N-methyl-4-(quinolin-2-yl)pyridin-2-amine compounds

Abstract

The present disclosure provides a compound of formula (I): TIFF2025529207000044.tif22161 (in the formula, R 1 , R 2 , and R 3 is as defined herein.) and pharmaceutically acceptable salts thereof. The compounds may be radiolabeled and are useful for diagnostic imaging using positron emission tomography (PET). The compounds of the present disclosure can be used, for example, in diagnostic imaging of 4R tau aggregates.

Description

[Technical Field]

[0001] The present disclosure relates to compounds that bind to 4-repeat (4R) tau, compositions containing such compounds, and methods of using these compounds in diagnostic imaging, including positron emission tomography (PET). [Background technology]

[0002] Tau is a microtubule-associated protein that is particularly abundant in neurons and regulates microtubule stability and axonal transport. Under physiological conditions, tau binding to microtubules is regulated by phosphorylation. However, under pathological conditions, increased tau phosphorylation leads to decreased microtubule binding, resulting in tau misfolding and self-aggregation, ultimately leading to the accumulation of insoluble paired helical filaments (PHFs) and other fibrillar structures.

[0003] Pathological accumulation of tau protein is a defining feature of neurodegenerative diseases called tauopathies, including Alzheimer's disease (AD), neurofibrillary tangle dementia (TD), argyrophilic grain dementia (AGD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), and familial frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17).

[0004] The heterogeneity of these disorders is closely related to the wide range of human tau isoforms and post-translational modifications. Six tau isoforms are expressed in the adult human brain, generated by alternative mRNA splicing of transcripts from the MAPT gene. These isoforms contain three or four microtubule-binding site repeats (3R or 4R tau, respectively) and zero to two N-terminal inserts (0N, 1N, or 2N tau). Depending on the predominant tau isoform present in the aggregates, tauopathies are typically classified as 3R tauopathies (predominantly 3R tau), 4R tauopathies (predominantly 4R tau), or 3R / 4R tauopathies (with approximately equal ratios of 3R and 4R tau). 4R tauopathies include PSP, CBD, and AGD.

[0005] The development of effective therapies for the treatment of tauopathies is hindered by the lack of reliable tools for early diagnosis, staging, and accurate monitoring of disease progression. There remains a need to identify effective means for measuring changes in tau pathology, and in particular, there remains a need for compounds that can reliably image tauopathies caused by aggregation, primarily composed of 4R tau isoforms. Summary of the Invention

[0006] The present disclosure provides a compound of formula (I):

[0007] [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 is CH3 or C( 3 H)3; R 2 is H, F or 18 F; R 3 is -O-CH2CH2-O-CH3, or

[0008] [ka] and; R 4 is F or 18 It's F.

[0009] In certain embodiments, the compound of formula (I) is a detectably labeled compound. In certain embodiments, the compound of formula (I) is 18 In certain embodiments, the compound of formula (I) is a [F] radiolabeled compound. 3 H] labeled compound.

[0010] In certain embodiments, the disclosure provides a compound of formula (I), wherein said compound is 4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-methylpyridin-2-amine; 4-[8-( 18 F) fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-methylpyridin-2-amine; 4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-( 3 H3) methylpyridin-2-amine; 3-(Fluoromethyl)-1-{2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl}azetidin-3-ol; 3-[( 18 F) fluoromethyl]-1-{2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl}azetidin-3-ol; and 3-(fluoromethyl)-1-(2-{2-[( 3 H3) Methylamino]pyridin-4-yl}quinolin-6-yl)azetidin-3-ol is selected from the group consisting of:

[0011] The present disclosure provides compound (I):

[0012] [ka] or a pharmaceutically acceptable salt thereof.

[0013] This disclosure 18 F] Compound (I):

[0014] [ka] is expressed as 18 The present invention provides an F-labeled compound, or a pharmaceutically acceptable salt thereof.

[0015] This disclosure 3 H] Compound (I):

[0016] [ka] is expressed as 3 3H-labeled compounds, or pharmaceutically acceptable salts thereof, are provided.

[0017] The present disclosure provides compound (II):

[0018] [ka] or a pharmaceutically acceptable salt thereof.

[0019] This disclosure 18 F] Compound (II):

[0020] [ka] is expressed as 18 The present invention provides an F-labeled compound, or a pharmaceutically acceptable salt thereof.

[0021] This disclosure 3 H] Compound (II):

[0022] [ka] is expressed as 33H-labeled compounds, or pharmaceutically acceptable salts thereof, are provided.

[0023] The present application also provides compositions containing the compounds of the present disclosure. In certain embodiments, the present disclosure provides pharmaceutical compositions containing a compound of Formula (I) or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable excipient.

[0024] The present disclosure further provides methods of using the compounds of the present disclosure, for example, as imaging agents in diagnostic imaging of 4R tau aggregates. In certain embodiments, the compounds can be used in diagnostic imaging using positron emission tomography. [Brief explanation of the drawings]

[0025] [Figure 1]

[0033] Figure 1 shows an autoradiograph depicting binding of radiolabeled compounds of the present disclosure in flash-frozen sections from PSP-affected and healthy brain tissue. The figure shows the distribution of [H]Compound (I) and [H]Compound (II) binding to FF sections of the globus pallidus (GP) and putamen (PUT) from a representative PSP brain and a representative normal brain. The figure shows binding of 3 nM radioligand alone (total binding) and binding of 3 nM radioligand in the presence of 10 μM of the corresponding non-radiolabeled compound (non-specific binding). [Figure 2] Analysis of binding of radiolabeled compounds of the present disclosure in sections derived from PSP-affected and healthy brain tissue. Quantitative analysis of [H]Compound (I) and [H]Compound (II) binding to gray matter in FF globus pallidus and putamen sections derived from PSP and normal brains. Black circles represent TB (total binding of radioligand) in PSP brain sections, white circles represent NSB (nonspecific binding of radioligand) in PSP brain sections, black squares represent TB in PSP brain sections, and white squares represent NSB in normal brain sections. [Figure 3]Autoradiograph showing binding of radiolabeled compounds of the present disclosure in deparaffinized sections from PSP-affected and healthy brain tissue. The figure shows the distribution of [H]Compound (I) and [H]Compound (II) binding to deparaffinized sections of FFPE globus pallidus (GP) and putamen (PUT) from a representative PSP brain and a representative normal brain. The figure shows binding of 3 nM radioligand alone (total binding) and binding of 3 nM radioligand in the presence of 10 μM of the corresponding non-radiolabeled compound (nonspecific binding). Scale bar is 2 mm. [Figure 4] Competitive binding curves showing the affinity of radiolabeled compounds of the present disclosure for PSP-affected brain tissue. The figure shows similar concentration-dependent inhibition of binding of [H]Compound (I) and [H]Compound (II) to FF globus pallidus and putamen slices from a representative PSP brain. The upper panel shows the inhibition of [H]Compound (I) from non-radiolabeled Compound (I), and the lower panel shows the inhibition of [H]Compound (II) from non-radiolabeled Compound (II). The dissociation constant (sdKD) of the radioligand was determined by self-displacement experiments. The sdKD values ​​shown in the figure are the average of values ​​obtained from three different PSP brains. [Figure 5] Time course of PET tracer uptake in the brain of non-human primates after intravenous administration. Time activity curves (TAC) of [18F]Compound (I) and [18F]Compound (II) in non-human primates. [Figure 6] Distribution of PET tracer in the brain of cynomolgus monkeys after injection of [18F]PET tracer or after blocking with unlabeled compound followed by injection of [18F]PET tracer. Total volume of distribution (VT) (baseline and blocking) of [18F]Compound (I) and [18F]Compound (II) in various brain regions of non-human primates. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present disclosure relates to compounds that exhibit high affinity for 4R tau aggregates. The detectably labeled compounds of the present disclosure can be used, for example, by using positron emission tomography imaging, to selectively detect disorders associated with tau aggregates, such as progressive supranuclear palsy (PSP) and other tauopathies.

[0027] The compounds disclosed herein may contain one or more variables that occur more than once in any substituent or formula described herein. The definition of a variable at each occurrence is independent of its definition at another occurrence. Furthermore, combinations of substituents are permissible if such combinations result in stable compounds. A stable compound is one that can be isolated from a reaction mixture.

[0028] compound In certain embodiments, the present disclosure provides a compound of formula (I):

[0029] [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 is CH3 or C( 3 H)3; R 2 is H, F or 18 F; R 3 is -O-CH2CH2-O-CH3, or

[0030] [ka] and; R 4 is F or 18 It's F.

[0031] In certain embodiments, the present disclosure provides R 1 However, CH3 or C( 3 H)3 and R 2 But F or 18 F and R3 is —O—CH 2 CH 2 —O—CH 3 , or a pharmaceutically acceptable salt thereof. 1 is CH3 and R 2 is F. In certain embodiments, R 1 is CH3 and R 2 teeth, 18 F. In certain embodiments, R 1 is C( 3 H)3 and R 2 is F.

[0032] In certain embodiments, the present disclosure provides R 1 However, CH3 or C( 3 H)3 and R 2 is H and R 3 but,

[0033] [ka] and R 4 But F or 18 F. In certain embodiments, R 1 is CH3 and R4 is F. In certain embodiments, R 1 is CH3 and R4 is 18 F. In certain embodiments, R 1 is C( 3 H)3 and R 4 is F.

[0034] Representative compounds of formula (I) include, for example, the compounds shown in Table 1 below, or pharmaceutically acceptable salts thereof.

[0035] [Table 1] TIFF2025529207000014.tif115161

[0036] The compounds of the present disclosure can be used as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" means salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and that are commensurate with a reasonable benefit / risk ratio.

[0037] In certain embodiments, the present disclosure provides compound (I):

[0038] [ka] or a pharmaceutically acceptable salt thereof.

[0039] In certain embodiments, the present disclosure provides a method for manufacturing a semiconductor device comprising: 18 F] Compound (I):

[0040] [ka] Represented by [ 18

[0013] F] radiolabeled compounds, or pharmaceutically acceptable salts thereof, are provided.

[0041] In certain embodiments, the present disclosure provides a method for manufacturing a semiconductor device comprising: 3 H] Compound (I):

[0042] [ka] Represented by [ 3 3H] radiolabeled compounds, or pharmaceutically acceptable salts thereof.

[0043] In certain embodiments, the present disclosure provides compound (II):

[0044] [ka] or a pharmaceutically acceptable salt thereof.

[0045] In certain embodiments, the present disclosure provides a method for manufacturing a semiconductor device comprising: 18 F] Compound (II):

[0046] [ka] Represented by [ 18

[0013] F] radiolabeled compounds, or pharmaceutically acceptable salts thereof, are provided.

[0047] In certain embodiments, the present disclosure provides a method for manufacturing a semiconductor device comprising: 3 H] Compound (II):

[0048] [ka] Represented by [ 3 3H] radiolabeled compounds, or pharmaceutically acceptable salts thereof.

[0049] Pharmaceutical Composition In certain embodiments, the compounds of the present disclosure can be administered as a pharmaceutical composition. By "pharmaceutical composition" is meant a composition suitable for medical administration. Such compositions can contain a therapeutically or diagnostically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable excipient.

[0050] In certain embodiments, there is provided a pharmaceutical composition comprising a therapeutically or diagnostically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable excipient.

[0051] How to use In certain embodiments, the radiolabeled compounds of the present disclosure can be used as imaging agents. In certain embodiments, the compounds of the present disclosure can be used as in vitro analytical references or in vitro screening tools. In certain embodiments, the compounds of the present disclosure can be used in in vivo diagnostic methods. In certain embodiments, the detectably labeled compounds of the present disclosure can be used for imaging tau aggregates, particularly 4R tau aggregates.

[0052] In certain embodiments, when used in imaging applications, the compounds of the present disclosure may be labeled, or the compounds may be unlabeled and conjugated to such molecules using a secondary labeling agent. In certain embodiments, the choice of label may depend on the detection means. For example, fluorescent labels may be suitable for optical detection. In certain embodiments, paramagnetic and radioisotope labels may be utilized and may be detected using, for example, positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0053] In certain embodiments, the present disclosure provides a method for imaging tau aggregates, comprising administering to a patient a compound according to the present disclosure (e.g., a compound of Formula (I), particularly a detectably labeled compound of Formula (I)). 18 F] Compound (I) or [ 18 F] Compound (II). In certain embodiments, the compound is 18 F] Compound (I). In certain embodiments, the compound is 18 F] Compound (II). In some embodiments, the compound is a detectably labeled compound, and a signal generated from the compound that specifically binds to the tau aggregates is detected. In some embodiments, at least one radiological image is generated.

