PET tracers for visualizing GABA A gamma 1 receptors

Abstract

The present invention provides a method for the preparation of radiolabeled GABA receptors useful in medical imaging. A Provided is a gamma 1 positive allosteric modulator (PAM).

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides radiolabeled GABA receptors useful in medical imaging, such as positron emission tomography (PET) and / or autoradiography. A This relates to gamma 1 positive allosteric modulators (PAMs). [Background technology]

[0002] 2. Background of the Invention Receptors for the major inhibitory neurotransmitter, γ-aminobutyric acid (GABA), are divided into two major classes: (1) GABA receptors, which are members of the ligand-binding ion channel superfamily; A receptors, and (2) GABA receptors, which are members of the G protein-coupled receptor family. B The receptors are divided into membrane-bound heteropentameric protein polymers, called GABA receptors. A The GABA receptor complex is composed primarily of α, β, and γ subunits. A The receptor is a ligand-bound chloride channel and is the primary mediator of inhibitory neurotransmission in the human brain.

[0003] GABA A There are 19 genes encoding the receptor subunits, with the most common stoichiometry being two α, two β, and one γ subunit assembled as a pentamer. A Subunit combinations give rise to functional, circuit and behavioral specificity, and their expression is heterogeneously distributed in the brain. A γ1 subunit-containing receptors are less abundant than γ2 subunit-containing receptors (GABA receptors in the brain A Approximately 5–10% of total receptor expression.

[0004] Positron emission tomography (PET), as a non-invasive imaging technique, aids in drug discovery and development by providing valuable information on drug-target engagement, assessing drug occupancy and monitoring treatment. Furthermore, PET can be used for neuroreceptor mapping in healthy subjects and disease states (Nasrallah I, Dubroff, J An overview of PET neuroimaging, Seminars in nuclear medicine, 2013, 43, 449-61. Hou L, Rong J, Haider A, et al. Positron Emission Tomography Imaging of the Endocannabinoid System: Opportunities and Challenges in Radiotracer Development, J. Med. Chem. 2021, 64, 1, 123-149). Alternatively, autoradiography can be used for in vitro neuroreceptor mapping on tissue sections obtained postmortem. In animal models, autoradiography can provide drug-target engagement information from an ex vivo readout. GABA A PET imaging of γ2 subunit-containing receptors is well established, and a number of radiotracers are routinely used in preclinical and clinical studies. The most prominent and important of these are 18 F]flumazenil (or the carbon-11 version [ 11 C]flumazenil) and [ 11 C]Ro15-4513 (Kassenbrock A, Vasdev N, Liang SH, Selected PET Radioligands for Ion Channel Linked Neuroreceptor Imaging: Focus on GABA, NMDA and nACh Receptors, Current Topics in Medicinal Chemistry, 2016, 16, 1830-1842). [ 18 [F]Flumazenil inhibits GABA receptors containing all combinations of α-subunits. AVisualize γ2 receptors, but 11 C]Ro15-4513 is GABA A Prefers α5γ2 receptors. GABA A In contrast to γ2 receptors, selective GABA receptors for in vitro applications A Although γ1 tracers have been described (see, for example, WO2021198124 and WO2021213952), no studies have been performed to detect GABA in vivo. A There is no PET tracer that targets and selectively visualizes γ1 receptor.Therefore, the need for such a PET tracer is unmet.The compounds of the present invention show increased affinity to GABAA γ1 receptor, allowing target imaging in vivo. Summary of the Invention

[0005] Summary of the Invention The radiolabeled compounds of the present invention are selective GABA A The compounds of the present invention are γ1 receptor positive allosteric modulators (PAMs) and are useful for visualizing the receptor in vivo and in vitro, for example by PET or autoradiography. The compounds of the present invention have high binding affinity and selectivity for γ1-containing subtypes (α5γ1, α2γ1, α1γ1) compared to γ2-containing subtypes (e.g., α1γ2, α2γ2, α3γ2 and α5γ2). Thus, the compounds of the present invention are similar to classical GABA receptors. A GABA not addressed by receptor tracers A Useful for visualizing γ1 receptors.

