (I) or administering a compound of formula (I) to a patient with said disease and a healthy sample or healthy subject; (b) monitoring whether binding of the compound of formula (I) has occurred in both samples; and (c) comparing the amount of compound of formula (I) bound to the CB2 receptor in both samples. The present invention relates to a method comprising:

[0031] Imaging techniques for carrying out the above steps include, but are not limited to, Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), particularly PET.

[0032] The present invention also relates to the method of the present invention, wherein autoradiography is used for monitoring.

[0033] The present invention further relates to pharmaceutical compositions comprising compounds of formula (I).

[0034] The present invention also relates to the compounds of formula (I) for use as diagnostic agents, i.e. for use in diagnosing disease.

[0035] The present invention also relates to a compound of formula (I) for use in the diagnosis of pain, atherosclerosis, age-related macular degeneration, diabetic retinopathy, glaucoma, diabetes, inflammation, inflammatory bowel disease, ischemia-reperfusion injury, acute liver failure, hepatic fibrosis, pulmonary fibrosis, renal fibrosis, systemic fibrosis, acute allograft rejection, chronic allograft nephropathy, diabetic nephropathy, glomerular nephropathy, cardiomyopathy, heart failure, myocardial ischemia, myocardial infarction, systemic sclerosis, thermal injury, scald, hypertrophic scar, keloid, gingivitis, fever, cirrhosis or liver tumour, regulation of bone mass, neurodegeneration, stroke, transient ischemic attack or uveitis.

[0036] The synthesis of compounds of formula (I) can be achieved, for example, by the following scheme.

[0037] [ka]

[0038] The target compound, 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid 3-[ 18 F]fluoropropyl (Ia) and 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid (1,1,2,2,3,3-hexadeuterio-3-[ 18 The carboxylic acid precursor of [F]fluoro-propyl) (16) can be generated from commercially available succinic anhydride (1), 1-menthol (2), and 5-bromo-6-chloropyridine-2-carboxylic acid (9) as described in Scheme 1 or by applying alternative synthetic strategies known to those skilled in the art.

[0039] [ka]

[0040] The target compound (Ia) can be obtained via a two-step procedure starting from carboxylic acid (16) and bis-tosylate (17) as described in Scheme 2. In the second step, [ 18 via unsupported nucleophilic substitution using [F]KF / Kryptofix 2.2.2, or any other method known to those skilled in the art; 18 F can be introduced at high specific activity.

[0041] [ka]

[0042] The deuterated target compound (Ib) can be obtained similarly to its non-deuterated analog (Ia) via a two-step procedure starting from carboxylic acid (16) and hexadeutero bis-tosylate (19) as described in Scheme 3. In the second step, [ 18via unsupported nucleophilic substitution using [F]KF / Kryptofix 2.2.2, or any other method known to those skilled in the art; 18 F can be introduced at high specific activity.

[0043] [ka]

[0044] The tritiated target compound (Ic) can be synthesized from carboxylic acid (16) and amine (24) by the procedure described in Scheme 4 or any other route known to one skilled in the art. In the radiolabeling step, bis-olefin (25) is subjected to reduction with tritium gas to give 3-fluoropropyl 2-(1,2-ditrithioethyl)-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]-3,4-ditrithio-butanoate.

[0045] The present invention therefore also provides a method for preparing a compound of formula (I), comprising the steps of: (a) Formula (A):

[0046] [ka] The compound represented by the formula [ 18 F] fluoride; or (b) Formula (B):

[0047] [ka] The compound represented by [ 3 reacting with H2 wherein LG is a leaving group and R 1 ~R 3 has the same meaning as above].

[0048] Leaving groups are, for example, p-tolylsulfonyloxy, 4-nitrobenzenesulfonyloxy, methanesulfonyloxy or trifluoromethanesulfonyloxy. In step (a), 18 [F] fluoride reagents, e.g., 18 F]KF / K 2.2.2 It may be. Step (a) may be carried out, for example, in acetonitrile. Step (a) can be carried out at a temperature between 25 and 200°C, but heating is not essential. The present invention further relates to compounds prepared by the methods of the present invention. The invention will now be illustrated by the following examples, which are not of a limiting nature. [Example]

[0049] Abbreviation 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 hexafluorophosphate 3-oxide; LAH = lithium aluminum hydride; LC = liquid chromatography LiTMP = lithium 2,2,6,6-tetramethylpiperidide; MS = mass spectrometry; NMR = nuclear magnetic resonance; NMR data are reported as parts per million (δ) relative to internal tetramethylsilane and referenced to the deuterium lock signal from the sample solvent (d6-DMSO unless otherwise noted); coupling constants (J) are expressed in Hertz; PTSA = p-toluenesulfonic acid; Rt = retention time; SOR = specific rotation; TBTU = O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate; THF = tetrahydrofuran.