[0054] In certain embodiments, the disclosure provides a method for diagnosing a tauopathy in a patient, the method comprising administering to the patient a compound according to the disclosure (e.g., a compound of Formula (I), particularly a detectably labeled compound of Formula (I)). 18 F] Compound (I) or [ 18F] Compound (II). Diagnosis of a tauopathy in a patient can be performed by detecting specific binding of a detectably labeled compound to 4R tau protein aggregates in the brain of an affected patient. In certain embodiments, the method may include the steps of: (a) contacting a patient suspected of having a tauopathy with a detectably labeled compound of the present disclosure, such as by injecting the patient with the compound; (b) allowing the detectably labeled compound to bind to 4R tau protein aggregates to form a compound / tau protein aggregate complex (hereinafter, "compound / tau protein aggregate complex" will be abbreviated as "compound / protein complex"); (c) detecting the formation of the compound / protein complex; (d) optionally, correlating the presence or absence of the compound / protein complex with the presence or absence of 4R tau protein aggregates in the subject's brain; and (e) optionally, comparing the amount of the compound / protein complex with a normal control value. If the amount of the compound / protein complex is increased compared to the normal control value, the patient can be determined to be suffering from or at risk of developing a 4R tau-associated disorder.

[0055] The compound bound to the 4R tau protein aggregates can then be detected by an appropriate method. One example of a detection method is positron emission tomography (PET). PET is a highly sensitive imaging technique that uses a small amount of a radioactively labeled compound called a "tracer" or "radiotracer." PET involves introducing a radiopharmaceutical (i.e., a drug bound to a radioisotope) into the body, for example by injection. The labeled compound is preferably transported, accumulated, and transformed in vivo in the same manner as a corresponding non-radioactive labeled compound. The labeled compound accumulates in the target tissue and releases a positron as it decays. The released positron instantly combines with a nearby electron, simultaneously emitting two distinguishable gamma rays in opposite directions. These gamma rays are detected by a gamma ray detector to form a three-dimensional image.

[0056] In certain embodiments, the presence or absence of the compound / protein complex can be correlated with the presence or absence of 4R tau protein aggregates in the patient's brain. Finally, in certain embodiments, the amount of the compound / protein complex can be compared to a baseline control value measured in a sample from healthy subjects or in a particular body part or region, and if the amount of the compound / protein complex is increased compared to the normal control value, the patient can be determined to be suffering from a tau-associated disorder.

[0057] In certain embodiments, diagnosis can be performed by comparing the number, size, and / or intensity of labeled tau conformers, aggregates, and / or neurofibrillary tangles in a sample taken from or within the subject with a corresponding baseline value. In certain embodiments, the baseline value can represent an average level in a population of non-diseased individuals. In certain embodiments, the baseline value can represent a previous level measured in the same subject.

[0058] In some embodiments, the methods of the present disclosure can be used to diagnose the presence of a tauopathy. In some embodiments, the tauopathy is a 4-repeat (4R) tauopathy characterized by cytoplasmic inclusions primarily composed of tau protein isoforms with four microtubule-binding domains. In some embodiments, the 4R tauopathy is progressive supranuclear palsy.

[0059] In some embodiments, the method of the present disclosure can also be used to monitor a subject's response to treatment. In some embodiments, the presence of 4R tau aggregates is quantified before treatment begins by administering a detectably labeled compound of the present disclosure. The level of 4R tau aggregates in the subject's body at this time point is used as a baseline value. A therapeutic drug is then administered to the subject. At various times during the course of administration of the therapeutic drug, the detection of tau aggregates is repeated, and the measured values ​​are compared to the baseline value.

[0060] The compounds of the present disclosure can also be incorporated into test kits for detecting tau protein aggregates. In certain embodiments, the test kit can include a container for housing one or more compounds of the present disclosure. In certain embodiments, the test kit can include instructions for using the compound to bind to tau protein aggregates, for detecting the formation of a compound / protein complex, and / or for assessing the presence or absence of the compound / protein complex to correlate with the presence or absence of the tau protein aggregates. [Example]

[0061] In order that the invention described herein may be more fully understood, the following examples are set forth. The synthetic and biological examples described herein are presented to illustrate the compounds and methods provided herein, but should not be construed as limiting the scope thereof.

[0062] Synthesis protocol The compounds provided herein can be prepared from readily available starting materials by varying the specific synthetic protocols described below, as will be known to those skilled in the art. It is understood that where typical or preferred process conditions (i.e., reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.) are described, other process conditions can also be used unless otherwise specified. Optimum reaction conditions may vary with the particular reactants or solvents used, although such conditions can be determined by one of ordinary skill in the art by routine optimization procedures.

[0063] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Selection of a suitable protecting group for a particular functional group and suitable conditions for protection and deprotection are well known to those skilled in the art. For example, Greene et al., Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references therein, describe numerous protecting groups and their introduction and removal.

[0064] Abbreviation API-ES: atmospheric pressure ionization electrospray; Boc: tert-butoxycarbonyl; (Boc2O): di-tert-butyl dicarbonate; t-Bu: tertiary butyl; tBuXPhosPdG3: methanesulfonato(2-di-t-butylphosphino-2',4',6'-tri-i-propyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II); DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DMAP: N,N-dimethylpyridin-4-amine; DMSO: dimethyl sulfoxide; ESI: elect HPLC: high performance liquid chromatography; ID: internal diameter; LCMS: liquid chromatography mass spectrometry; MS: mass spectrum; m / z: mass to charge ratio; NMR: nuclear magnetic resonance; ppm: parts per million; PVDF: polyvinylidene fluoride; RuPhosPdG4: methanesulfonato(2-dicyclohexylphosphino-2',6'-di-i-propoxy-1,1'-biphenyl)(2'-methylamino-1,1'-biphenyl-2-yl)palladium(II); SFC: supercritical fluid chromatography; TLC: thin layer chromatography; and WFI: sterile water for injection.

[0065] [Example 1] Preparation of Compound (I), 4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-methylpyridin-2-amine

[0066] [ka]

[0067] Example 1 - Step 1: 3-chloro-N-(2-fluoro-4-hydroxyphenyl)propanamide To a solution of 3-chloropropanoyl chloride (22.0 g, 173 mmol) in acetone (200 mL) was added 4-amino-3-fluorophenol (20 g, 157 mmol). The mixture was degassed with argon three times. The reaction mixture was then heated to 60 °C for 3 h. Nine additional vials were prepared and treated as above, and all 10 reaction mixtures were combined. The combined mixture was poured into water (1 L). Ethyl acetate (1 L) was added, and the two phases were separated. The aqueous phase was extracted with ethyl acetate (2 × 500 mL). The combined organic phases were washed with brine (500 mL), dried over anhydrous NaSO, filtered, and concentrated. The crude residue was purified by silica gel column chromatography using petroleum ether / ethyl acetate = 2:1 as eluent to give the title compound (220 g, 1.01 mmol, 64% yield). 1 H NMR (400MHz, DMSO-d6) δppm9.75(s,1H),9.56(s,1H),7.44(t,J=9.0Hz,1H),6.64-6.52(m,2H),3.84(t,J=6.3Hz,2H),2.81(t,J=6.3Hz,2H).

[0068] Example 1 - Step 2: 8-Fluoro-6-hydroxy-3,4-dihydroquinolin-2(1H)-one A mixture of aluminum chloride (103 g, 772 mmol) and 3-chloro-N-(2-fluoro-4-hydroxyphenyl)propanamide (28 g, 129 mmol) was stirred at 160 °C for 4 hours. Two additional vials were prepared and treated as above, and all three reaction mixtures were combined. The combined mixture was added to 1 N HCl (800 mL) at 0 °C, and the mixture was stirred at 0 °C for 10 minutes. Ethyl acetate (1 L) was added, and the two phases were separated. The aqueous phase was extracted with ethyl acetate (2 × 1 L). The combined organic phases were washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude residue was purified by precipitation from tert-butyl methyl ether (200 mL). The mixture was filtered, and the filter cake was dried under vacuum to give the title compound (56.5 g, 312 mmol, 81% yield). 1H NMR (400MHz, DMSO-d6) δppm 9.78 (s, 1H), 9.55-9.40 (m, 1H), 6.49-6.39 (m, 2H), 2.86-2.74 (m, 2H), 2.45-2.35 (m, 2H).

[0069] Example 1 - Step 3: 8-Fluoro-6-methoxy-3,4-dihydroquinolin-2(1H)-one To a solution of 8-fluoro-6-hydroxy-3,4-dihydroquinolin-2(1H)-one (20 g, 88 mmol) and potassium carbonate (18.3 g, 132 mmol) in acetone (30 mL) and N,N-dimethylformamide (30 mL) was added methyl iodide (6.63 mL, 106 mmol) at 0 °C. The mixture was stirred at 25 °C for 12 h. Four additional vials were prepared and treated as above, and all five reaction mixtures were combined. The combined mixture was poured into water (500 mL). Ethyl acetate (500 mL) was added, and the two phases were separated. The aqueous phase was extracted with ethyl acetate (2 × 250 mL). The combined organic phases were washed with brine (150 mL), dried over anhydrous NaSO, filtered, and concentrated. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate=1:1 as the eluent to give the title compound (52 g, 266 mmol, 60% yield). 1 H NMR(400MHz,DMSO-d6)δppm9.90(s,1H),6.71(dd,J=2.6,12.3Hz,1H),6.66(s,1H),3.71(s,3H),2.91-2.84(m,2H),2.43(dd,J=6.5,8.2Hz,2H).

[0070] Example 1 - Step 4: 8-Fluoro-6-methoxyquinolin-2(1H)-one To a solution of 8-fluoro-6-methoxy-3,4-dihydroquinolin-2(1H)-one (10 g, 51.2 mmol) in dichloromethane (100 mL) was added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (34.9 g, 154 mmol). The mixture was stirred at 90 °C for 12 h. Three additional vials were prepared and treated as above, and all four reaction mixtures were combined. The combined mixture was adjusted to pH = 9 with 1 M aqueous NaOH. Ethyl acetate (500 mL) was added, and the two phases were separated. The aqueous phase was extracted with ethyl acetate (2 × 500 mL). The combined organic phases were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography using petroleum ether:ethyl acetate=100 / 1→0 / 1 as an eluent to give the title compound (16 g, 83 mmol, 40% yield). 1 H NMR(400MHz,DMSO-d6)δppm11.77-11.52(m,1H),7.87(dd,J=1.5,9.5Hz,1H),7.16-7.07(m,2H),6.56(d,J=9.5Hz,1H),3.81-3.78(m,3H).

[0071] Example 1 - Step 5: 2-Chloro-8-fluoro-6-methoxyquinoline 8-Fluoro-6-methoxyquinolin-2(1H)-one (4 g, 20.7 mmol) was dissolved in POCl3 (16 mL, 172 mmol). The solution was heated to 100 °C for 2 h. Two more vials were prepared and treated as above. All three reaction mixtures were combined. The combined reaction mixture was concentrated under reduced pressure to remove POCl3. The reaction residue was then diluted with ethyl acetate (150 mL) and poured into water. Ethyl acetate (150 mL) was added, and the two phases were separated. The aqueous phase was extracted with ethyl acetate (2 × 100 mL). The combined organic phases were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate = 1:1 as the eluent. The crude residue was purified by precipitation from petroleum ether (5 mL) and n-hexane (5 mL). The mixture was filtered. The filter cake was washed with petroleum ether (5 mL) and dried under vacuum to give the title compound (5.2 g, 22.1 mmol, 35% yield). 1 H NMR(400MHz,DMSO-d6)δppm8.39(dd,J=1.7,8.6Hz,1H),7.64(d,J=8.8Hz,1H),7.42(dd,J=2.4,12.2Hz,1H),7.34(d,J=2.0Hz,1H),3.91(s,3H);MS(ESI + ) m / z 211.9 [M+1] + .

[0072] Example 1 - Step 6: 2-Chloro-8-fluoroquinolin-6-ol 2-Chloro-8-fluoro-6-methoxyquinoline (1000 mg, 4.73 mmol) was dissolved in dichloromethane (25 mL) and cooled in an ice-water bath. Boron tribromide (14 mL, 14 mmol, 1 mol / L dichloromethane solution) was then added slowly under an argon stream. The reaction mixture was allowed to warm to room temperature and stirred overnight. Excess reagent was quenched by the slow addition of saturated NaHCO3 solution, followed by dilution with methanol. The crude reaction mixture was poured into 10% NH4Cl solution and extracted with dichloromethane / methanol (5:1). The combined extracts were dried over MgSO4 and concentrated under reduced pressure to give the title compound (699 mg, 3.54 mmol, 75% yield), which was used without further purification. MS (ESI) + ) m / z 198 [M+H] + .

[0073] Example 1 - Step 7: 2-Chloro-8-fluoro-6-(2-methoxyethoxy)quinoline 2-Chloro-8-fluoroquinolin-6-ol (231 mg, 1.17 mmol), 1-bromo-2-methoxyethane (0.166 mL, 1.75 mmol, 1.5 equiv.), and KCO (485 mg, 3.51 mmol, 3 equiv.) were mixed in N,N-dimethylformamide (5 mL). The reaction mixture was heated to 80 °C for 2 h to achieve complete conversion. The resulting solution of 2-chloro-8-fluoro-6-(2-methoxyethoxy)quinoline (299 mg; 1.17 mmol; 100% crude yield) was used directly in the subsequent Suzuki reaction step (one-pot reaction).