[0006] In a first aspect, the present invention provides a compound of formula (I) or (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a radiolabel.

[0007] In a further aspect, the present invention provides a method for the preparation of a GABA A Radiolabeled compounds as described herein for use in γ1 occupancy studies are provided.

[0008] In a further aspect, the present invention provides a method for the treatment of GABA in a mammal. A Provided are radiolabeled compounds as described herein for use in diagnostic imaging of γ1. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows an in vitro autoradiogram of [3H]-(I) and [3H]-(II) in coronal mouse brain sections (see Example 5 for details). [Diagram 2] FIG. 2 shows diagrams of regional time-activity curves (TAC) of [11C]-(I) and [11C]-(II) in baboons (see Example 6 for details). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] definition It should be understood that features, integers, characteristics, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the invention are applicable to any other aspect, embodiment or example described herein, except where inconsistent therewith. All of the features disclosed herein (including any accompanying claims, abstracts and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel or any novel combination of features disclosed herein (including any accompanying claims, abstracts and drawings), or any novel or any novel combination of steps of any method or process so disclosed.

[0011] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid, without being biologically or otherwise undesirable. Salts are formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, especially hydrochloric acid, and organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, and the like. These salts may also be prepared by adding inorganic or organic bases to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyimine resins, and the like.

[0012] The term "mammal" includes humans, non-human primates, such as chimpanzees and other ape and monkey species, farm animals, such as cows, horses, sheep, goats and pigs, domestic animals, such as rabbits, dogs and cats, and laboratory animals, such as rodents, such as rats, mice and guinea pigs. In certain embodiments, the mammal is a human. The term mammal does not denote a particular age or sex.

[0013] The term "radiolabel" refers to radioactive isotopes, e.g., of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine. In some embodiments, radiolabels used in the context of the present invention are useful for PET imaging and / or autoradiography. Examples of isotopes that can be incorporated into compounds of formulas (I) and (II) as radiolabels include, respectively: 3H, 11 C. 14 C. 13 N, 15 O. 18 F, and 36 Preferred radioactive labels include: 3 H, 11 C. 13 N, 15 O, and 18 F. A further preferred radiolabel is 3 H, 11 C and 18 F. Particularly preferred radiolabels are 3 H and 11 It's C.

[0014] Compounds of the Invention In a first aspect, the present invention provides a compound of formula (I) or (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a radiolabel.

[0015] The compounds of formula (I) and (II) of the present invention are radiolabeled (i.e., isotopically labeled) by replacing one or more atoms therein with an isotope having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of formula (I) and (II) include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, including, but not limited to, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, respectively. 3 H, 11 C. 14 C. 13 N, 15 O. 18 F, and 36 Certain isotopically labeled compounds of formula (I) and (II), for example those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 3 H, carbon-11, i.e. 11C, and fluorine-18, i.e. 18 F are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. For example, compounds of formula (I) can be enriched with 1, 2, 5, 10, 25, 50, 75, 90, 95, or 99 percent of a given isotope.

[0016] 11 C. 18 F, 15 O and 13 Substitution with positron emitting isotopes, such as N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

[0017] 3 Substitution with beta negative radiation emitting isotopes, such as H, can be useful in autoradiography studies, for example, for examining substrate receptor occupancy.

[0018] In one embodiment, the radiolabel is 3 H, 11 C. 13 N, 15 O. 18 F, and 36 Cl.

[0019] In a preferred embodiment, the radiolabel is 11 C. 18 F and 3 H.

[0020] In a preferred embodiment, the radiolabel is 11 C and 3 H.

[0021] In a particularly preferred embodiment, the radiolabel is 11 It's C.

[0022] In a particularly preferred embodiment, the radiolabel is 3 It's H.

[0023] In a particularly preferred embodiment, the radiolabel is 18 It's F.

[0024] In one embodiment, the compound of formula (I) or (II) of the present invention is [ka] or a pharma- ceutically acceptable salt thereof.

[0025] In a preferred embodiment, the compound of formula (I) or (II) of the present invention is [ka] or a pharma- ceutically acceptable salt thereof.