[0050] experiment All reactions were carried out in flame-dried glassware. Analytical grade solvents were used in the reactions, and anhydrous solvents were used without further purification where necessary. Reagents were purchased from reputable suppliers and used without further purification unless otherwise stated. All 1 H NMR spectra were recorded on a Bruker Advance Ultra Shield 300 MHz spectrometer. Chemical shifts relative to the designated deuterated solvents are reported. Mass spectra were recorded on a PE Sciex API 150EX LC / MS Turbo Spray System. Flash chromatography was performed using an Isco Combi Flash® Companion using pre-packed silica columns of various sizes (230-400 mesh, 40-63 μm) from various suppliers. Thin-layer chromatography was performed on pre-coated plates (20 × 20 cm, silica gel F254) purchased from Merck KgaA and visualized using a 254 nm CAMAG UV lamp or basic potassium permanganate solution. All reactions were analyzed by thin-layer chromatography, LCMS, and HPLC. 1 The reaction was monitored using a combination of 1 H NMR. Example 1

[0051] 2-Ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid 3-[ 18 F]Fluoropropyl

[0052] [ka]

[0053] a) bis(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl butanedioate

[0054] [ka]

[0055] A 2 L single-neck round-bottom flask was equipped with a stirrer, Dean-Stark trap, and condenser. The flask was charged with succinic anhydride (64 g, 0.64 mol, 1 equiv.), 1-menthol (200 g, 1.3 mol, 2 equiv.), p-toluenesulfonic acid monohydrate (1.1 g, 6.39 mmol, 0.01 equiv.), and toluene (576 mL). The mixture was heated to reflux for 24 h, cooled to 25 °C, diluted with hexane (640 mL), and poured into a mixture of saturated aqueous 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 sulfate, 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 hours to form colorless crystals, which were collected by suction filtration and purified by recrystallization from methanol (240 mL) to give pure bis(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl butanedioate (212 g, 84%). SOR value: [-87.64°] at t ≈ 25°C, 1.0132% solution in CHCl3.

[0056] b) (1S,2S)-cyclopropane-1,2-dicarboxylic acid 1,2-bis(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl

[0057] [ka]

[0058] 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 N2 atmosphere. With stirring, lithium tetramethylpiperidide (28.2 mL, 167 mmol) was added dropwise over 20 minutes. Stirring was continued at 0 °C for 1 hour. The reaction mixture was then cooled to -78 °C. A solution of bis(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl butanedioate (30 g, 76.1 mmol) in THF (60 mL) was added dropwise over 20 minutes. The yellow solution was stirred for 1 hour. Bromochloromethane (4.08 mL, 60.91 mmol) was added dropwise over 20 minutes. The mixture was stirred at -78 °C for 3 hours. A saturated aqueous solution of NH4Cl (120 mL) was added. After stirring at 25 °C for 30 min, the mixture was extracted with EtOAc (3 × 150 mL). The combined organic layers were washed with brine (200 mL), dried over NaSO, filtered, and concentrated in vacuo. The crude product was purified by column chromatography (SiO, 100–200 mesh, 0.5–1% ethyl acetate and hexanes) to give the title compound (38 g, 42%) as colorless crystals. This material was recrystallized from methanol (380 mL) to give pure 1,2-bis(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (1S,2S)-cyclopropane-1,2-dicarboxylate (27 g, 36%). SOR value: [+18.18°] at t ≈ 25°C, 1.0288% solution in CHCl3.

[0059] c) (1S,2S)-cyclopropane-1,2-dicarboxylic acid mono-((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl) ester

[0060] [ka]

[0061] 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 (200 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 NaSO, filtered, and concentrated under reduced pressure to give (1S,2S)-2-({[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]oxy}carbonyl)cyclopropane-1-carboxylic acid (11.4 g, 69%) as an off-white semi-solid.

[0062] d) (1S,2S)-2-(hydroxymethyl)cyclopropane-1-carboxylic acid (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl

[0063] [ka]

[0064] To a stirred solution of (1S,2S)-cyclopropane-1,2-dicarboxylic acid mono-((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl) ester (20 g, 74.63 mmol) in THF (200 mL) was added dropwise a 1 M solution of borane in THF (56 mL) at −78° C. The mixture was stirred at 25° C. for 1 h and quenched with aqueous NH4Cl (150 mL). The organic solvents were removed under reduced pressure. Water (50 mL) was added and the mixture was extracted with ethyl acetate (2×100 mL). The combined organic layers were washed with brine (80 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (15–19% ethyl acetate / hexane) to give (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (1S,2S)-2-(hydroxymethyl)cyclopropane-1-carboxylate (13.7 g, 72%) as a yellowish semi-solid.