[0074] Isolation procedure: The crude reaction mixture was poured into 10% NH₄Cl and extracted with dichloromethane. The combined organic extracts were washed with saturated NaCl, dried over MgSO₄, and concentrated in vacuo to give the title compound. MS (ESI) + ) m / z 256 [M+H] + .

[0075] Example 1 - Step 8: Compound (I) 4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-methylpyridin-2-amine To a crude solution of 2-chloro-8-fluoro-6-(2-methoxyethoxy)quinoline (299 mg; 1.17 mmol) in N,N-dimethylformamide (5 mL) was added N-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (329 mg, 1.4 mmol; 1.2 equiv.), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (96 mg; 0.12 mmol; 0.1 equiv.), and CsCO (381 mg; 1.17 mmol, 1 equiv.), and the mixture was further diluted with dioxane (5 mL) and water (2 mL). After sparging with argon, the reaction mixture was heated to 80 °C for 1 h to achieve complete conversion. The cooled crude mixture was poured into saturated NaHCO solution and extracted with dichloromethane. The combined organic extracts were washed with saturated NaCl solution, dried over MgSO4, and concentrated in vacuo. The residue was pre-purified by silica gel flash chromatography using 0 to 100% ethyl acetate / cyclohexane as eluent, followed by SFC (column: VDS 100 Diol, 150 × 32 mm, 5 μm; eluents: (A) supercritical CO2, (B) methanol + 30% dichloromethane + 0.2% NH4OH; isocratic 90 / 10 (A / B); flow rate: 120 mL / min) to give the title compound (219 mg, 0.67 mmol, 57% yield). 1 H NMR(500MHz,DMSO-d6)δppm8.41(dd,J=8.8,1.5Hz,1H),8.14(d,J=5.3Hz,1H),8.11(d,J=8.7Hz,1H),7.38(dd,J=12.2,2.6Hz,1H),7.32(d,J=2.6Hz,1 H),7.27(d,J=1.0Hz,1H),7.25(dd,J=5.3,1.5Hz,1H),6.70(d,J=4.9Hz,1H ),4.30-4.24(m,2H),3.77-3.71(m,2H),3.34(s,3H),2.85(d,J=4.8Hz,3H); 19 F NMR(471MHz,DMSO-d6)δppm-123.58(d,J=12.0Hz);MS(ESI + ) m / z 328 [M+H] + .

[0076] Preparative separations were performed on a Waters Precolumn 100q SFC system controlled by Waters MassLynx™ software. The system consisted of an open-bed injector / collector, a heated column compartment with a six-column switch, a CO2 booster pump, and a modifier pump module. Detection was performed by UV and a quadrupole mass spectrometer (Waters Aquity QDa®, ESI-ionization). To enable quantitative separations, methanol was used at a makeup flow rate of 30 mL / min to drive the gas-liquid separator. The backpressure regulator was set to 120 bar and heated to 60 °C. The column was maintained at 30 °C throughout the separation.

[0077] [Example 2] 18 F] Compound (I) 4-[8-( 18 F) Preparation of fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-methylpyridin-2-amine

[0078] [ka]

[0079] Example 2 - Step 1: 6-Methoxy-1-methyl-8-nitroquinolin-1-ium iodide 6-Methoxy-8-nitroquinoline (17 g, 83 mmol) was dissolved in dimethyl sulfate (31.5 g, 250 mmol). The mixture was stirred at 100 °C for 12 hours. Another vial was prepared and treated as above, and the two reaction mixtures were combined. The combined mixture was added to water (17 mL). Sodium iodide was then added to the mixture to precipitate. The precipitate was collected by filtration, rinsed with water, and dried under reduced pressure to give the title compound (47 g, 90% yield). 1 H NMR (400MHz, DMSO-d6) δppm9.47(d,J=5.5Hz,1H),9.28(d,J=8.0Hz,1H),8.61(d,J=3.0Hz,1H),8.35-8.24(m,2H),4.38(s,3H),4.06(s,3H).

[0080] Example 2 - Step 2: 6-Methoxy-1-methyl-8-nitroquinolin-2(1H)-one To a solution of 6-methoxy-1-methyl-8-nitroquinolin-1-ium iodide (32 g, 146 mmol) in ethanol (60 mL) was added 2 M NaOH (64 mL, 128 mmol). 30% HO (128 mL, 1.25 mol) was slowly added to the mixture at 50 °C. Once the entire hydroxide had been added, the suspension was cooled and filtered. An additional vial was prepared and treated as above, and the two precipitates were combined. The precipitate was washed with water and dried under reduced pressure to give the title compound (8 g, 34.2 mmol, 23% yield). 1 H NMR (400MHz, DMSO-d6) δppm7.98(d,J=9.5Hz,1H),7.73(d,J=3.0Hz,1H),7.67(d,J=3.0Hz,1H),6.77(d,J=9.5Hz,1H),3.87(s,3H),3.27(s,3H).

[0081] Example 2 - Step 3: 2-Chloro-6-methoxy-8-nitroquinoline To a solution of 6-methoxy-1-methyl-8-nitroquinolin-2(1H)-one (6 g, 25.6 mmol) in phosphorus oxychloride (120 mL, 1.28 mol) was added phosphorus pentachloride (6.40 g, 30.7 mmol). The mixture was stirred at 100°C for 3 hours. An additional vial was prepared and treated as above, and the two reaction mixtures were combined. The phosphorus oxychloride was removed under reduced pressure. The crude residue was poured into water, resulting in the formation of a precipitate. The precipitate was collected by filtration, rinsed with water, and dried under reduced pressure to give the title compound (10 g, 87% yield). 1 H NMR (400MHz, DMSO-d6) δppm8.50(d,J=8.6Hz,1H),8.13(d,J=2.6Hz,1H),7.80(d,J=2.6Hz,1H),7.74(d,J=8.8Hz,1H),3.96(s,3H).

[0082] Example 2 - Step 4: 8-Nitroquinoline-2,6-diol 2-Chloro-6-methoxy-8-nitroquinoline (3 g, 12.6 mmol) was dissolved in hydrobromic acid (60 mL, 442 mmol). The mixture was stirred at 130 °C for 16 hours. An additional vial was prepared and treated as above, and the two reaction mixtures were combined. The combined mixture was cooled in an ice bath. Insoluble by-products were removed from the reaction mixture by filtration and discarded. The filtrate was concentrated under reduced pressure to give the title compound (6 g, 93% yield). 1 H NMR (400MHz, DMSO-d6) δppm8.01(brd,J=9.5Hz,1H),7.84(brs,1H),7.57(brs,1H),6.66(brd,J=9.0Hz,1H).

[0083] Example 2 - Step 5: 2-Chloro-8-nitroquinolin-6-ol 8-Nitroquinoline-2,6-diol was dissolved in phosphorus oxychloride (35 mL, 375 mmol). The mixture was stirred at 100 °C for 1 hour. An additional vial was prepared and treated as above, and the two reaction mixtures were combined. The reaction mixture was distilled under reduced pressure to remove phosphorus oxychloride. The residue was then diluted with ethyl acetate (50 mL) and poured into water. Ethyl acetate (50 mL) was added, and the two phases were separated. The aqueous phase was extracted with ethyl acetate (2 × 30 mL). The combined organic phases were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate = 3:1 as eluent. The crude residue was purified by precipitation from tert-butyl methyl ether (5 mL) and acetonitrile (0.5 mL). The mixture was filtered. The filter cake was washed with petroleum ether and dried under reduced pressure to give the title compound (3.19 g, 32% yield). 1 H NMR (400MHz, DMSO-d6) δppm 10.98 (br s, 1H), 8.45 (d, J = 8.8 Hz, 1 H), 7.88 (d, J = 2.4 Hz, 1 H), 7.66 (d, J = 8.8 Hz, 1 H), 7.53 (d, J = 2.4 Hz, 1 H).

[0084] Example 2 - Step 6: 2-Chloro-6-(2-methoxyethoxy)-8-nitroquinoline To a solution of 2-chloro-8-nitroquinolin-6-ol (200 mg, 0.89 mmol) and K2CO3 (369 mg, 2.67 mmol) in N,N-dimethylformamide (2 mL), 1-bromo-2-methoxyethane (0.25 mL, 2.67 mmol, 1.0 equiv.) was slowly added, and the mixture was stirred at room temperature overnight. The reaction mixture was poured into saturated NaHCO3 solution and extracted with dichloromethane. The organic fraction was concentrated to give the title compound (280 mg, 0.89 mmol, 100% yield). MS (ESI) + ) m / z 283 [M+H] + .

[0085] Example 2 - Step 7: tert-Butyl {4-[6-(2-methoxyethoxy)-8-nitroquinolin-2-yl]pyridin-2-yl}methylcarbamate In a microwave synthesis vial, 2-chloro-6-(2-methoxyethoxy)-8-nitroquinoline (153 mg, 0.54 mmol), tert-butyl methyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)carbamate (253 mg, 0.76 mmol, 1.4 equiv.), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (44 mg, 0.054 mmol, 0.1 equiv.), and CsCO (529 mg, 1.62 mmol, 3 equiv.) were dissolved in 1,4-dioxane (4 mL) and water (0.4 mL). The reaction mixture was sparged with argon and then heated to 80 °C for 3 h (microwave synthesizer: Biotage® Initiator+, 400 W). The cooled crude reaction mixture was diluted with dichloromethane and the insoluble material was removed by filtration. The organic fraction was washed with water, dried over MgSO4, and purified by silica gel flash chromatography eluting with 0 to 50% ethyl acetate in cyclohexane to give the title compound (134 mg, 0.30 mmol, 55% yield). 1H NMR(500MHz,DMSO-d6)δppm8.59(d,J=8.9Hz,1H),8.55(d,J=5.2Hz,1H),8.35(d,J=8.7Hz,1H),8.33(d,J=1.6Hz,1H),8.13(d,J=2. MS(ESI) + ) m / z 455 [M+H] + .

[0086] Example 2 - Step 8: 18 F] Compound (I) 4-[8-( 18 F) Fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-methylpyridin-2-amine At the Wisconsin Medical Cyclotron (Milwaukee, WI) 18 O(p,n) 18 In the F reaction, 18 [F] fluoride was synthesized and delivered to the radiochemistry laboratory the morning of the day of use. 18 The [F]fluoride was transferred to a GE TRACERlab™ FX FN synthesis module and trapped on an ion exchange cartridge (Waters Sep-Pak® Accell QMA Carbonate Plus Light Cartridge, catalog number WAT186004540) preconditioned with TRACESELECT™ Ultra ACS water (5 mL). 18 O]H2O was removed. 18 [F] fluoride was eluted with a solution of 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (Kryptofix® 2.2.2, 7.5 mg, 20 μmol) and potassium carbonate (0.75 mg, 5.4 μmol) in acetonitrile / water for ultra-trace analysis (0.4 mL each) and transferred to a reaction vessel. 18The [F] fluoride was dried under full vacuum by heating (70 °C) and a stream of nitrogen or helium for 5 minutes, followed by drying at 100 °C for 5 minutes under full vacuum. After drying, a solution of tert-butyl {4-[6-(2-methoxyethoxy)-8-nitroquinolin-2-yl]pyridin-2-yl}methylcarbamate (4 mg, 8.80 μmol) in anhydrous dimethyl sulfoxide (1.0 mL) was added, and the resulting solution was heated and stirred at 140 °C for 20 minutes. The reaction mixture was then cooled to 100 °C, and 3 N HCl (1.0 mL) was added. After stirring at 100 °C for 5 minutes to remove the tert-butoxycarbonyl protecting group, the reaction mixture was cooled to 50 °C and diluted with dimethyl sulfoxide (1.0 mL), 4 N NaOH (0.9 mL), and sterile water for injection (WFI, 1.0 mL). The resulting mixture was transferred to an HPLC loading vial. The contents of the loop-loading vial were transferred to semi-preparative HPLC (Phenomenex® Gemini® NX-C18 5 μm, 110 Å 10 × 250 mm column with a UV setting of 275 nm) and purified using a mobile phase of 65% 10 mM ammonium acetate and 35% acetonitrile at a flow rate of 4 mL / min.

[0087] The title compound peak (retention time approximately 27 minutes) was collected in an HPLC dilution flask and diluted with 40 mL of WFI. The purified title compound was then trapped on a preconditioned 50 mg Phenomenex® Strata C18-E cartridge (catalog no. 8B-S001-DAK) and washed with 5 mL of WFI. The trapped title compound was eluted with 1.0 mL of ethanol into a formulation flask and then diluted with 9 mL of 0.9% USP-grade sodium chloride injection (Hospira, catalog no. 0409-4888-02). The title compound formulation was then passed through a 13 mm 0.22 μm Millex-GV PVDF filter (Millipore, catalog no. SLGVR13SL) and transferred to a sterile, empty vial for quality control testing.