[0026] In a preferred embodiment, the compound of formula (I) or (II) of the present invention is [ka] or a pharma- ceutically acceptable salt thereof.

[0027] In a preferred embodiment, the compound of formula (I) or (II) of the present invention is [ka] or a pharma- ceutically acceptable salt thereof.

[0028] In a preferred embodiment, the compound of formula (I) or (II) of the present invention is [ka] or a pharma- ceutically acceptable salt thereof.

[0029] Uses of the Compounds of the Invention The radiolabeled compounds of the present invention are potent GABA Aγ1 positive allosteric modulators (PAMs), which are used to treat, for example, therapeutic GABA A To validate target engagement of γ1 modulators and GABA under normal and disease conditions A GABA to investigate the function of γ1 receptors A It can be used as a PET tracer for the γ1 receptor.

[0030] Thus, in one aspect, the present invention provides a method for the treatment of GABA in a mammal. A A method for imaging diagnosis of γ1, comprising: (a) administering to a mammal a detectable amount of a radiolabeled compound described herein, or a pharma- ceutically acceptable salt thereof; (b) GABA A detecting the radiolabeled compound when associated with γ1.

[0031] In a preferred embodiment, the imaging is brain imaging.

[0032] In a preferred embodiment, the detecting is via autoradiography and / or PET.

[0033] In a preferred embodiment, the detecting is via autoradiography.

[0034] In a preferred embodiment, the detecting is detecting via PET.

[0035] In a further aspect, the present invention provides a radiolabeled compound as described herein, or a pharma- ceutically acceptable salt thereof, for use in the diagnostic imaging methods described herein.

[0036] In a further aspect, the present invention provides the use of a radiolabeled compound as described herein, or a pharma- ceutically acceptable salt thereof, in the diagnostic imaging methods described herein.

[0037] In a further aspect, the present invention provides a method for the preparation of a GABA A Provided herein is a radiolabeled compound or a pharma- ceutically acceptable salt thereof for use in gamma 1 occupancy studies.Such occupancy studies can be carried out, for example, as described in Scientific Reports (2021), 11(1), 7700.

[0038] In a further aspect, the present invention provides a method for the preparation of a GABA A The use of the radiolabeled compounds disclosed herein in γ1 occupancy studies is provided.

[0039] In one embodiment, the GABA A γ1 occupancy studies have shown that GABA A The method includes contacting γ1 with a radiolabeled compound disclosed herein, or a pharma- ceutically acceptable salt thereof.

[0040] In a further aspect, the present invention provides a method for the preparation of a GABA A A method for studying γ1 receptor occupancy comprising administering to said GABA receptor A The method includes contacting a γ1 receptor with a radiolabeled compound described herein.

[0041] In one embodiment, the GABA A The γ1 occupancy study is an in vitro occupancy study. EXAMPLES

[0042] The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the claims.

[0043] Unless otherwise noted, all reactions and intermediates were prepared under an argon atmosphere.

[0044] Synthesis of building blocks The building blocks can be prepared according to the following synthetic procedures.

[0045] Component A 6,7-Dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one [ka]

[0046] a) 5-chloro-2-methyl-3,1-benzoxazin-4-one A solution of 2-amino-6-chlorobenzoic acid (15.0 g, 87.4 mmol) in acetic anhydride (200 mL) was stirred at 140° C. for 2 hours. The reaction solution was concentrated in vacuo. The residue was suspended in acetonitrile, the solid was filtered, and the filter cake was dried in vacuo to give the title compound (11.3 g, 66%) as a white solid. 1 H NMR(400 MHz,CDCl3)δ ppm 2.47(3 H,s)7.47(1 H,dd,J=8.1,0.9 Hz)7.53(1 H,dd,J=7.9,1.0 Hz)7.67(1 H,dd,J=8.1,8.0 Hz).