[0065] e) (1S,2S)-2-[(benzyloxy)methyl]cyclopropane-1-carboxylic acid (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl

[0066] [ka]

[0067] To a stirred solution of (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (1S,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. Benzyl bromide (18.7 mL, 157.5 mmol) was added and stirring was continued at 25 °C for 30 min. Aqueous NH4Cl (150 mL) was added and the mixture was extracted with EtOAc (2 × 150 mL). The combined organic layers were washed with water (3 × 120 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (1.9% EtOAc / hexane) to afford (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (1S,2S)-2-[(benzyloxy)methyl]cyclopropane-1-carboxylate (22 g, 8%) as a pale yellow oil.

[0068] f) [(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methanol

[0069] [ka]

[0070] 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 at 0 °C for 40 min and quenched with aqueous NHCl (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 dried, and the crude product was purified using silica gel column chromatography (30–35% ethyl acetate / hexane) to give [(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methanol (5.33 g, 95%) as a pale yellow oil.

[0071] g) 6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridine-2-carboxylic acid

[0072] [ka]

[0073] 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) portionwise at 0 °C and stirred at 0 °C for 20 min. [(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methanol (4.18 g, 21.78 mmol) in DMF (15 mL) was added dropwise at 0 °C. The mixture was stirred at 25 °C for 15 min, heated to 80 °C for 3 h, cooled to 25 °C, and quenched with 2 N aqueous HCl until 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 NaSO, and concentrated under reduced pressure to give 6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridine-2-carboxylic acid (7.7 g, 99%) as an off-white viscous liquid.

[0074] LCMS: Column: Zorbax Ext C 18 (50 × 4.6 mm), 5μ (Mobile phase: 90% [10 mM NHOAc aqueous solution] + 10% [CH3CN] ~ 70% [10 mM NHOAc aqueous solution] + 30% [CH3CN] over 1.5 min, then ~ 10% [10 mM NHOAc aqueous solution] + 90% [CH3CN] over 3.0 min, hold this mobile phase composition for 4 min, and finally return to initial conditions over 5 min). Purity: 76.8%, Rt = 2.60 min, MS calculated: 391, MS found: 391.8 ([M+H] + ).

[0075] h) Ethyl 2-[(6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridin-2-yl)formamido]-2-ethylbutanoate

[0076] [ka]

[0077] To a solution of 6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridine-2-carboxylic acid (15.5 g, 39.54 mmol) in DMF (100 mL) was 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 SO , filtered, and dried. The crude product was purified via silica gel column chromatography (25% ethyl acetate / hexanes) to afford ethyl 2-[(6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridin-2-yl)formamido]-2-ethylbutanoate (20.5 g, 97%) as a light brown oil.

[0078] LCMS: Column: Zorbax Ext C 18 (50 × 4.6 mm), 5μ (mobile phase: 90% [10 mM NHOAc aqueous solution] + 10% [CH3CN] ~ 70% [10 mM NHOAc aqueous solution] + 30% [CH3CN] over 1.5 min, then ~ 10% [10 mM NHOAc aqueous solution] + 90% [CH3CN] over 3.0 min, hold this mobile phase composition for 4 min, and finally return to initial conditions over 5 min). Purity: 91.47%, Rt = 2.58 min, MS calculated: 533, MS found: 533.0 ([M+H] + ).

[0079] i) 2-[(6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]-2-ethylbutanoate

[0080] [ka]

[0081] To a solution of ethyl 2-[(6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-bromopyridin-2-yl)formamido]-2-ethylbutanoate (4.0 g, 7.5 mmol) in toluene (160 mL) was 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 minutes. 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 hours, diluted with EtOAc (100 mL), filtered through a bed of Celite, and washed with EtOAc (3×100 mL). The filtrate was concentrated, and the crude product was purified by silica gel column chromatography (42–50% ethyl acetate / hexane) to give ethyl 2-[(6-{[(1S,2S)-2-[(benzyloxy)methyl]cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]-2-ethylbutanoate (3.1 g, 76%) as a light brown oil.

[0082] LCMS: Column: Zorbax Ext C 18 (50 × 4.6 mm), 5μ (mobile phase: 90% [10 mM NHOAc aqueous solution] + 10% [CH3CN] ~ 70% [10 mM NHOAc aqueous solution] + 30% [CH3CN] over 1.5 min, then ~ 10% [10 mM NHOAc aqueous solution] + 90% [CH3CN] over 3.0 min, hold this mobile phase composition for 4 min, and finally return to initial conditions over 5 min). Purity: 96.7%, Rt = 2.37 min, MS calculated: 539, MS found: 539.9 ([M+H] + ).

[0083] j) 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoate

[0084] [ka]

[0085] A stirred solution of ethyl 2-[(6-{[(1S,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 of 10:1 EtOAc:MeOH was degassed for 30 min. Pd / C (10%) (6.5 g) was added. The mixture was hydrogenated under 40 PSI of hydrogen atmosphere at 25° C. for 28 h, filtered through a bed of Celite, and washed with 10% MeOH / EtOAc (4×200 mL). The filtrate was evaporated under reduced pressure to give the crude product. The crude product was purified using silica gel column chromatography (10-50% EtOAc:hexanes) to afford 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 a colorless viscous liquid.