[0088] Analytical HPLC (Agilent 1260) was performed using a Phenomenex® Luna® C18(2) analytical column (5 μm, 110 Å, 4.6 × 150 mm, part number 00F-4251-E0) and eluted with a mobile phase of 55% 10 mM ammonium acetate and 45% acetonitrile at a flow rate of 1 mL / min. CsI(Tl) scintillation crystals were randomly placed in 1 cm increments. 2 Chemical and radiochemical purity / identity were analyzed using a Carroll-Ramsey Model 105S-1 single-channel high-sensitivity radiation detector coupled with a silicon pin diode. The identity of the labeled compound was confirmed by co-injection of standards into the HPLC. Specific radioactivity was determined by injecting an aliquot of the final solution with known radioactivity into the analytical HPLC system. The area of ​​the UV peak corresponding to the carrier material was measured and compared with a calibration curve showing the relationship between mass and UV absorbance. Radioactivity was measured using a Capintec CRC®-15 PET dose calibrator. The radiochemical purity of the dose was >99%, and identity was confirmed by comparing the retention time of the radiolabeled product with that of the corresponding unlabeled reference standard. The average results of four experiments are summarized below. Total synthesis time: 80.8 + 1.3 minutes Decay-corrected yield: 13.8 + 1.3% Specific radioactivity: 5980+1880Ci / mmol (at the end of synthesis) Radiochemical purity:>99%.

[0089] [Example 3] 3 H] Compound (I), 4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-( 3 H3) Preparation of methylpyridin-2-amine

[0090] [ka]

[0091] Example 3 - Step 1: 4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]pyridin-2-amine 2-Chloro-8-fluoro-6-(2-methoxyethoxy)quinoline (77 mg, 0.3 mmol), 2-aminopyridine-4-boronic acid pinacol ester (79 mg, 0.36 mmol, 1.2 equiv.), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (24.5 mg, 0.03 mmol, 0.01 equiv.), and CsCO (195 mg, 0.600 mmol) were dissolved in dioxane (2 mL) and water (0.3 mL) (microwave synthesis vial). After sparging with argon, the reaction mixture was heated to 100 °C for 2 h (microwave synthesis system: Biotage® Initiator+, 400 W). The crude reaction mixture was poured into saturated NaHCO solution and extracted with dichloromethane. The combined organic fractions were washed with saturated NaCl solution, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with 0 to 100% ethyl acetate / cyclohexane to give the title compound (60 mg, 0.18 mmol, 60% yield). 1 H NMR(500MHz,DMSO-d6)δppm8.41(dd,J=8.8,1.5Hz,1H),8.11-8.04(m,2H),7.39(dd,J=12.2,2.6Hz,1H),7.34-7 .28(m,2H),7.25(dd,J=5.4,1.6Hz,1H),6.11(s,2H),4.32-4.24(m,2H),3.78-3.71(m,2H),3.35(s,3H);MS(ESI + ) m / z 314 [M+H] + .

[0092] Example 3 - Step 2: tert-Butyl {4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]pyridin-2-yl}carbamate 4-[8-Fluoro-6-(2-methoxyethoxy)quinolin-2-yl]pyridin-2-amine (27 mg, 0.086 mmol) was dissolved in dichloromethane (5 mL), and di-tert-butyl dicarbonate (BocO) (56 mg, 0.26 mmol, 3 equiv.) and N,N-dimethylpyridin-4-amine (31 mg, 0.26 mmol, 3 equiv.) (dissolved in 0.5 mL of dichloromethane) were added at room temperature. Stirring was continued overnight to give a mixture of starting material, tert-butoxycarbonylamino product, and bis-tert-butoxycarbonylamino product. An additional 1.5 equivalents of di-tert-butyl dicarbonate and N,N-dimethylpyridin-4-amine were added, and stirring continued at room temperature, resulting in complete conversion to the bis-tert-butoxycarbonylamino product (LCMS: Column—YMC Meteoric Core C18, 50 × 2.1 mm, 2.7 μm; 50 to 100% acetonitrile / water (0.1% formic acid) in 1.8 min, 50 °C, flow rate: 1 mL / min). 1 M NaOH (3 mL) and methanol (5 mL) were added to the reaction mixture, which was stirred vigorously until complete hydrolysis to the desired tert-butoxycarbonylamino product was confirmed (LCMS). The crude reaction mixture was then poured into saturated NaCl solution and extracted with dichloromethane. The combined organic fractions were dried over MgSO4 and concentrated to dryness. The residue was pre-purified by silica gel flash chromatography using 0→80% ethyl acetate / cyclohexane as eluent, followed by HPLC (Column: Waters® XBridge™ OBD™ Precolumn C8, 5 μm, 150 × 30 mm; Eluents: (A) water + 0.2% NH4OH, (B) acetonitrile + 0.2% NH4OH; Gradient: 0→0.73 min (50.3% A / 49.7% B), 8.12 min (30.3% A / 69.7% B), 8.13 min (100% B); Flow rate: 80 mL / min) to give the title compound (20 mg, 0.046 mmol, 30% yield). 1H NMR(500MHz,DMSO-d6)δppm9.88(s,1H),8.60(dd,J=1.6,0.8Hz,1H),8.47(dd,J=8.9,1.5Hz,1H),8.41(dd,J=5.2,0.8Hz,1H),8.17(d,J=8.7Hz,1H) ),7.80(dd,J=5.2,1.6Hz,1H),7.42(dd,J=12.1,2.6Hz,1H),7.35(d,J=2. 6Hz,1H),4.33-4.25(m,2H),3.80-3.70(m,2H),3.35(s,3H),1.52(s,9H); 19 F NMR(471MHz,DMSO-d6)δppm-123.30(d,J=7.9Hz);MS(ESI + ) m / z 414 [M+H] + .

[0093] Example 3 - Step 3: tert-Butyl{4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]pyridin-2-yl}( 3 H3) Methyl carbamate A suspension of tert-butyl {4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]pyridin-2-yl}carbamate (1.7 mg, 4.1 μmol) in anhydrous N,N-dimethylformamide (0.2 mL) was added to a nitrogen-flushed 4 mL vial equipped with a stir bar. Sodium hydride (57%, 0.173 mg, 4.11 μmol) was added to this mixture at 0°C, and the solution was stirred at room temperature for 30 minutes. A separate 4 mL vial equipped with a stir bar was charged with 4-nitrobenzene-1-sulfonic acid ( 3 A solution of methyl 4-nitrobenzene-1-sulfonic acid (H3) (10 mCi, 0.121 μmol) in acetonitrile (0.1 mL) was added and concentrated to dryness using a rotary evaporator. 3H3) The solution of tert-butyl {4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]pyridin-2-yl}carbamate was transferred to a vial containing methyl methyl, and the mixture was stirred at room temperature for 2 hours. The reaction was worked up by first quenching with brine (0.1 mL) and then extracting with ethyl acetate (2 × 3 mL). The combined organic fractions were concentrated under reduced pressure to give 10 mCi (4.11 μmol) of the crude title compound.

[0094] Example 3 - Step 4: 3 H] Compound (I), 4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-( 3 H3) Methylpyridin-2-amine Crude tert-butyl{4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]pyridin-2-yl}( 3 H3) Methyl carbamate was dissolved in dichloromethane (0.5 mL) and the solution was transferred to a 5 mL flask equipped with a stir bar. Trifluoroacetic acid (0.4 mL) was added and the solution was stirred at room temperature for 20 hours. The reaction mixture was concentrated to dryness. The crude title compound (10 mCi) was dissolved in a mixture of acetonitrile (0.8 mL) and water (0.1 mL) and used for preparative HPLC purification.

[0095] Approximately 0.05 mL of the crude title compound solution was injected onto a Phenomenex® Luna® C18 column (5 μm, 250 mm x 10 mm ID) using an Agilent 1200 Series HPLC system. Using an isocratic solvent flow of 48% mobile phase B (mobile phase A = 10 mM ammonium acetate and mobile phase B = acetonitrile), the title compound was eluted at a flow rate of approximately 4.7 mL / min for 15 min. Peak detection and chromatograms were acquired using an Agilent variable wavelength UV detector set at 254 nm and ChemStation software. Fractions containing the title compound were collected around 10 min using an Agilent fraction collector, pooled, and concentrated under reduced pressure to yield 0.3 mCi at 95% radiochemical purity. The specific radioactivity of the title compound was determined to be 81.8 Ci / mmol by LCMS (LC-Agilent 1260 using an Ascentis Express C18, 150 x 4.6 mm, 2.7 μm column, flow rate 0.8 mL / min, detection wavelength 254 nm, column temperature 30 °C, mobile phase solution A 0.1% aqueous formic acid and B acetonitrile, gradient 20 to 95% acetonitrile in 10 min, followed by equilibration to 20% acetonitrile in 2 min). Using an Agilent 6130 quadrupole mass spectrometry unit coupled with LC in API-ES ionization mode, the standard unlabeled compound (I) was determined to have m / z 327 [M+H]. + shows a peak at [ 3 H] Compound (I) has m / z 333 [M+H] + A peak was observed at .

[0096] [Example 4] Preparation of Compound (II), 3-(fluoromethyl)-1-{2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl}azetidin-3-ol

[0097] [ka]

[0098] Example 4 - Step 1: 2-[2-(methylamino)pyridin-4-yl]quinolin-6-ol 2-Chloroquinolin-6-ol (453 mg, 2.52 mmol), N-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (650 mg, 2.78 mmol, 1.1 equiv.), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (206 mg, 0.25 mmol, 0.1 equiv.), and Cs2CO3 (2.47 g, 7.57 mmol, 3 equiv.) were dissolved in 1,4-dioxane (5 mL) and water (0.8 mL) (microwave synthesis vial). The reaction mixture was sparged with argon and heated to 70 °C for 3 h (microwave synthesis system: Biotage® Initiator+, 400 W). The reaction mixture was diluted with ethyl acetate, and the insoluble material was removed by filtration. The organic fraction was washed with water, dried over MgSO4, and concentrated in vacuo to give the crude title compound (618 mg, 2.46 mmol, 97% yield), which was used in the next step without further purification. MS (ESI + ) m / z 252 [M+H] + .

[0099] Example 4 - Step 2: 2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl trifluoromethanesulfonate 2-[2-(methylamino)pyridin-4-yl]quinolin-6-ol (450 mg, 1.79 mmol) was dissolved in tetrahydrofuran (10 mL) and K2CO3 (742 mg, 5.37 mmol, 3 equiv.) was added. After cooling in an ice-water bath, 1,1,1-trifluoro-N-phenyl-N-(trifluoromethanesulfonyl)methanesulfonamide (640 mg, 1.79 mmol, 1 equiv.) was added, and the reaction mixture was stirred and allowed to warm to room temperature overnight. The reaction mixture was diluted with dichloromethane, poured into saturated NaHCO3 solution, and extracted with dichloromethane. The combined organic layers were dried over Na2SO4 and concentrated to dryness. The residue was purified by silica gel flash chromatography using 5% methanol in dichloromethane as eluent to give the title compound (68 mg, 0.93 mmol, 52% yield). MS (ESI) + ) m / z 384 [M+H] + .

[0100] Example 4 - Step 3a: tert-Butyl 3-methylideneazetidine-1-carboxylate To a solution of methyltriphenylphosphonium bromide (49.0 g, 137 mmol) in tetrahydrofuran (400 mL) was added potassium tert-butoxide (15.4 g, 137 mmol) at 25 °C. After stirring the reaction solution at 25 °C for 1 h, tert-butyl 3-oxoazetidine-1-carboxylate (10 g, 58.4 mmol) was added at 25 °C. The mixture was stirred at 35 °C for 2 h. TLC (petroleum ether / ethyl acetate = 4 / 1) confirmed the consumption of the starting material, and a new spot was detected. 16 additional reactions were prepared and processed as described above. All 17 reaction mixtures were then combined and filtered through a pad of diatomaceous earth, washing the pad with tetrahydrofuran (500 mL). The filtrate was washed with water (1 L), dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash column chromatography using petroleum ether / ethyl acetate=100 / 1→10 / 1 as the eluent to give the title compound (130 g, 77% yield). 1 H NMR (400MHz, CDCl3) δppm 1.40 (s, 9H), 4.43 (s, 4H), 4.94 (t, J = 2.43Hz, 2H).

[0101] Example 4 - Step 3b: tert-Butyl 1-oxa-5-azaspiro[2.3]hexane-5-carboxylate To a solution of tert-butyl 3-methylideneazetidine-1-carboxylate (30 g, 177 mmol) in CHCl3 (180 mL) was added 3-chloroperoxybenzoic acid (141 g, 515 mmol, 85 wt%) at 0 °C. The resulting mixture was stirred at 25 °C for 2 days. TLC (petroleum ether / ethyl acetate = 4 / 1) confirmed the consumption of the starting material, and a new spot was detected. Two more reactions were prepared and run as described above. All three reaction mixtures were then combined and quenched with a mixture of 10% sodium thiosulfate solution and saturated sodium bicarbonate solution (250 mL, 1:1). The organic layer was separated, dried over sodium sulfate, filtered, and concentrated to give the title compound (40 g, 40.6% yield), which was used directly in the next step. 1 H NMR (400MHz, CDCl3) δppm 4.27-4.22 (m, 2H), 4.21-4.13 (m, 2H), 2.88-2.74 (m, 2H), 1.49-1.36 (m, 9H).