[0047] b) N-[3-chloro-2-(2-fluoro-5-methoxy-benzoyl)phenyl]acetamide To a solution of 2-bromo-1-fluoro-4-methoxybenzene (5.45 g, 26.6 mmol) in THF (200 mL) was added n-butyllithium (2.5 m in hexane, 12.8 mL, 31.9 mmol) at -78 °C. After stirring for 1 h, 5-chloro-2-methyl-3,1-benzoxazin-4-one (5.20 g, 26.6 mmol) was added to the mixture and stirring was continued for another 1 h at -78 °C. The mixture was quenched with saturated aqueous NH4Cl and extracted with ethyl acetate. The organic layer was dried (Na2SO4), filtered and concentrated in vacuo. The residue was purified by preparative HPLC (Phenomenex luna C18, 10 μm, 250 × 50 mm, 0.05% HCl in water / acetonitrile) to give the title compound (3.63 g, 42%) as a pale yellow solid. MS: 322.1 ([{ 35 Cl}M+H] + ),324.1([{37 Cl}M+H] + ),ESI pos.

[0048] c) (2-amino-6-chloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone To a solution of N-[3-chloro-2-(2-fluoro-5-methoxy-benzoyl)phenyl]acetamide (4.00 g, 12.4 mmol) in ethanol (50 mL) was added aqueous HCl (37%, 53.3 mL, 640 mmol). The mixture was stirred at 100° C. for 2 h and then concentrated in vacuo. The residue was dissolved in DCM and washed successively with saturated aqueous NaHCO3 and water. The organic layer was dried (Na2SO4), filtered and concentrated in vacuo to give the title compound (2.87 g, 83%) as an off-white solid. MS: 280.0 ([{ 35 Cl}M+H] + ),282.0([{ 37 Cl}M+H] + ),ESI pos.

[0049] d) (6-amino-2,3-dichloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone A solution of (2-amino-6-chloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone (1.00 g, 3.58 mmol) and N-chlorosuccinimide (430 mg, 3.22 mmol) in DMF (20 mL) was stirred at 0° C. for 2 h. The mixture was quenched with water and extracted with DCM. The organic layer was dried (Na2SO4), filtered and concentrated in vacuo. The residue was purified by preparative HPLC (Phenomenex Synergi C18, 10 μm, 150×25 mm, 0.1% trifluoroacetic acid in water / acetonitrile) to give the title compound (367 mg, 33%) as a pale yellow solid. MS: 313.9 ([{ 35 Cl, 35 Cl}M+H] + ),315.9([{ 35 Cl, 37 Cl}M+H] + ),ESI pos.

[0050] e) 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one A solution of glycine ethyl ester hydrochloride (2.89 g, 20.7 mmol) and (6-amino-2,3-dichloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone (650 mg, 2.07 mmol) in pyridine (30 mL) was stirred at 100° C. for 16 h. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (Phenomenex Synergi C18, 10 μm, 150×25 mm, 0.1% trifluoroacetic acid in water / acetonitrile) to give the title compound (280 mg, 38%) as a yellow solid. MS: 353.0 ([{ 35 Cl, 35 Cl}M+H] + ),355.0([{ 35 Cl, 37 Cl}M+H] + ),ESI pos.

[0051] Component B 6-Chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one [ka]

[0052] a) (6-amino-3-bromo-2-chloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone Analogously to the experiment for building block A d, (2-amino-6-chloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone (building block A c) was converted to the title compound (1.63 g, 64%) using N-bromosuccinimide instead of N-chlorosuccinimide, affording a pale yellow solid. MS: 357.9 ([{ 79 Br, 35 Cl}M+H] + ),359.9([{ 81 Br, 35 Cl or79 Br, 37 Cl}M+H] + ),ESI pos.

[0053] b) 7-Bromo-6-chloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one Analogously to the experiment for building block Ae, (6-amino-3-bromo-2-chloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone was converted to the title compound (1.63 g, 64%) as a pale yellow solid (860 mg, 38%). MS: 396.9 ([{ 79 Br, 35 Cl}M+H] + ),398.9([{ 81 Br, 35 Cl or 79 Br, 37 Cl}M+H] + ),ESI pos.