[0086] SOR value: [+15.51°] at t≈20°C, 0.2514% in MeOH. LCMS: Column: Zorbax Ext C 18 (50 × 4.6 mm), 5μ (mobile phase: 90% [10 mM NHOAc aqueous solution] + 10% [CH3CN] ~ 70% [10 mM NHOAc aqueous solution] + 30% [CH3CN] over 1.5 min, then ~ 10% [10 mM NHOAc aqueous solution] + 90% [CH3CN] over 3.0 min, hold this mobile phase composition for 4 min, and finally return to initial conditions over 5 min). Purity: 98.9%, Rt = 3.26 min, MS calculated: 449, MS found: 449.9 ([M+H] + ).

[0087] k) 2-Ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoic acid

[0088] [ka]

[0089] In a 25 mL round-bottom 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 pale yellow solution. KOH pellets (62 mg, 1.11 mmol) were added. The mixture was heated to 90° C. After 18 h, the organic solvents were 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 concentrated under reduced pressure to give 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 a colorless sticky mass.

[0090] LCMS: Column: Zorbax Ext C 18 (50 × 4.6 mm), 5μ (mobile phase: 90% [10 mM NHOAc aqueous solution] + 10% [CH3CN] ~ 70% [10 mM NHOAc aqueous solution] + 30% [CH3CN] over 1.5 min, then ~ 10% [10 mM NHOAc aqueous solution] + 90% [CH3CN] over 3.0 min, hold this mobile phase composition for 4 min, and finally return to initial conditions over 5 min). Purity: 95.5%, Rt = 2.00 min, MS calculated: 419, MS found: 420.4 ([M+H] + ).

[0091] l) 2-ethyl-2-[(6-{[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamido]butanoate 3-{[(4-methylbenzene)sulfonyl]oxy}propyl

[0092] [ka]

[0093] 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) was added KCO (256 mg, 1.85 mmol) and 3-{[(4-methylbenzene)sulfonyl]oxy}propyl 4-methylbenzene-1-sulfonate (711 mg, 1.85 mmol). The reaction mixture was stirred at 25 °C for 16 h, poured into water, quenched with 1 (N) aqueous 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 give the crude product, which was purified by CombiFlash using a silica column and 20-80% EtOAc in hexanes to give 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 a colorless sticky mass.

[0094] LCMS: Column: Zorbax Ext C 18 (50 × 4.6 mm), 5μ (mobile phase: 90% [10 mM NHOAc aqueous solution] + 10% [CH3CN] ~ 70% [10 mM NHOAc aqueous solution] + 30% [CH3CN] over 1.5 min, then ~ 10% [10 mM NHOAc aqueous solution] + 90% [CH3CN] over 3.0 min, hold this mobile phase composition for 4 min, and finally return to initial conditions over 5 min). Purity: 90.7%, Rt = 3.48 min, MS calculated: 633, MS found: 634.4 ([M+H] + ).

[0095] m) 2-Ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid 3-[ 18 F]Fluoropropyl [ 18[F] fluoride ions were generated using the Cyclone 18 / 9 cyclotron (18 MeV, IBA Belgium). 18 O(p,n) 18 98% enrichment via F nuclear reaction 18 O- was obtained by bombardment with water and trapped on an anion exchange cartridge (Waters SepPak Accell QMA Cartridge Carbonate), followed by K2CO3 (1 mg / mL) in water / MeCN (1:3) and Krypofix 222 The mixture was eluted with a solution of 2.5 mg / mL of 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 of MeCN) and the reaction mixture was stirred at 90°C for 10 min. The volatiles were removed under reduced pressure at 110°C with a gentle stream of nitrogen. Azeotropic drying was performed using MeCN (3 x 1 mL). 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 of MeCN) was added and the reaction mixture was stirred at 90°C for 10 min. The reaction mixture was then diluted with water (2.5 mL), and the crude product was purified by semi-preparative HPLC (Merck-Hitachi L2130 system) equipped with a radiation detector VRM 202 (Comecer, Netherlands) coupled to an ACE 5 C-18-300 (250 × 10.0 mm, 5 μm) column and a gradient solvent system: 0.1% HPO in HO (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; and 37.1–43.0 min, 20% B. A flow rate of 4 mL / min was used, and UV signal detection was performed at 230 nm. The semi-preparative HPLC product fraction was collected in 35 mL of water and passed through a C18 cartridge (Waters, preconditioned with 5 mL of EtOH and 5 mL of water). The cartridge was washed with water (5 mL), and the title compound was then eluted using 0.5 mL of EtOH. The radioligand 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid 3-[18 F]fluoropropyl was formulated with 5% EtOH in water for injection (WFI). 2-Ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate 3-[ 18 [F]fluoropropyl was obtained with molar activities ranging from 52 to 65 GBq / µmol and excellent radiochemical purity (>99%). The decay-corrected radiochemical yield was 9.0 ± 0.4%. Analytical quality control was performed on an Agilent 1100 series HPLC system equipped with a UV detector and a GabiStar radiation detector (Raytest) to determine radiochemical and chemical purity, specific activity, and chemical identity. A reversed-phase ACE C18-AR column (50 × 4.6 mm, 3 mm) was used in combination with the following separation conditions: 0.1% TFA in HO (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; and 15.1–20.0 min, 20% B. A flow rate of 1 mL / min was used and the UV signal was recorded at 230 nm. The molar activity was calculated by comparing the UV intensity of the formulated product with a calibration curve of the corresponding non-radioactive standard. Example 2