[0102] Example 4 - Step 3c: tert-Butyl 3-(fluoromethyl)-3-hydroxyazetidine-1-carboxylate A solution of tert-butyl 1-oxa-5-azaspiro[2.3]hexane-5-carboxylate (5 g, 27.0 mmol) in triethylamine trihydrofluoride (30 mL, 27.0 mmol) was stirred at 25 °C for 4 days. TLC (petroleum ether / ethyl acetate = 2:1) confirmed the consumption of the starting material, and a new spot was detected. Seven additional reactions were prepared and run as described above. All eight reaction mixtures were then combined and concentrated. The residue was diluted with ethyl acetate (1 L) and adjusted to pH 8 with saturated aqueous NaHCO3. The mixture was extracted with ethyl acetate (3 × 500 mL). The combined organic layers were washed with brine (3 × 500 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50:1 → 2:1) to give the title compound (16 g, 41.3% yield). 1H NMR (400MHz, CDCl3) δppm 4.53 (s, 1H), 4.42 (s, 1H), 3.95-3.80 (m, 4H), 1.49-1.32 (m, 9H).

[0103] Example 4 - Step 3d: 3-(Fluoromethyl)azetidin-3-ol To a solution of tert-butyl 3-(fluoromethyl)-3-hydroxyazetidine-1-carboxylate (7 g, 34.1 mmol) in 1,4-dioxane (50 mL) was added 4N HCl / dioxane (100 mL). The reaction solution was stirred at 25 °C for 6 h. An additional reaction mixture was prepared and processed as described above. The two reaction mixtures were then combined and concentrated at 40 °C. The residue was diluted with water (70 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with water (2 × 20 mL). The combined aqueous phases were lyophilized to give the title compound (6 g, 41.4% yield) as the hydrochloride salt. 1 H NMR(400MHz,DMSO-d6)δppm9.64-9.32(m,1H),8.22(br s,1H),6.76-6.29(m,1H),4.65-4.38(m,2H),3.88(br t,J=5.6Hz,2H),3.74(s,1H),2.95(br d,J=5.5Hz,1H).

[0104] Example 4 - Step 3e: Compound (II) 3-(fluoromethyl)-1-{2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl}azetidin-3-ol 2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl trifluoromethanesulfonate (300 mg, 0.78 mmol), 3-(fluoromethyl)azetidin-3-ol hydrochloride (166 mg, 1.17 mmol, 1.5 equiv.), KPO (997 mg, 4.7 mmol, 6 equiv.), and RuPhosPdG (133 mg, 0.16 mmol, 0.2 equiv.) were suspended in tetrahydrofuran (8 mL) under Ar. After sparging with argon, the reaction mixture was capped and heated to 70–80 °C for 5 h. The reaction mixture was diluted with dichloromethane and washed sequentially with water and saturated NaCl solution. The organic layer was dried over MgSO and concentrated under reduced pressure. The residue was pre-purified by silica gel flash chromatography using 0→70% ethyl acetate / cyclohexane as eluent, followed by HPLC (Column: Waters® XSelect CSH C18 OBD precolumn, 5 μm, 150 × 30 mm; Eluents: (A) water + 0.1% formic acid, (B) acetonitrile + 0.1% formic acid; Gradient: 0→0.1 min (94.8% A / 5.2% B), 7.40 min (74.8% A / 25.2% B), 7.41 min (100% B), 9.24 min (100% B); Flow rate: 80 mL / min) to give the title compound (68 mg, 0.177 mmol, 22% yield). 1 H NMR(500MHz,DMSO-d6)δppm8.18(dd,J=8.8,0.7Hz,1H),8.13-8.06(m,1H),7.91(dd,J=8.9,6.7Hz,2H),7.22(d,J=4.9Hz,2H),7.14(dd,J=9.1,2.6Hz ,1H),6.78(d,J=2.6Hz,1H),6.59(q,J=4.8Hz,1H),4.57(d,J=47.5Hz,2H) ,4.07(d,J=8.2Hz,2H),3.78(dd,J=8.4,3.0Hz,2H),2.84(d,J=4.8Hz,3H); 19 F NMR(471MHz,DMSO-d6)δppm-226.25(t,J=47.3Hz);MS(ESI + ) m / z 339 [M+H] + .

[0105] [Example 5] 18F] Compound (II), 3-[( 18 F) Preparation of fluoromethyl]-1-{2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl}azetidin-3-ol

[0106] [ka]

[0107] Example 5 - Step 1: tert-butyl [4-(6-hydroxyquinolin-2-yl)pyridin-2-yl]methylcarbamate 2-Chloroquinolin-6-ol (400 mg, 2.23 mmol), tert-butyl methyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)carbamate (819 mg, 2.45 mmol, 1.1 equiv.), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (182 mg, 0.22 mmol, 0.1 equiv.), and CsCO (2.18 g, 6.68 mmol, 3 equiv.) were dissolved in 1,4-dioxane (8 mL) and water (0.9 mL). After sparging with argon, the reaction vial was capped and the mixture was heated to 70 °C for 1 h. The crude reaction mixture was diluted with dichloromethane, and the insoluble material was removed by filtration. The organic layer was washed with water, dried over MgSO4, and concentrated in vacuo. The residue was purified by silica gel flash chromatography eluting with 0 to 50% ethyl acetate / cyclohexane to give the title compound (648 mg, 1.84 mmol, 83% yield). MS (ESI) + ) m / z 352 [M+H] + .

[0108] Example 5 - Step 2: 2-{2-[(tert-butoxycarbonyl)(methyl)amino]pyridin-4-yl}quinolin-6-yl trifluoromethanesulfonate tert-Butyl [4-(6-hydroxyquinolin-2-yl)pyridin-2-yl]methylcarbamate (648 mg, 1.84 mmol) and K2CO3 (765 mg, 5.53 mmol, 3 equiv.) were suspended in tetrahydrofuran (10 mL) and cooled in an ice-water bath. N,N-Bis(trifluoromethylsulfonyl)aniline (659 mg, 1.84 mmol, 1 equiv.) was added, and the reaction mixture was allowed to warm to room temperature. Stirring was continued for 2 h, and the precipitate was collected by filtration and washed with dichloromethane and methanol. Drying under vacuum gave the title compound (671 mg, 1.39 mmol, 75% yield), which was used without further purification. MS (ESI) + ) m / z 484 [M+H] + .

[0109] Example 5 - Step 3a: tert-Butyl 3-cyano-3-[(trimethylsilyl)oxy]azetidine-1-carboxylate To a solution of tert-butyl 3-oxoazetidine-1-carboxylate (25 g, 146 mmol) in tetrahydrofuran (250 mL) was added trimethylsilyl cyanide (47.3 mL, 353 mmol) and lithium chloride (1.060 mg, 0.025 mmol). The reaction mixture was stirred at 25 °C for 12 h. TLC (petroleum ether / ethyl acetate = 5 / 1) confirmed the consumption of the starting material, and a new spot was detected. An additional vial was prepared and the reaction mixture was treated as described above. The two reaction mixtures were then combined. The mixture was concentrated under reduced pressure to give the title compound (80 g, 91% yield). 1 H NMR (400MHz, CDCl3) δppm0.22-0.30(m,9H)1.44(s,9H)4.02(d,J=9.88Hz,2H)4.34(d,J=9.88Hz,2H).

[0110] Example 5 - Step 3b: tert-Butyl 3-cyano-3-hydroxyazetidine-1-carboxylate To a HCl / methyl alcohol solution (200 mL, 0.5 M) was added tert-butyl 3-cyano-3-[(trimethylsilyl)oxy]azetidine-1-carboxylate (20 g, 74.0 mmol). The reaction mixture was stirred at 25 °C for 12 h. TLC (petroleum ether / ethyl acetate = 5 / 1) confirmed the consumption of the starting material, and a new spot was detected. Three more vials were prepared and run as above. All four reaction mixtures were then combined. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with petroleum ether / ethyl acetate = 100 / 1 → 0 / 1) to give the title compound (51 g, 78% yield). 1 H NMR(400MHz,DMSO-d6)δppm1.39(s,9H)3.89(d,J=9.54Hz,2H)4.28(d,J=9.54Hz,2H)7.52(s,1H);MS(ESI + ) m / z 143.1 [M-100] + .

[0111] Example 5 - Step 3c: tert-butyl 3-cyano-3-[(oxan-2-yl)oxy]azetidine-1-carboxylate To a solution of tert-butyl 3-cyano-3-hydroxyazetidine-1-carboxylate (22.5 g, 114 mmol) in dichloromethane (65 mL) were added 3,4-dihydro-2H-pyran (17.00 g, 202 mmol) and 4-methylbenzenesulfonic acid (0.344 mg, 0.002 mmol). The reaction mixture was stirred at 25 °C for 12 h. TLC (petroleum ether / ethyl acetate = 5 / 1) confirmed the consumption of the starting material, and a new spot was detected. An additional vial was prepared and processed as described above. The two reaction mixtures were then combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with petroleum ether / ethyl acetate = 100 / 1 → 5 / 1) to give the title compound (45 g, 63.2% yield). 1H NMR(400MHz,CDCl3)δppm1.45(s,9H)1.55-1.67(m,4H)1.73-1.90(m,2H)3.52-3.65(m,1H)3.94(ddd,J=10.82,7 MS(ESI + ) m / z 227.1 [M-100] + .

[0112] Example 5 - Step 3d: 1-(tert-butoxycarbonyl)-3-[(oxan-2-yl)oxy]azetidine-3-carboxylic acid To a solution of tert-butyl 3-cyano-3-[(oxan-2-yl)oxy]azetidine-1-carboxylate (22.5 g, 80 mmol) in ethanol (200 mL) and water (200 mL) was added KOH (17.88 g, 319 mmol). The reaction mixture was stirred at 100 °C for 12 h. TLC (petroleum ether / ethyl acetate = 5 / 1) confirmed the consumption of the starting material and a new spot was detected. An additional vial was prepared and processed as described above. After cooling to 25 °C, the two reaction mixtures were combined. The ethanol was removed under reduced pressure, and the aqueous phase was acidified to pH = 5 with 2 N HCl and extracted with ethyl acetate (3 × 200 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the title compound (35 g, 65.6% yield). 1 H NMR(400MHz,DMSO-d6)δppm1.38(s,9H)1.42-1.55(m,4H)1.64-1.78(m,2H)3.37-3.46(m,1H)3.79-3.94(m,2H)3.99-4.20(m,3H)4.74(br d,J=1.38Hz,1H)12.84(br s,1H);MS(ESI + ) m / z 202.1 [M-100] + .

[0113] Example 5 - Step 3e: tert-butyl 3-(hydroxymethyl)-3-[(oxan-2-yl)oxy]azetidine-1-carboxylate To a solution of 1-(tert-butoxycarbonyl)-3-[(oxan-2-yl)oxy]azetidine-3-carboxylic acid (7.5 g, 24.89 mmol) in tetrahydrofuran (60 mL) was added N,N'-carbonyldiimidazole (4.44 g, 27.4 mmol), and the mixture was stirred at 25 °C for 10 min. Next, a solution of NaBH (1.883 g, 49.8 mmol) in water (30 mL) was added. The reaction mixture was stirred at 25 °C for 12 h. TLC (petroleum ether / ethyl acetate = 1 / 1) confirmed the consumption of the starting material, and a new spot was detected. An additional vial was prepared and treated as described above. Next, the two reaction mixtures were combined, ethyl acetate (300 mL) was added, and the organic layer was separated. The organic fraction was washed with 1N NaHCO solution (200 mL) and brine (200 mL), dried over NaSO, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with petroleum ether / ethyl acetate = 100 / 1 → 1 / 1) to give the title compound (5 g, 66.4% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3)δppm1.45(s,9H)1.51-1.67(m,4H)1.72-1.94(m,2H)3.4 3-3.60(m,2H)3.70-3.89(m,6H)3.92-4.06(m,1H)4.55-4.69(m,1H);MS(ESI + ) m / z 188.1 [M+1] + .

[0114] Example 5 - Step 3f: {3-[(oxan-2-yl)oxy]azetidin-3-yl}methanol In a microwave synthesizer vial, tert-butyl 3-(hydroxymethyl)-3-[(oxan-2-yl)oxy]azetidine-1-carboxylate (177 mg, 0.62 mmol) and KOH (104 mg, 1.85 mmol, 3 equiv.) were dissolved in water (2 mL) and methanol (3 mL). The reaction mixture was heated to 100–120 °C (microwave synthesizer: Biotage® Initiator+, 400 W) for 6 h. Additional equivalents of KOH were added portionwise (up to a total of 10 equiv.) until complete tert-butoxycarbonyl deprotection was observed by LCMS (column: YMC Meteoric Core C18, 50 × 2.1 mm, 2.7 μm; 50 → 100% acetonitrile / water (0.1% formic acid) over 1.8 min, 50 °C, flow rate: 1 mL / min). The crude mixture was manually passed through a Chromabond® C18 cartridge, first with water to reduce the total salt loading, then switched to water / methanol (1:1), and the product-containing fractions were collected. The combined water / methanol fractions were concentrated under reduced pressure at 50°C, and the residue was dissolved in methanol. Concentration under reduced pressure gave the title compound (132 mg, 0.612 mmol, 100% crude yield), which was used without further purification. MS (ESI) + ) m / z 188 [M+H] + .