[0054] c) 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one A solution of 7-bromo-6-chloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one (600 mg, 1.51 mmol), methylboronic acid (117 mg, 1.96 mmol), potassium phosphate (641 mg, 3.02 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.10 g, 1.51 mmol) in DMF (12 mL) was stirred at 80° C. under nitrogen for 6 h. The reaction was diluted with methanol, filtered through a Celite plug and the filtrate was concentrated in vacuo. The residue was treated with water and extracted with ethyl acetate. The organic layer was dried (Na2SO4), filtered and concentrated in vacuo. The residue was purified by preparative HPLC (Phenomenex Synergi C18, 10 μm, 150×25 mm, 0.1% trifluoroacetic acid in water / acetonitrile) to give the title compound (230 mg, 45%) as a light brown solid. MS: 333.1 ([{ 79 Br, 35 Cl}M+H] + ),335.1([{ 81 Br, 35 Cl or 79 Br, 37 Cl}M+H] + ),ESI pos.

[0055] 11 C radiolabeled precursor 1 5-[5-[tert-butyl(dimethyl)silyl]oxy-2-fluoro-phenyl]-6,7-dichloro-1,3-dihydro-1,4-benzodiazepin-2-one [ka]

[0056] a) tert-Butyl N-[3,4-dichloro-2-[(2-fluoro-5-methoxy-phenyl)-hydroxy-methyl]phenyl]carbamate To a solution of tert-butyl N-(3,4-dichlorophenyl)carbamate (5.82 g, 22.2 mmol) in THF (64 mL) was added tert-butyllithium, 1.7 ml, in pentane (28.7 ml, 48.8 mmol) dropwise from a dry ice-cooled dropping funnel at -90 °C, and the resulting mixture was stirred at -85 °C for an additional 0.5 h. Then, a solution of 2-fluoro-5-methoxybenzaldehyde (3.76 g, 24.4 mmol) in THF (16 ml) was added dropwise from a dry ice-cooled dropping funnel at -85 to -90 °C. After stirring at -90 to -85 °C for an additional 0.5 h, the mixture was warmed to -65 °C and then quenched by the dropwise addition of saturated aqueous NH4Cl. The mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was suspended in DCM and the solid was filtered and dried under high vacuum to give the title compound (4.29 g, 46%) as a white solid. MS: 414.2 ([{ 35 Cl, 35 Cl}MH] - ),416.2([{ 35 Cl, 37 Cl}MH] - ),ESI neg.

[0057] b) tert-Butyl (3,4-dichloro-2-(2-fluoro-5-methoxybenzoyl)phenyl)carbamate To a suspension of tert-butyl N-[3,4-dichloro-2-[(2-fluoro-5-methoxy-phenyl)-hydroxy-methyl]phenyl]carbamate (9.58 g, 23 mmol) in DCM (95 ml) was added water (95 ml), potassium bromide (383 mg, 3.22 mmol) and sodium bicarbonate (773 mg, 9.21 mmol) at 22° C., giving two layers cooled to 0° C. TEMPO (75.5 mg, 483 μmol) was then added and sodium hypochlorite (21.4 g, 17.8 ml, 34.5 mmol) was added dropwise (over 1 h) with vigorous stirring, maintaining the internal temperature below 2° C. The mixture was warmed to 22° C. and extracted with DCM (3×100 ml). The organic layer was washed with water (1×100 ml) and half-saturated NaCl (1×100 ml), dried over Na2SO4, filtered and evaporated. The residue (light brown foam) was treated with EtOAc (50 ml) to give a precipitate, the suspension was stirred for 30 min, the solid was filtered off, washed with EtOAc (2×20 ml) and dried to give the product (5.602 g, 59%) as a white solid. The filtrate was concentrated, adsorbed onto Isolute sorbent and purified by flash chromatography (silica gel, 330 g, adsorbed onto Isolute HM-N, 5% to 10% EtOAc in heptane) to give additional product (3.00 g, 31%) as a white solid. 412.2([{ 35 Cl, 35 Cl}MH] - ),414.2([{ 35 Cl, 37 Cl}MH] - ),ESI neg.