[0096] 2-Ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid (1,1,2,2,3,3-hexadeuterio-3-[ 18 F]fluoro-propyl)

[0097] [ka]

[0098] a) 4-methylbenzenesulfonic acid [1,1,2,2,3,3-hexadeuterio-3-(p-tolylsulfonyloxy)propyl]

[0099] [ka]

[0100] To a solution of 1,1,2,2,3,3-hexadeuteriopropane-1,3-diol (73 mg, 0.87 mmol) in DCM (1 mL) was added 2,6-lutidine (0.5 mL, 4.34 mmol) and tosyl chloride (496 mg, 2.60 mmol, 3 equiv.). The reaction mixture was stirred at 25 °C for 17 h, diluted with DCM (20 mL), washed with 1 N aqueous HCl (10 mL), dried, filtered, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (5–30% EtOAc in hexanes) to afford the title compound (205 mg, 61%) as a white solid.

[0101] LCMS: Column: Zorbax Ext C 18 (50 × 4.6 mm), 5μ (mobile phase: 90% [10 mM aqueous NHOAc] + 10% [CHCN] ~ 70% [10 mM aqueous NHOAc] + 30% [CHCN] over 1.5 min, then ~ 10% [10 mM aqueous NHOAc] + 90% [CHCN] over 3.0 min, held at this mobile phase composition for 4 min, and finally returned to initial conditions over 5 min). Purity: 99.84%, Rt = 3.48 min, MS calculated: 390, MS found: 408.1 ([M + NH4] + ).

[0102] b) 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-(p-tolylsulfonyloxy)propyl]

[0103] [ka]

[0104] 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 1k, 50 mg, 0.12 mmol) in DMF (5.0 mL) was added KCO (49 mg, 0.35 mmol) and 4-methylbenzenesulfonate [1,1,2,2,3,3-hexadeuterio-3-(p-tolylsulfonyloxy)propyl] (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 product was purified by column chromatography on silica gel (30-80% EtOAc in hexanes) to afford the title compound (50 mg, 67%) as a colorless liquid.

[0105] LCMS: Column: Zorbax Ext C 18 (50 × 4.6 mm), 5μ (mobile phase: 90% [10 mM NHOAc aqueous solution] + 10% [CH3CN] ~ 70% [10 mM NHOAc aqueous solution] + 30% [CH3CN] over 1.5 min, then ~ 10% [10 mM NHOAc aqueous solution] + 90% [CH3CN] over 3.0 min, hold this mobile phase composition for 4 min, and finally return to initial conditions over 5 min). Purity: 95.5%, Rt = 3.47 min, MS calculated: 639, MS found: 640.3 ([M+H] + ).

[0106] c) 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid (1,1,2,2,3,3-hexadeuterio-3-[ 18 F]fluoro-propyl) [ 18 [F] fluoride ions were generated using the Cyclone 18 / 9 cyclotron (18 MeV, IBA Belgium). 18 O(p,n) 18 98% enrichment via F nuclear reaction 18It was obtained from the impact of O-water. 18 [F]Fluoride was trapped on an anion exchange cartridge (Waters SepPak Accell QMA Cartridge Carbonate) followed by K2CO3 (1 mg / mL) in water / MeCN (1:3) and Krypofix 222 The resulting solution was eluted with a solution of 2.5 mg / mL of 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-(p-tolylsulfonyloxy)propyl] (1 mg in 0.5 mL of MeCN) and the reaction mixture was stirred at 90°C for 10 minutes. The reaction mixture was then diluted with water (2.5 mL), and the crude product was purified by semi-preparative HPLC (Merck-Hitachi L2130 system) equipped with a radiation detector VRM 202 (Comecer, Netherlands) coupled to an ACE 5 C-18-300 (250 × 10.0 mm, 5 μm) column and a gradient solvent system: 0.1% HPO in HO (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; and 37.1–43.0 min, 20% B. A flow rate of 4 mL / min was used, and UV signal detection was performed at 230 nm. The semi-preparative HPLC product fraction was collected in 35 mL of water and passed through a C18 cartridge (Waters, preconditioned with 5 mL EtOH and 5 mL water). The cartridge was washed with water (5 mL), and the title compound was then eluted using 0.5 mL EtOH. The radioligand, 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid (1,1,2,2,3,3-hexadeuterio-3-[ 18The radioligand 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid (1,1,2,2,3,3-hexadeuterio-3-[[F]fluoro-propyl) was formulated with 5% EtOH in water for injection (WFI). 18 [F]fluoropropyl) was obtained with molar activities ranging from 27 to 44 GBq / µmol and excellent radiochemical purity (>99%). The decay-corrected radiochemical yield was 5.0 ± 1.1%. Analytical quality control was performed on an Agilent 1100 series HPLC system equipped with a UV detector and a GabiStar radiation detector (Raytest) to determine radiochemical and chemical purity, specific activity, and chemical identity. A reversed-phase ACE C18-AR column (50 × 4.6 mm, 3 mm) was used in combination with the following separation conditions: 0.1% TFA in HO (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; and 15.1–20.0 min, 20% B. A flow rate of 1 mL / min was used and the UV signal was recorded at 230 nm. The molar activity was calculated by comparing the UV intensity of the formulated product with a calibration curve of the corresponding non-radioactive standard. Example 3