[0115] Example 5 - Step 3g: tert-butyl [4-(6-{3-(hydroxymethyl)-3-[(oxan-2-yl)oxy]azetidin-1-yl}quinolin-2-yl)pyridin-2-yl]methylcarbamate A microwave synthesis vial was charged with 2-{2-[(tert-butoxycarbonyl)(methyl)amino]pyridin-4-yl}quinolin-6-yl trifluoromethanesulfonate (163 mg, 0.34 mmol), KPO (340 mg, 1,602 mmol), and tBuXPhosPdG (26 mg, 0.032 mmol, 0.1 equiv). A solution of {3-[(oxan-2-yl)oxy]azetidin-3-yl}methanol (60 mg, 0.32 mmol, 1 equiv) in 1,4-dioxane (2 mL) and N,N-dimethylformamide (0.5 mL) was added. After sparging with argon, the reaction vial was capped and the reaction mixture was heated to 100 °C and stirred for 4 h. The crude reaction mixture was poured into water and extracted with dichloromethane. The combined organic extracts were dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel flash chromatography eluting with 0 to 60% ethyl acetate / cyclohexane to give the title compound (72 mg, 0.138 mmol, 43% yield). MS (ESI+) m / z 521 [M+H] + .

[0116] Example 5 - Step 4: {1-(2-{2-[(tert-butoxycarbonyl)(methyl)amino]pyridin-4-yl}quinolin-6-yl)-3-[(oxan-2-yl)oxy]azetidin-3-yl}methyl 4-methylbenzene-1-sulfonate tert-Butyl [4-(6-{3-(hydroxymethyl)-3-[(oxan-2-yl)oxy]azetidin-1-yl}quinolin-2-yl)pyridin-2-yl]methylcarbamate (72 mg, 0.138 mmol) was dissolved in dichloromethane (3 mL). N,N-Dimethylpyridin-4-amine (25 mg, 0.21 mmol, 1.5 equiv.) and p-toluenesulfonyl chloride (30 mg, 0.16 mmol, 1.15 equiv.) were added, and the resulting reaction mixture was stirred at room temperature overnight. An additional 0.5 equiv. of both reagents was added, and stirring was continued for 3 days to achieve complete conversion. The crude reaction mixture was poured into 10% citric acid solution and extracted with dichloromethane. The combined organic extracts were dried over MgSO4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with 0→20% ethyl acetate / dichloromethane to give the title compound (45 mg, 0.067 mmol, 48% yield). 1 H NMR(500MHz,CDCl3)δppm8.49(d,J=5.2Hz,1H),8.39-8.35(m,1H),8.01(t,J=7.8Hz,2H),7.85-7.74(m,4H), 7.34(d,J=8.1Hz,2H),6.99(dd,J=9.1,2.6Hz,1H),6.59(d,J=2.6Hz,1H),4.81(dd,J=4.8,2.8Hz,1H),4.47(d ,J=10.4Hz,1H),4.39(d,J=10.4Hz,1H),4.05-3.94(m,4H),3.89(ddd,J=11.0,6.9,3.4Hz,1H),3.46(s,3H), 3.45-3.40(m,1H),2.44(s,3H),1.85-1.75(m,1H),1.73-1.64(m,1H),1.56(s,9H),1.61-1.46(m,4H);MS(ESI + ) m / z 675 [M+H] + .

[0117] Example 5 - Step 5: 18 F] Compound (II), 3-[( 18 F) Fluoromethyl]-1-{2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl}azetidin-3-ol At the Wisconsin Medical Cyclotron (Milwaukee, WI) 18 O(p,n) 18 In the F reaction, 18 [F] fluoride was synthesized and delivered to the radiochemistry laboratory the morning of use. Approximately 1–4 mL of [ 18 O] dissolved in H2O [ 18 The [F]fluoride was transferred to a GE TRACERlab™ FX FN synthesis module and trapped on an ion exchange cartridge (Waters Sep-Pak® Accell QMA Carbonate Plus Light Cartridge, catalog number WAT186004540) preconditioned with TRACESELECT™ Ultra ACS water (5 mL). 18 A solution of KCO (0.75 mg, 5.43 μmol) in ultratrace analytical grade water (0.4 mL) and a solution of 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (Kryptofix® 2.2.2, 7.5 mg, 19.9 μmol) in acetonitrile (0.4 mL) were passed through the cartridge to remove [O]H2O. 18 [F] fluoride was then eluted into the reaction vessel. 18The [F] fluoride was dried under full vacuum by heating (70 °C) and a stream of nitrogen or helium for 5 minutes, followed by drying at 100 °C for 5 minutes under full vacuum. After drying, a solution of {1-(2-{2-[(tert-butoxycarbonyl)(methyl)amino]pyridin-4-yl}quinolin-6-yl)-3-[(oxan-2-yl)oxy]azetidin-3-yl}methyl 4-methylbenzene-1-sulfonate (2 mg, 2.96 μmol) in anhydrous dimethyl sulfoxide (1.0 mL) was added, and the resulting solution was heated and stirred at 100 °C for 10 minutes. 1N HCl (1.0 mL) was added to the reaction mixture, which was then heated to 100 °C for 3 minutes to remove the tert-butoxycarbonyl and tetrahydropyranyl protecting groups. The reaction mixture was then cooled to 50 °C and neutralized with 1N NaOH (1.1 mL) and sterile water for injection (WFI, 2.0 mL). The resulting mixture was transferred to an HPLC loading vial. The contents of the loop loading vial were transferred to semi-preparative HPLC (Phenomenex® Gemini® NX-C18 5 μm, 110 Å 10 × 250 mm column with a UV setting of 260 nm) and purified using a mobile phase of 72% 10 mM ammonium acetate and 28% acetonitrile at a flow rate of 4 mL / min.

[0118] The title compound peak (retention time approximately 26 minutes) was collected in an HPLC dilution flask and diluted with WFI (40 mL). The purified title compound was then trapped on a preconditioned 50 mg Phenomenex® Strata C18-E cartridge (catalog no. 8B-S001-DAK) and washed with WFI (5 mL). The trapped title compound was eluted with ethanol (1.0 mL) into a formulation flask and then diluted with 0.9% USP-grade sodium chloride injection (9 mL) (Hospira, catalog no. 0409-4888-02). The title compound formulation was then passed through a 13 mm 0.22 μm Millex-GV PVDF filter (Millipore, catalog no. SLGVR13SL) and transferred to a sterile, empty vial for quality control testing.

[0119] Analytical HPLC (Agilent 1260) was performed using a Phenomenex® Luna® C18(2) analytical column (5 μm, 110 Å, 4.6 × 150 mm, part number 00F-4251-E0) with a mobile phase of 65% 10 mM ammonium acetate and 35% acetonitrile at a flow rate of 1 mL / min. CsI(Tl) scintillation crystals were randomly placed in 1 cm increments. 2 Chemical and radiochemical purity / identity were analyzed using a Carroll-Ramsey Model 105S-1 single-channel high-sensitivity radiation detector coupled with a silicon pin diode. The identity of the labeled compound was confirmed by co-injection of standards into the HPLC. Specific radioactivity was determined by injecting an aliquot of the final solution with known radioactivity into the analytical HPLC system. The area of ​​the UV peak corresponding to the carrier material was measured and compared with a calibration curve showing the relationship between mass and UV absorbance. Radioactivity was measured using a Capintec CRC®-15 PET dose calibrator. The radiochemical purity of the dose was >99%, and identity was confirmed by comparing the retention time of the radiolabeled product with that of the corresponding unlabeled reference standard. The average results of four experiments are summarized below. Total synthesis time: 66.8 + 0.4 min Decay-corrected yield: 35.3 + 3.0% Specific radioactivity: 2649+944Ci / mmol (at the end of synthesis) Radiochemical purity:>99%.

[0120] [Example 6] 3 H] Compound (II), 3-(fluoromethyl)-1-(2-{2-[( 3 H3) Preparation of methylamino)pyridin-4-yl}quinolin-6-yl)azetidin-3-ol

[0121] [ka]

[0122] Example 6 - Step 1: tert-butyl [4-(6-hydroxyquinolin-2-yl)pyridin-2-yl]carbamate In a microwave synthesis vial, 2-chloroquinolin-6-ol (200 mg, 1.11 mmol), tert-butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)carbamate (465 mg, 1.45 mmol, 1.3 equiv.), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (91 mg, 0.11 mmol, 0.1 equiv.), and CsCO (1.1 g, 3.34 mmol, 3 equiv.) were dissolved in 1,4-dioxane (8 mL) and water (0.9 mL). After sparging with argon, the vial was capped and the reaction mixture was heated to 80 °C for 3 h. The crude reaction mixture was diluted with dichloromethane, and the insoluble material was removed by filtration. The organic filtrate was washed with water, dried over MgSO4, and concentrated in vacuo. The residue was purified by silica gel flash chromatography eluting with 0 to 40% ethyl acetate / cyclohexane to give the title compound (251 mg, 0.74 mmol, 67% yield). MS (ESI) + ) m / z 338 [M+H] + .

[0123] Example 6 - Step 2: 2-{2-[(tert-butoxycarbonyl)amino]pyridin-4-yl}quinolin-6-yl trifluoromethanesulfonate tert-Butyl [4-(6-hydroxyquinolin-2-yl)pyridin-2-yl]carbamate (251 mg, 0.74 mmol) and K2CO3 (308 mg, 2.23 mmol, 3 equiv.) were suspended in tetrahydrofuran and cooled in an ice-water bath. N,N-Bis(trifluoromethylsulfonyl)aniline (266 mg, 0.74 mmol, 1 equiv.) was added, and the reaction mixture was allowed to warm to room temperature and stirred overnight. The precipitate was filtered off and washed with dichloromethane and methanol. Drying under vacuum gave the title compound (380 mg, 0.73 mmol, 98% yield), which was used without further purification. MS (ESI) + ) m / z 470 [M+H] + .

[0124] Example 6 - Step 3: tert-butyl (4-{6-[3-(fluoromethyl)-3-hydroxyazetidin-1-yl]quinolin-2-yl}pyridin-2-yl)carbamate 2-{2-[(tert-butoxycarbonyl)amino]pyridin-4-yl}quinolin-6-yl trifluoromethanesulfonate (211 mg, 0.45 mmol), 3-(fluoromethyl)azetidin-3-ol hydrochloride (127 mg, 0.90 mmol, 2 equiv.), KPO (572 mg, 2.70 mmol, 6 equiv.), tBuXPhosPdG (71 mg, 0.09 mmol, 0.2 equiv.), and RuPhosPdG (76 mg, 0.09 mmol, 0.2 equiv.) were suspended in tetrahydrofuran (5 mL) in a microwave synthesis vial. After sparging with argon, the vial was capped and the reaction mixture was heated to 80 °C for 16 h (microwave synthesis system, Biotage® Initiator+, 400 W). The crude reaction mixture was poured into saturated NaHCO3 and extracted with ethyl acetate. The combined extracts were dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel flash chromatography eluting with 0 to 10% ethyl acetate-methanol (4:1) / dichloromethane to give the title compound (58 mg, 0.137 mmol, 30% yield). MS (ESI) + ) m / z 425 [M+H] + .

[0125] Example 6 - Step 4: tert-butyl [4-(6-{3-(fluoromethyl)-3-[(oxan-2-yl)oxy]azetidin-1-yl}quinolin-2-yl)pyridin-2-yl]carbamate tert-Butyl (4-{6-[3-(fluoromethyl)-3-hydroxyazetidin-1-yl]quinolin-2-yl}pyridin-2-yl)carbamate (58 mg, 0.137 mmol) was dissolved in tetrahydrofuran (2 mL). 3,4-Dihydro-2H-pyran (0.135 mL, 1.37 mmol, 10 equiv.) and pyridinium p-toluenesulfonate (7 mg, 0.027 mmol, 0.2 equiv.) were then added. The reaction mixture was heated to 50-60 °C until complete conversion (LCMS: Column - YMC Meteoric Core C18, 50 × 2.1 mm, 2.7 μm; 50 → 100% acetonitrile / water (0.1% formic acid) in 1.8 min, 50 °C, flow rate: 1 mL / min). Addition of an additional equivalent of 3,4-dihydro-2H-pyran may be necessary. The crude reaction mixture was concentrated in vacuo and the residue was purified by silica gel flash chromatography eluting with 0→10% ethyl acetate / dichloromethane to give the title compound (48 mg, 0.094 mmol, 69% yield). 1 H NMR(500MHz,DMSO-d6)δppm9.84(s,1H),8.59(dd,J=1.6,0.8Hz,1H),8.36(dd,J=5.2,0.8Hz,1H),8.26-8.20(m,1H),7 .99(d,J=8.7Hz,1H),7.95(d,J=9.1Hz,1H),7.76(dd,J=5.2,1.6Hz,1H),7.18(dd,J=9.1,2.6Hz,1H),6.83(d,J=2.6Hz ,1H),4.98(dd,J=5.1,2.7Hz,1H),4.88-4.67(m,2H),4.07(t,J=8.7Hz,2H),4.03-3.98(m,2H),3.87(ddd,J=11.1,7.2 ,3.8Hz,1H),3.49(ddd,J=10.9,6.4,4.0Hz,1H),2.53-2.51(m,1H),1.82-1.64(m,2H),1.52(s,9H),1.56-1.41(m,3H); 19 F NMR(471MHz,DMSO-d6)δppm-226.78(t,J=47.0Hz);MS(ESI + ) m / z 509 [M+H] + .