[0058] c) (6-amino-2,3-dichloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone To a solution of tert-butyl N-[3,4-dichloro-2-[(2-fluoro-5-methoxy-phenyl)-hydroxymethyl]phenyl]carbamate (8.60 g, 20.8 mmol) in DCM (200 mL) at 22° C. was added trifluoroacetic acid (47.3 g, 415 mmol) and the mixture was stirred at 22° C. for 2 h. The solution was concentrated in vacuo. The residue (combined with another batch-17.9 mmol-scale) was treated with saturated aqueous NaHCO3 and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo to give the title compound (11.0 g, 90%) as a yellow solid. MS: 314.0 ([{ 35 Cl, 35 Cl}M+H] + ),316.0([{ 35 Cl, 37 Cl}M+H] + ),ESI pos.

[0059] d) 6,7-Dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one (Building Block A, alternative synthesis) A solution of (6-amino-2,3-dichloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone (8.35 g, 26.6 mmol) in pyridine (165 mL) was heated to 90° C., then ethyl glycinate hydrochloride (26.0 g, 186 mmol) was added in one portion, and the resulting mixture was stirred at 110° C. for 4 h. The mixture was cooled to 90° C., then more ethyl glycinate hydrochloride (14.8 g, 106 mmol) was added, and stirring at 110° C. was continued for 16 h. The mixture was cooled to room temperature and concentrated in vacuo. The residue was treated with saturated aqueous NaHCO3 and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (silica, 10-100% ethyl acetate in heptane) to give the title compound (5.47 g, 58%) as a yellow solid. MS:353.0([{ 35 Cl, 35 Cl}M+H] + ),355.0([{ 35Cl, 37 Cl}M+H] + ),ESI pos.

[0060] e) 6,7-dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one To a pale yellow solution of 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one (500 mg, 1.42 mmol) in DCM (15 mL) was added boron tribromide (1.77 g, 7.08 mmol) dropwise at -65 °C. The mixture was warmed to -20 °C and stirred for 0.5 h. The mixture was quenched with half-saturated aqueous NaHCO3 and extracted with DCM. The organic layer was washed with half-saturated aqueous NaHCO3, dried over sodium sulfate, filtered and concentrated in vacuo. The aqueous layer was extracted again with ethyl acetate and the organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. Both residues were combined and purified by flash column chromatography (silica, 10-100% ethyl acetate in heptane) followed by crystallization from MTBE / heptane to give the title compound (220 mg, 46%) as a pale yellow solid. MS:339.0([{ 35 Cl, 35 Cl}M+H] + ),341.0([{ 35 Cl, 37 Cl}M+H] + ),ESI pos.

[0061] f) 5-[5-[tert-butyl(dimethyl)silyl]oxy-2-fluoro-phenyl]-6,7-dichloro-1,3-dihydro-1,4-benzodiazepin-2-one To a solution of 6,7-dichloro-5-(2-fluoro-5-hydroxyphenyl)-1,3-dihydro-1,4-benzodiazepin-2-one (175 mg, 0.516 mmol) in DMF (1.75 mL) was added imidazole (77.3 mg, 1.14 mmol) at 22° C., followed by tert-butyldimethylchlorosilane (85.5 mg, 0.568 mmol), and the resulting mixture was stirred at 22° C. for 0.5 h. The mixture was concentrated in vacuo. The residue was treated with aqueous NaOH (0.1 m) and extracted with ethyl acetate. The organic layer was washed successively with aqueous NaOH (0.1 m) and brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (silica, 0-30% ethyl acetate in heptane) followed by crystallization from ethyl acetate / heptane to give the title compound (122 mg, 52%) as a white solid. MS:453.2([{ 35 Cl, 35 Cl}M+H] + ),455.1([{ 35 Cl, 37 Cl}M+H] + ),ESI pos.