[0107] 2-(1,2-ditrithioethyl)-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]-3,4-ditrithiobutanoate 3-fluoropropyl

[0108] [ka]

[0109] a) 2-ethyl-2-(6-(((1S,2S)-2-(hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinamido)butanoyl azide

[0110] [ka]

[0111] In a 30 mL round-bottom flask, 2-ethyl-2-(6-(((1S,2S)-2-(hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinamido)butanoic acid (Example 1k, 338 mg, 802 μmol, 1 equiv.) was dissolved in toluene (14 mL). Triethylamine (81 mg, 116 μL, 802 μmol, 1 equiv.) and DPPA (221 mg, 173 μL, 802 μmol, 1 equiv.) were added. The reaction mixture was stirred at ambient temperature for 24 hours, poured into 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 (SiO2, 120 g, 10-70% AcOEt in heptane) to afford the title compound (177 mg, 0.396 mmol, 48%) as an off-white solid.

[0112] 1 H NMR (600 MHz, CDCl3): δ ppm 8.32 (s, 2 H, NH), 7.54 - 7.59 (d, 3 J = 7.9 Hz, 1 H, N Py -C q -CH-CH), 6.46 - 6.53 (d, 3 J = 7.9 Hz, 1 H, N Py -C q -CH), 4.09 - 4.30 (m, 8 H, m, O-CH2, CH2-N-CH2, PO 3-O-CH2, O-CH), 3.72 - 3.84 (m, 2 H, CH2-N-CH2), 3.23 (s, 3 H, O-CH3), 2.35 - 2.51 (m, 2 H, N3-CO-C q -CH2), 1.68 - 1.89 (m, 2 H, N3-CO-C q -CH2), 1.24 - 1.34 (m, 2 H, CH-CH2-CH), 0.74 (t, 3 J = 7.5 Hz, 6 H, N3-CO-C q -CH2-CH3), 0.63 - 0.72 (m, 2 H, CH-CH2-CH) HRMS(ESI):C 21 H 30 N6O5[M+H] + Calculated value = 447.2304; Measured value = 447.2296.

[0113] b) 6-(((1S,2S)-2-(hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinamide

[0114] [ka]

[0115] In a 25 mL round-bottom flask, 2-ethyl-2-(6-(((1S,2S)-2-(hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinamido)butanoyl azide (177 mg, 0.396 mmol, 1 equiv) was dissolved in toluene (10.0 mL). The reaction mixture was heated to 110° C. with stirring for 3 h and then concentrated in vacuo. THF (3 mL) and 3 N NaOH (7 mL) were added. The reaction mixture was heated to 90° C. with stirring for 1 h, poured into 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 a pale orange oil. The crude material was used in the next step without further purification.

[0116] 1 H NMR (600 MHz, CDCl3): δ ppm 8.18(CO-NH2), 7.74 (d, 3 J = 8.0 Hz, 1 H, N Py -C q -CH-CH), 6.56 (d, 3 J = 8.0 Hz, 1 H, N Py -C q -CH), 3.99 - 4.41 (m, 7 H, O-CH2, CH2-N-CH2, O-CH, HO-CH2), 3.95 - 4.00 (m, 2 H, CH2-N-CH2), 3.29 (m, 3 H, O-CH3), 1.20 - 1.36 (CH-CH2-CH), 0.54 - 0.79 (m, 2H, CH-CH2-CH) MS(ESI):C 15 H 21 N3O4[M+H] + Calculated value = 308.14; Measured value = 308.20.

[0117] c) 6-(((1S,2S)-2-(hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinic acid

[0118] [ka]

[0119] 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 equiv.) was dissolved in methanol (3 mL) and water (5 mL). Sodium hydroxide (55 mg, 1.38 mmol, 5 equiv.) was added. The reaction mixture was heated to 85° C. with stirring for 12 h, poured into water (10 mL) and 1 N 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, 12 g, 40–100% AcOEt in heptane) to give the title compound (64 mg, 0.207 mmol, 75%) as a pale orange oil.