[0126] Example 6 - Step 5: tert-Butyl [4-(6-{3-(fluoromethyl)-3-[(oxan-2-yl)oxy]azetidin-1-yl}quinolin-2-yl)pyridin-2-yl]( 3 H3) Methyl carbamate To a 4 mL septum-capped vial was added sodium hydride (0.5 mg, 13 μmol) and tert-butyl [4-(6-{3-(fluoromethyl)-3-[(oxan-2-yl)oxy]azetidin-1-yl}quinolin-2-yl)pyridin-2-yl]carbamate (1.0 mg, 2 μmol). The vial was evacuated and refilled with N2 three times. Anhydrous N,N-dimethylformamide (0.15 mL) was then added and the reaction was stirred at room temperature for 30 minutes. In another 4 mL septum-capped vial, 4-nitrobenzene-1-sulfonic acid ( 3 A solution of methyl 4-nitrobenzene-1-sulfonate (H3) (10 mCi, 77 Ci / mmol) in acetonitrile was obtained, and the solvent was removed under reduced pressure. 3 The vial containing H3) methyl was transferred to a Schlenk manifold and evacuated and backfilled with nitrogen three times. The N,N-dimethylformamide solution containing tert-butyl [4-(6-{3-(fluoromethyl)-3-[(oxan-2-yl)oxy]azetidin-1-yl}quinolin-2-yl)pyridin-2-yl]carbamate was then transferred to the vial, and the reaction was stirred at room temperature for 16 hours. HPLC analysis (column: Phenomenex® Luna® C18, 25 mm × 4.6 mm, 5 μm, flow rate: 1.5 mL / min, detection wavelength: 254 nm, column temperature: 40°C; mobile phase B: gradient of 10 to 50% acetonitrile over 9 min, followed by gradual increase to 95% acetonitrile over 1 min, hold at 95% acetonitrile for 3 min, and equilibrate to 10% acetonitrile for 3 min; mobile phase A: 0.1% aqueous trifluoroacetic acid) revealed 4-nitrobenzene-1-sulfonic acid ( 3The H3) methyl was judged to be completely consumed. The reaction mixture was then diluted with dichloromethane (4 mL) and quenched with brine (0.25 mL), after which the phases were separated. The aqueous phase was further extracted with dichloromethane (4 mL). The organic fractions were combined, and the solution was dried by rotary evaporation to give the title compound.

[0127] Example 6 - Step 6: 3 H] Compound (II), 3-(fluoromethyl)-1-(2-{2-[( 3 H3) Methylamino]pyridin-4-yl}quinolin-6-yl)azetidin-3-ol Crude tert-butyl[4-(6-{3-(fluoromethyl)-3-[(oxan-2-yl)oxy]azetidin-1-yl}quinolin-2-yl)pyridin-2-yl]( 3 H3) The methyl carbamate was dissolved in dichloromethane (0.5 mL), trifluoroacetic acid (0.3 mL) was added, and the mixture was stirred at room temperature for 4 h. HPLC analysis (HPLC conditions: Phenomenex® Luna® C18, 25 mm x 4.6 mm, 5 μm column, flow rate 1.5 mL / min, detection wavelength 254 nm, column temperature 40 °C; mobile phase B: 10 → 50% acetonitrile over 9 min, ramped to 95% acetonitrile over 1 min, held at 95% acetonitrile for 3 min, equilibrated to 10% acetonitrile for 3 min; mobile phase A: 0.1% aqueous trifluoroacetic acid) confirmed the reaction was complete. The reaction mixture was concentrated by rotary evaporation, and the crude residue was dissolved in 1.5 mL of a 2:1 water (10 mM ammonium acetate) / acetonitrile solution for HPLC purification. The radioactivity of the crude solution was 6.1 mCi.

[0128] The crude solution was purified by semi-preparative high-performance liquid chromatography (Agilent 1260) using a Phenomenex® Luna® C18 column (250 x 10 mm, 5 μm) at a column temperature of 40°C, with a mobile phase solution of solvent A: 10 mM ammonium acetate and B: acetonitrile delivered at a flow rate of 4.5 mL / min. The crude product was purified in two batches using a mobile phase gradient of 30 to 50% acetonitrile over 16 minutes. 3-(fluoromethyl)-1-(2-{2-[( 3 H3)methylamino]pyridin-4-yl}quinolin-6-yl)azetidin-3-ol began to elute at 9.3 minutes and was collected using an Agilent fraction collector. The purified fractions were combined, and the acetonitrile was removed by rotary evaporation to give purified 3-(fluoromethyl)-1-(2-{2-[( 3 A 6.2 mL solution of 10 mM H3)methylamino[pyridin-4-yl}quinolin-6-yl]azetidin-3-ol in ammonium acetate was obtained. To this solution was added 2 mL of ethanol containing 16 mg of ascorbic acid as a stabilizer. The total radioactivity was 3.1 mCi, and the radiochemical purity was 95%. The specific radioactivity was determined to be 76.1 Ci / mmol by LCMS (Ascentis® Express C18, 2.7 μm, 4.6 × 150 mm, flow rate 1.4 mL / min, detection wavelength 254 nm, column temperature 30°C; mobile phase B: 10 → 50% acetonitrile gradient over 8 min, followed by a gradual increase to 95%, a 2.5 min hold at 95% acetonitrile, and a 2.5 min equilibration to 10% acetonitrile; mobile phase A: 0.1% formic acid in water).

[0129] [Example 7] In vitro biological test The following abbreviations were commonly used in the in vitro biological tests:

[0130] [Table 2]

[0131] Brain tissue. Snap-frozen (FF) tissue blocks from the globus pallidus and putamen of normal or progressive supranuclear palsy (PSP) brains, and in some cases corresponding formalin-fixed, paraffin-embedded (FFPE) tissue blocks, were purchased from Tissue Solutions Ltd (TS; Glasgow, UK). Both FF and FFPE tissue blocks were used to combine normal and PSP brain samples. 3 H] Compound (I) and [ 3 The distribution pattern of [H] Compound (II) was evaluated by in vitro autoradiography. Only FF tissue blocks were used, and [H] Compound (II) distribution pattern in PSP brain was evaluated by in vitro autoradiography. 3 H] Compound (I) and [ 3 The affinity of [H] Compound (II) was measured by in vitro autoradiography. A single FF tissue block from the frontal cortex of an Alzheimer's disease (AD) Braak stage V brain, purchased from Analytical Biological Services Inc. (ABS; Wilmington, DE, USA), was used to measure the binding affinity of the compound to AD tau and amyloid beta (Aβ) protein aggregates by radioligand binding assays on tissue homogenates.

[0132] Brain tissue preparation. For in vitro autoradiography experiments, FF tissue blocks were sectioned into 14 μm-thick brain sections using a Leica (Wetzlar, DE) CM3050S cryostat (specimen head temperature: −18°C; chamber temperature: −20°C), mounted on Superfrost™ Plus glass slides (Gerhard Menzel GmbH, Braunschweig, DE), and stored at −80°C until the day of experimentation. FFPE blocks were sectioned into 14 μm-thick brain sections at ambient temperature using a ThermoFisher Scientific (Waltham, MA, USA) HM355S microtome (specimen head temperature: 4°C), mounted on glass slides, and stored at ambient temperature until the day of experimentation. On the day of experimentation, FF brain sections were allowed to return to ambient temperature for at least 1 h before incubation with radioligand. On the day of the experiment, before incubation with the radioligand, FFPE slices were deparaffinized and rehydrated as follows: heating at 70°C for 30 min, immersion in xylene at ambient temperature for 2 × 10 min, 100% ethanol at ambient temperature for 2 × 10 min, 96% ethanol at ambient temperature for 2 min, 70% ethanol at ambient temperature for 1 min, Milli-Q® water at ambient temperature for 1 min, and Dulbecco's phosphate-buffered saline (DPBS) at ambient temperature for 5 min.

[0133] The AD brain tissue blocks selected for radioligand binding studies were brought back to ambient temperature, homogenized in ice-cold DPBS buffer, pH 7.4, without calcium and magnesium, divided into aliquots, and stored at −80°C until the day of the experiment.

[0134] In vitro autoradiography. 3 H] Compound (I) and [ 3For in vitro autoradiography of [H] compound (II), 14 μm-thick FF brain slices or 14 μm-thick deparaffinized FFPE brain slices were incubated with 3 nM radioligand in the presence or absence of the corresponding non-radiolabeled compound in DPBS buffer supplemented with 0.1% bovine serum albumin (BSA) for 90 min at ambient temperature. Radioligand binding was terminated by washing the tissue slices with ice-cold DPBS buffer (3 × 3 min). After rinsing with ice-cold Milli-Q® water (2 × 5 s), the slices were air-dried and attached to a tritium-sensitive BAS-TR2025 imaging plate (Fujifilm Corporation, Tokyo, Japan) for 7 days. The plate was read using a Phosphoimager Typhoon™ FLA-7000IP (GE Healthcare, Chicago, IL, USA). Total binding (TB) was defined as radioligand binding in the presence of radioligand alone, and nonspecific binding (NSB) was defined as radioligand binding in the presence of 10 μM of the corresponding non-radiolabeled compound (Figure 1, Figure 3).

[0135] Quantitative analysis of in vitro autoradiography results. Quantitative analysis was performed by computer-assisted microdensitometry (MCD; MCID Basic, Interfocus, Devon, UK). 1 mm was measured by referencing a tritium standard (American Radiolabeled Chemicals, Saint Louis, MO, USA) on the same plate. 2 The MCD values ​​per unit area were converted to the corresponding radioligand concentration (fmol / mg). Regions of interest were identified by referencing the Atlas of the Human Brain (Mai et al., 2015, Elsevier, ISBN: 9780128028018). Radioligand binding in normal and PSP brains was quantified by selecting the entire gray matter region in the section, including the globus pallidus and putamen, and measuring the average binding intensity in the selected region (Figure 2).

[0136] Measurement of the affinity of Compound (I) and Compound (II) for PSP tissue by in vitro autoradiography. To measure the affinity of Compound (I) and Compound (II) for PSP brain tissue, 3 nM radioligand ([ 3 H] Compound (I) or [ 3 H] Compound (II)) alone or in the presence of increasing concentrations (10 -10 ~10 -4 Homologous displacement (also known as self-displacement) curves were obtained by incubating serial FF brain slices in the presence of the corresponding non-radiolabeled ligand (compound (I) or compound (II), respectively) of compound (M). The concentration required to inhibit 50% of total binding (IC) was calculated by fitting the binding intensity data with the "One site-fit logIC" function in GraphPad Prism (GraphPad Software, San Diego, CA, USA). 50 The dissociation constant (K D ) is K i (i.e., K D =K i The inhibition constant (K) was calculated using the Cheng-Prusoff equation (Cheng and Prusoff, Biochem Pharmacol 1973 Dec 1;22(23):3099). i The affinity values ​​obtained in such self-substitution experiments were then calculated using the K i values ​​and K obtained in radioligand saturation experiments D To distinguish the affinity values ​​obtained in self-displacement experiments from the sd K D (K obtained by self-replacement experiments D )

[0137] The globus pallidus and putamen in the PSP brain and normal brain [ 3 H] Compound (I) and [ 3 H] Binding of Compound (II). 3 H] Compound (I) and [ 3[H] Compound (II) bound to FF tissue sections of the globus pallidus and putamen from PSP donor brains in a heterogeneous distribution pattern. These tissue sections were characterized by punctate binding in the globus pallidus and putamen of PSP brains, with significantly higher density in the globus pallidus. In contrast, binding was homogeneous in the same regions of age-matched normal donors and in the presence of 10 μM of the corresponding non-radiolabeled compound (Figure 1). The mean binding density in the gray matter of the globus pallidus and putamen was higher in PSP brains than in normal brains (Figure 2). At a radioligand concentration of 3 nM, [H] compound (II) bound to the tissue. 3 The amount of [H] Compound (I) bound to the tissue after incubation at a radioligand concentration of 3 nM was 2.1-fold higher in PSP brains than in normal brains. 3 The amount of [H] Compound (II) was 2.2-fold higher in PSP brains than in normal brains (Figure 2). The distribution pattern of radioligand binding was consistent with the loading and distribution of 4R tau in PSP as reported by Williams (Brain, 2007, 130, (6):1566-1576). A similar distribution pattern was observed when FFPE sections were used, i.e., punctate binding was observed in PSP brains but not in normal brains (Figure 3).