[0062] 11 C radiolabeled precursor 2 5-[5-[tert-butyl(dimethyl)silyl]oxy-2-fluoro-phenyl]-6-chloro-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one [ka]

[0063] a) 6-chloro-5-(2-fluoro-5-hydroxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one To a solution of 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one (500 mg, 1.50 mmol) in DCM (15 mL) at -65°C was added boron tribromide (1.88 g, 7.51 mmol). The mixture was stirred at -60°C for 0.5 h. The mixture was then warmed to -20°C and stirred for 0.5 h. The mixture was quenched with saturated aqueous NaHCO3 and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica, 10-100% ethyl acetate in petroleum ether) followed by crystallization from ethyl acetate / heptane to give the title compound (177 mg, 37%) as a pale yellow solid. MS: 319.1 ([{ 79 Br, 35 Cl}M+H] + ),321.1([{ 81 Br, 35 Cl or 79 Br, 37 Cl}M+H] + ),ESI pos.

[0064] b) 5-[5-[tert-butyl(dimethyl)silyl]oxy-2-fluoro-phenyl]-6-chloro-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one To a solution of 6-chloro-5-(2-fluoro-5-hydroxyphenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one (184 mg, 0.577 mol) in DMF (1.8 mL) at 22° C. was added imidazole (86.5 mg, 1.27 mmol) followed by tert-butyldimethylchlorosilane (95.7 mg, 0.635 mmol) and the mixture was stirred at 22° C. for 1 h. The mixture was concentrated in vacuo, treated with aqueous NaOH (0.1 m) and extracted with ethyl acetate. The organic layer was washed with aqueous NaOH (0.1 m) and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica, 0-45% ethyl acetate in petroleum ether) followed by crystallization from ethyl acetate / heptane to give the title compound (110 mg, 44%) as a white solid. MS: 433.2 ([{ 79 Br, 35 Cl}M+H] + ),435.2([{ 81 Br, 35 Cl or 79 Br, 37 Cl}M+H] + ),ESI pos.

[0065] Example 1 6,7-Dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1-methyl-3H-1,4-benzodiazepin-2-one (I) [ka]

[0066] a) 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1-methyl-3H-1,4-benzodiazepin-2-one A solution of 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one (Building A, 120 mg, 0.340 mmol), iodomethane (1.00 g, 7.05 mmol) and potassium carbonate (70 mg, 0.51 mmol) in DMF (3 mL) was stirred at 25° C. for 0.5 h. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by preparative HPLC (Phenomenex Synergi C18, 10 μm, 150×25 mm, 0.1% trifluoroacetic acid in water / acetonitrile) to give the title compound (114 mg, 91%) as a white solid. MS: 367.1 ([{ 35 Cl, 35 Cl}M+H] + ),369.1([{ 35 Cl, 37 Cl}M+H] + ),ESI pos.

[0067] b) 6,7-dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1-methyl-3H-1,4-benzodiazepin-2-one To a solution of 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1-methyl-3H-1,4-benzodiazepin-2-one (80 mg, 0.22 mmol) in DCM (9 mL) at 0° C. was added boron tribromide (273 mg, 1.09 mmol) dropwise. The reaction mixture was stirred at 0° C. for 1 h, warmed to room temperature and stirred for an additional 5 h. The reaction was quenched with ice water and extracted with DCM. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by preparative HPLC (Phenomenex Synergi C18, 10 μm, 150×25 mm, 0.225% formic acid in water / acetonitrile) to give the title compound (59 mg, 77%) as a white solid. MS: 353.1 ([{ 35 Cl, 35 Cl}M+H] + ),355.1([{ 35 Cl, 37 Cl}M+H] + ),ESI pos.

[0068] Example 2 6-Chloro-5-(2-fluoro-5-hydroxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one (II) [ka]

[0069] a) 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one Analogously to the experiment in Example 1a, 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one (building block B) was converted to the title compound (76 mg, 36%) as a yellow solid. MS: 347.1 ([{ 35 Cl}M+H] + ),349.1([{ 37 Cl}M+H] + ),ESI pos.

[0070] b) 6-chloro-5-(2-fluoro-5-hydroxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one As in the experiment in Example 1b, 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one was converted to the title compound (72 mg, 83%) as an off-white solid. MS: 333.0 ([{ 35 Cl}M+H] + ),335.0([{ 37 Cl}M+H] + ),ESI pos.