[0120] 1 H NMR (600 MHz, CDCl3): δ ppm 7.72 (dd, 3 J = 7.9 Hz, 4 J = 2.9 Hz, 1 H, N Py -C q -CH-CH), 6.56 (d, 3 J = 7.9 Hz, 1 H, N Py -C q -CH), 3.99 - 4.41 (m, 7 H, O-CH2, CH2-N-CH2, O-CH, HO-CH2), 3.97 - 3.99 (m, 2 H, CH2-N-CH2), 3.28 (m, 3 H, O-CH3), 1.18 - 1.32 (CH-CH2-CH), 0.56 - 0.81 (m, 2H, CH-CH2-CH) HRMS(ESI):C 15 H 20 N2O5[M+H] + Calculated value = 309.1379; Measured value = 309.1451.

[0121] d) 3-fluoropropyl 2-amino-2-vinylbut-3-enoate

[0122] [ka]

[0123] 3-Fluoropropan-1-ol (1.55 g, 1.61 mL, 19.8 mmol, equiv.: 18) and 2-amino-2-vinylbut-3-enoic acid hydrochloride (CAN 1865695-91-5, 180 mg, 1.1 mmol, equiv.: 1) were added to a round-bottom flask. Thionyl chloride (1.31 g, 798 μL, 11 mmol, equiv.: 10) was added. The reaction mixture was stirred at 80 °C for 1 h, poured into water (10 mL), and extracted with CHCl (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 a colorless oil. LC-MS (UV peak area / ESI) 94%, 187.1083 [MH + ].

[0124] e) 2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]-2-vinyl-but-3-enoic acid 3-fluoropropyl ester

[0125] [ka]

[0126] 6-(((1S,2S)-2-(hydroxymethyl)cyclopropyl)methoxy)-5-(3-methoxyazetidin-1-yl)picolinic acid (19.8 mg, 64.1 μmol, equivalent weight: 0.8) and 3-fluoropropyl 2-amino-2-vinylbut-3-enoate (15 mg, 80.1 μmol, equivalent weight: 1) were dissolved in CHCl (1.34 mL). N-ethyl-N-isopropylpropan-2-amine (41.4 mg, 55.2 μL, 320 μmol, equivalent weight: 4) was added, followed by 1-(bis(dimethylamino)methylene)-1H-[1,2,3]triazolo[4,5-b]pyridin-1-ium 3-oxide hexafluorophosphate (V) (36.6 mg, 96.1 μmol, equivalent weight: 1.2). The reaction mixture was stirred at ambient temperature for 1 h, poured into water (10 mL) and extracted with CHCl (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 a colorless oil. LC-MS (UV peak area / ESI) 98%, 478.2399 [MH + ].

[0127] f) 2-(1,2-ditrithioethyl)-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]-3,4-ditrithiobutanoate 3-fluoropropyl In a 2 mL tritiation 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 equiv.) and Pd / C (10%) (0.89 mg, 0.84 μmol, 0.2 equiv.) were suspended in dimethylformamide (0.4 mL). The flask was attached to a tritium manifold (RC-TRITEC) and subjected to freeze-pump-thaw. Tritium gas was introduced, and the black suspension was vigorously stirred under a tritium atmosphere at 560 mbar for 3 h. The solution was cooled with liquid nitrogen, and excess tritium gas in the reaction vessel was reabsorbed in a uranium trap for waste tritium. The solvent was lyophilized, and the labile tritium was removed by lyophilization with methanol (3 × 1 ml). The remaining black residue was suspended in methanol (10 ml) and filtered through a 17 mm Titan HPLC filter (0.45 μm, PTFE) to give 8.21 GBq (222 mCi) of crude product with a purity of >90%. The crude product was concentrated and purified by preparative HPLC (SunFire C18, 5 μm, 4.6 × 250 mm) using acetonitrile [A] and 5% acetonitrile [B] in water as eluents (gradient: 10% [A], 90% [B] to 99% [A], 1% [B] in 12 min, hold for 3 min, then return to initial conditions in 5 min). The title compound (4.59 GBq (124 mCi)) was obtained with a radiochemical purity of 98.7% and a specific activity of 4.18 TBq / mmol (113 Ci / mmol) as determined by MS analysis. The compound was stored as an ethanol solution. MS m / z: 482.3 [M+H] + (1%), 484.3 [M( 3 H)+H] + (5%), 486.3 [M( 3 H2)+H] + (13%), 488.3 [M( 3 H3)+H] + (21%), 490.3[M( 3 H4)+H] + (12%), 492.3[M( 3 H5)+H]+ (20%), 494.3 [M( 3 H6)+H] + (12%), 496.3 [M( 3 H7)+H] + (4%). Example 4