[0138] Affinity of Compound (I) and Compound (II) for tau aggregates in the globus pallidus and putamen of PSP brain. Unlabeled Compound (I) and Compound (II) were administered to PSP brain slices of the globus pallidus and putamen at high concentrations, respectively. 3 H] Compound (I) and [ 3 H] compound (II) could be substituted (see Figure 4). sd K D The value is 3 H] Compound (I) and [ 3 1H] Compound (II) and 12.4 nM and 16.0 nM, respectively (Figure 4).

[0139] In summary, the analysis of the in vitro data revealed several important properties of Compound (I) and Compound (II). First, 3 H] Compound (I) and [ 3Both H] compounds (II) showed high selectivity for tau aggregates in the globus pallidus and putamen in PSP-affected brain tissue compared to healthy brain tissue. 3 H] Compound (I) and [ 3 Total binding of [H] Compound (II) was significantly higher in the globus pallidus and putamen of PSP-affected brain tissue, which could be titrated with unlabeled Compound (I) or Compound (II), respectively. The specificity of the interaction of these compounds with PSP-affected brain tissue was demonstrated by their sd K D The binding activity of Compound (I) was 12.4 nM and that of Compound (II) was 16.0 nM. Overall, these data indicate that Compound (I) and Compound (II) were highly specific for binding to tau in the globus pallidus and putamen of PSP-affected brain tissue.

[0140] Example 8: Positron Emission Tomography (PET) Imaging and Interpretation The following abbreviations were commonly used in PET imaging studies:

[0141] [Table 3]

[0142] PET imaging procedures in cynomolgus monkeys were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. Anesthesia was maintained using isoflurane gas at 1.25–3.0% throughout the imaging procedure. Prior to PET imaging, head computed tomography (CT) images were acquired using a Neurologica CereTom® CT system (Neurologica, Danvers, MA, USA) for the purposes of animal positioning in the PET system and subsequent attenuation correction of the PET data.

[0143] The radiotracer was administered as a bolus injection (3 mL over 2 minutes), and data acquisition was initiated for 120 minutes using a Siemens Concorde microPET® Focus™ 220 (Siemens USA, Washington DC, USA) with dynamic list-mode emission acquisition. Arterial blood samples were obtained throughout the PET acquisition period to measure radioactivity in whole blood and plasma and assess the percentage of parent compound and metabolites to generate input functions for kinetic modeling analysis. Arterial whole blood samples were initially obtained rapidly (every 30 seconds) and then reduced in frequency to every 30 minutes toward the end of PET acquisition. Radioactivity concentrations in arterial whole blood samples were measured using a well counter (PerkinElmer Wizard® 1470; PerkinElmer, Waltham, MA, USA). Samples were then centrifuged (3000 RCF for 5 minutes) to allow for plasma extraction, after which radioactivity concentrations were measured. To determine the percentage of unchanged radiotracer, selected plasma samples (1, 2, 3, 4, 7, 9, 15, 20, 40, 60, 90, and 120 min) were extracted with an equal volume of acetonitrile and then injected onto a Phenomenex® Onyx™ C18 10 × 100 mm column (Torrance, CA, USA), an Eckert and Ziegler BGO coincidence detector (model B-FC-4100, Eckert and Ziegler, Atlanta, GA, USA), and a Dionex / Thermo Ultimate 3000 radio-HPLC system (ThermoFisher, Waltham, MA, USA) consisting of a mobile phase of (A) 10 mM ammonium acetate and (B) acetonitrile. 18 F] Compound (I) conditions are: 0 → 2.5 min, 4 mL / min, 95% A; 2.5 → 2.6 min, 4 mL / min, 3 mL / min, 95% A → 50% A; 2.6 → 10 min, 3 mL / min, 50% A; 10 → 10.1 min, 3 mL / min, 4 mL / min, 50% A → 95% A; 10.1 → 13 min, 4 mL / min, 95% A. 18 F] The retention time of compound (I) was 8.4 minutes. 18F] Compound (II) conditions are: 0 → 2.5 min, 4 mL / min, 95% A; 2.5 → 2.6 min, 4 mL / min, 3 mL / min, 95% A → 65% A; 2.6 → 10 min, 3 mL / min, 65% A; 10 → 10.1 min, 3 mL / min, 4 mL / min, 65% A → 95% A; 10.1 → 13 min, 4 mL / min, 95% A. 18 The retention time of [F] Compound (II) was 9.3 min. Arterial sampling data were used to generate input functions for kinetic modeling. Potential off-target specific binding in the brain was assessed for each lead-based radiotracer using a pre-dosing paradigm in which a 1 mg / kg equivalent dose of non-radioactive tracer was administered via a 15-minute infusion 20 min prior to radiotracer injection, followed by PET.

[0144] After PET image acquisition was completed, anesthesia was terminated, and the primates were monitored until fully awake and then returned to their cages. In addition to PET imaging, each primate underwent separate MRI imaging (GE Discovery™ 3T MR750 MRI scanner and GE Signa™ HDxt 1.5T MRI scanner, GE Healthcare, Chicago, IL, USA) to acquire structural T1w images for coregistration and transformation to standard atlas space. PET data analysis was performed, and images were reconstructed using Siemens MicroPET® Manager and AsiPro (Siemens) software. PET list-mode data were binned into sinograms with 6 × 30 s, 3 × 1 min, 2 × 2 min, and 22 × 5 min time windows. Images were reconstructed using filtered backprojection with a Hanning filter cutoff of 0.5 cycles per pixel. Attenuation correction (using the acquired CT), scatter correction, and dead-time correction were applied. Additionally, dynamic images were attenuation-corrected at the start of PET acquisition, which coincided with the time of radiotracer injection. The final reconstructed dynamic image had a voxel size of 1.898 × ​​1.898 × ​​0.796 mm and a matrix size of 128 × 128 × 95 (x, y, z). Dynamic PET imaging data, structural CT and MRI images, and ancillary data (arterial blood data, injected radiotracer information, and subject information) were processed to obtain dynamic PET images in atlas space, enabling regional and voxel assessment of radiotracer dynamics. Briefly, to enable coregistration, unattenuation-corrected PET images reconstructed from the entire scan period were tightly registered to the subject's head CT image, followed by tight registration to the subject's structural MRI image. Next, linear and nonlinear warping methods were used to transform the MRI dataset into standard space. Registration to standard space allowed for the comparison and extraction of time-activity curves (TACs) within and between subjects using atlas-based regions of interest (ROIs). MRI-based subject-specific gray matter segmentation was applied to the atlas, excluding any portion of the ROI outside of what was considered the gray matter of this specific subject. The ROIs tested in this task included gray matter, cerebral white matter, cerebellum, frontal lobe, parietal lobe, temporal lobe, occipital lobe, globus pallidus, and putamen.Total volume of distribution (V T Regional TACs were fitted using standard one-tissue (1T) and two-tissue reversible (2T) and irreversible (2T) compartmental models to estimate kinetic parameters, including . Global compartmental configurations were assessed using a fixed ratio of 5% and the cerebrovascular ratio as the fitted model parameter (v). The Akaike Information Criterion (AIC), which penalizes models for increasing complexity and poor fit, was used to assess the validity of compartmental configurations. Global coregistration and kinetic modeling were performed using MIAKAT™ (Invicro, Konica-Minolta, Boston, MA, USA), which runs within the MATLAB environment (MATLAB 2019b, MathWorks, Natick, MA, USA). Reference region analysis was performed using the cerebellar gray matter as the reference region to estimate V under baseline and corresponding block conditions. T This allows for comparison of the blood distribution volume ratio (DVR) and the serotonin concentration.

[0145] PET imaging results shown in Figure 5 are time-activity curves (TACs) extracted from the gray matter by compartmental modeling fit using a two-tissue compartment model, which was the preferred model by Akaike Information Criterion (AIC). Figure 6 shows the regional V at baseline and after self-blocking administration of the corresponding non-radiolabeled compound. T After injection of the radiotracer, the radioactivity concentration in the brain was initially high (SUV 2-4) and [ 18 [F] Compound (I) showed the highest uptake, reaching a peak brain uptake at approximately 5 minutes, followed by a rapid decline. Near the end of the PET measurements, no significant differences in regional uptake were observed. The distribution of radioactivity was relatively uniform across all brain regions examined, consistent with the absence of target-specific binding of either tracer in these healthy nonhuman primates. Furthermore, V was significantly higher in baseline and self-block experiments. T The lack of statistical difference in values ​​indicates little to no off-target binding of these tracers.

[0146] In summary, the results of the PET imaging study in cynomolgus monkeys demonstrated several important findings related to Compound (I) and Compound (II). First, 18 F] Compound (I) and [ 18 Both compounds (II) exhibited the ability to cross the blood-brain barrier and were characterized by rapid uptake and clearance. Healthy cynomolgus monkeys do not exhibit 4R tauopathy, but [ 18 F] Compound (I) and [ 18 F]Compound (II) loading was uniform across all brain regions and showed no significant off-target binding.

Claims

1. Formula (I): 【Chemical 1】 [In the formula, R １ is CH ３ Or C ( ３ H) ３ and R ２ is H, F or １８ F; R ３ is -O-CH ２ CH ２ -O-CH ３ , or 【Chemistry 2】 and R ４ is F or １８ F], or a pharmaceutically acceptable salt thereof.

2. R １ But CH ３ Or C ( ３ H) ３ and R ２ But F or １８ F; R ３ But -O-CH ２ CH ２ -O-CH ３ 2. The compound of claim 1, wherein:

3. R １ But CH ３ and R ２ The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein:

4. R １ But CH ３ and R ２ but, １８ 3. The compound of claim 2, wherein R is H, or a pharmaceutically acceptable salt thereof.

5. R １ But C ( ３ H) ３ and R ２ The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein:

6. R １ But CH ３ Or C ( ３ H) ３ and R ２ is H; R ３ but, 【Chemistry 3】 and R ４ But F or １８ 2. The compound of claim 1, wherein R is H, or a pharmaceutically acceptable salt thereof.

7. R １ But CH ３ and R ４ The compound of claim 6, wherein is F, or a pharmaceutically acceptable salt thereof.

8. R １ But CH ３ and R ４ but, １８ 7. The compound of claim 6, wherein R is H, or a pharmaceutically acceptable salt thereof.

9. R １ But C ( ３ H) ３ and R ４ The compound of claim 6, wherein is F, or a pharmaceutically acceptable salt thereof.

10. 4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-methylpyridin-2-amine; 4-[8-( １８ F) fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-methylpyridin-2-amine; 4-[8-fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-( ３ H ３ ) methylpyridin-2-amine; 3-(fluoromethyl)-1-{2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl}azetidin-3-ol; 3-[( １８ F) fluoromethyl]-1-{2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl}azetidin-3-ol; and 3-(fluoromethyl)-1-(2-{2-[( ３ H ３ ) methylamino]pyridin-4-yl}quinolin-6-yl)azetidin-3-ol 10. The compound of claim 1, selected from the group consisting of:

11. The compound is 【Chemistry 4】 2. The compound of claim 1, wherein:

12. The compound is 【Chemistry 5】 2. The compound of claim 1, wherein:

13. The compound is 【Chemistry 6】 2. The compound of claim 1, wherein:

14. The compound is 【Chemistry 7】 2. The compound of claim 1, wherein:

15. The compound is 【Chemistry 8】 2. The compound of claim 1, wherein:

16. The compound is 【Chemistry 9】 2. The compound of claim 1, wherein:

17. The compound is 【Chemistry 10】 2. The compound of claim 1, wherein:

18. The compound is 【Chemistry 11】 2. The compound of claim 1, wherein:

19. The compound is 【Chemistry 12】 2. The compound of claim 1, wherein:

20. The compound is 【Chemistry 13】 2. The compound of claim 1, wherein:

21. The compound is 【Chemistry 14】 2. The compound of claim 1, wherein:

22. The compound is 【Chemistry 15】 2. The compound of claim 1, wherein:

23. 10. A pharmaceutical composition comprising a therapeutically or diagnostically effective amount of a compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable excipient.

24. 10. A method for imaging tau aggregates in a subject, comprising administering to a patient a diagnostically effective amount of the radiolabeled compound of claim 1 and imaging tau aggregates in the patient.

25. The radiolabeled compound is 4-[8-( １８ F) fluoro-6-(2-methoxyethoxy)quinolin-2-yl]-N-methylpyridin-2-amine.

26. The radiolabeled compound is 3-[( １８ F) fluoromethyl]-1-{2-[2-(methylamino)pyridin-4-yl]quinolin-6-yl}azetidin-3-ol.

27. 25. The method of claim 24, wherein the subject is afflicted with or suspected of being afflicted with a tauopathy.

28. 28. The method of claim 27, wherein the tauopathy is progressive supranuclear palsy.

29. 25. The method of claim 24, wherein the tau aggregates are 4R tau aggregates.

Images