[0128] Radioligand binding assays and microPET studies Stably transfected cells or spleen tissue were homogenized in 15 mmol / L Hepes, 0.3 mmol / L EDTA, 1 mmol / L EGTA, 2 mmol / L MgCl, cOmplete EDTA-free protease inhibitors (Roche Applied Science, Rotkreuz, Switzerland) (pH 7.4) using a glass potter and centrifuged at 47,800 g for 30 min at 4°C. The pellet was then rehomogenized and centrifuged twice in the same buffer (47,800 g, 4°C, 30 min). The final pellet was then resuspended in 75 mmol / L Tris, 0.3 mmol / L EDTA, 1 mmol / L EGTA, 12.5 mmol / L MgCl2, and 250 mmol / L sucrose (pH 7.4) at a protein concentration of 1–3 mg / mL, aliquoted, frozen on dry ice, and stored at −80°C.

[0129] Saturation binding was performed using 0.05–2.4 nM of compounds of Formula (I) and 40 μg of membrane protein. Nonspecific binding was defined using CP55940 (10 μM). Assay buffer consisted of 50 mmol / L Tris-HCl, 5 mmol / L MgCl2, 2.5 mmol / L EGTA, and 0.1% fatty acid-free BSA (pH 7.4). The assay was initiated by adding membranes to a final volume of 250 μl per well. The assay was incubated for 2 hours at room temperature in a Filtermate cell harvester through Packard GF / B filters presoaked in 0.3% polyethyleneimine, then vacuum filtered and rinsed with wash buffer (50 mmol / L Tris-HCl, 5 mmol / L MgCl2, 2.5 mmol / L EGTA, and 0.5% fatty acid-free BSA (pH 7.4)).

[0130] For competitive binding, unlabeled (R 1 =R 2 =CH2CH3, R 3 =CH2CH2CH2F) in the presence or absence of increasing concentrations of the compound of formula (I) [ 3 H]-CP55940 0.3 nM, or in the presence or absence of CP55940 (10 μM). 1 =R 2 =CHTCH2T,R 3 Plates were incubated with 1.5 nM (=CH2CH2CH2F) and increasing amounts of membrane preparations (2.5–80 μg) for 60 min at 30°C in a final volume of 0.2 mL of 50 mmol / L Tris-HCl, 5 mmol / L MgCl2, 2.5 mmol / L EGTA, 0.1% fatty acid-free BSA, and 1% DMSO (pH 7.4) buffer with gentle shaking. All binding reactions were terminated by vacuum filtration onto GF / B filter plates (Packard) presoaked in 0.5% polyethyleneimine, followed by seven short 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 measure bound radiolabel. IC 50Values ​​and Hill slopes were determined by a four-parameter logistic model using ActivityBase (ID Business Solution, Ltd.).

[0131] The results are shown in Table 1 and FIG. [Brief explanation of the drawings]

[0132] [Figure 1] Figure 2 Time radioactivity curves in rat spleen and muscle after intravenous administration of 3-[F]fluoropropyl 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate (Example 1, non-deuterated) and 1,1,2,2,3,3-hexadeuterio-3-[F]fluoro-propyl 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoate (Example 2, deuterated). Displacement in follow-up experiments was performed by administering GW405833 (CAS 180002-83-9) (intravenous, 1.5 mg / kg) 10 min after tracer injection.

[0133] [Table 1]

[0134] Table 1 shows the activity of non-selective CB1 / CB2 radioligands in cells recombinantly expressing human CB1 receptors and human CB2 receptors. 3 3H]CP55940, the unlabeled compound of formula (I) exhibits high binding selectivity to the human CB2 receptor (Ki 0.7 nM) over the human CB1 receptor (Ki > 10,000 nM).

Claims

[Claim 1] 2-Ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy]-5-(3-methoxyazetidin-1-yl)pyridine-2-carbonyl]amino]butanoic acid (1,1,2,2,3,3-hexadeuterio-3-[ １８ F]fluoro-propyl): 【Chemical 21】 A method for producing a compound comprising the steps of: (a) 1,1,2,2,3,3-hexadeuteriopropane-1,3-diol is reacted with tosyl chloride in the presence of 2,6-lutidine to give [1,1,2,2,3,3-hexadeuterio-3-(p-tolylsulfonyloxy)propyl] 4-methylbenzenesulfonate: 【Chemical 22】 obtaining (b) The compound obtained in step (a) is treated with K ２ CO ３ to give 2-ethyl-2-[[6-[[(1S,2S)-2-(hydroxymethyl)cyclopropyl]methoxy}-5-(3-methoxyazetidin-1-yl)pyridin-2-yl)formamide]butanoic acid [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: 【Chemical 23】 obtaining (c) reacting the compound obtained in step (b) with [ 18 F]KF / Kryptofix 2.2.2 in acetonitrile; A method comprising:

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