[0071] Example 3 H]1 6,7-Dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1-([ 3 H3]methyl)-3H-1,4-benzodiazepin-2-one [ka]

[0072] a) 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1-([ 3 H3]methyl)-3H-1,4-benzodiazepin-2-one [ 3 To [H3]methyl nosylate (1.85 GBq, 50 mCi, 0.61 μmol) was added a solution of 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one (Building A) (443 μg, 1.25 μmol, 2.0 equiv.) in THF (120 μL). A 0.5 M solution of sodium tert-butoxide (6.3 μL, 3.1 μmol, 5.0 equiv.) in THF was then added and the reaction mixture was stirred at room temperature for 2 h. The reaction was quenched by adding water (20 μL). The solvent was evaporated under a stream of argon and the remaining solid was dissolved in MeCN / H2O 1:1 (180 μL). The crude product was purified by HPLC (Sunfire C18 OBD, 4.6 × 250 mm, MeCN[A] / HO+5% MeCN[B], gradient: 1–18 min 10:90–90:10 [A]:[B], 18.0–18.1 min 90:10–95:5, 21.0–21.1 min 95:5–10:90, run time 24 min, flow rate 1 mL / min, 236 nm, oven temperature 40 °C; 5 injections). Pure fractions were combined, frozen, and lyophilized under vacuum for 1.5 h. The product was used directly in the next step without further characterization.

[0073] b) 6,7-dichloro-5-(2-fluoro-5-hydroxyphenyl)-1-([ 3 H3]methyl)-3H-1,4-benzodiazepin-2-one 6,7-Dichloro-5-(2-fluoro-5-methoxy-phenyl)-1-([ 3[H3]methyl)-3H-1,4-benzodiazepin-2-one (expected from previous experiment: 131 μg, 0.35 μmol, 1 equiv, 28 mCi) was dissolved in dichloromethane (0.5 mL) and transferred to a 1 mL Alltech tube. The solvent was evaporated under a stream of argon. This was repeated three times with a total of 1.5 mL of dichloromethane. The residue was dissolved in dichloromethane (extra dried, over molecular sieves, 0.15 mL) and treated with a 1 M solution of boron tribromide (5.3 μL, 5.3 μmol, 15 equiv). The tube was closed with a Teflon sealed plastic cap. The solution turned yellow and was stirred at 40 °C (oil bath temperature) for 4 h. Radioactive HPLC analysis revealed nearly complete consumption of the starting material. At room temperature, the reaction was quenched by adding water (20 μL). The solvent was evaporated under a stream of argon and the remaining solid was dissolved in MeCN / HO 1:1 (150 μL). The crude product was purified by HPLC (Sunfire C18 OBD, 4.6 × 250 mm, MeCN[A] / HO+5% MeCN[B], gradient: 1–18 min 10:90–90:10[A]:[B], 18.0–18.1 min 90:10–95:5, 21.0–21.1 min 95:5–10:90, run time 24 min, flow rate 1 mL / min, 236 nm, oven temperature 40 °C; 5 injections). Pure fractions were combined, frozen, and lyophilized under vacuum for 2 h. The pure tritium-labeled compound (337 MBq, 9.1 mCi) was dissolved in ethanol (10 mL) and stored. A radiochemical purity of 96% was determined by radio-HPLC and a specific activity of 3.0 TBq / mmol (81 Ci / mmol) was determined by mass spectrometry (MS). The identity of the labeled compound was confirmed by HPLC (by co-injecting an unlabeled reference standard) and MS. MS: m / z=353.0 [M(H)+H] + (3%), 355.0 [M( 3 H)+H] + (0%), 357.0 [M( 3 H2)+H] + (6%), 359.0.1 [M( 3 H3)+H] + (90%).

[0074] Example 3 H]2 6-Chloro-5-(2-fluoro-5-hydroxy-phenyl)-7-methyl-1-([ 3 H3]methyl)-3H-1,4-benzodiazepin-2-one [ka]

[0075] a) 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1-([ 3 H3]methyl)-3H-1,4-benzodiazepin-2-one [ 3 To [H3]methyl nosylate (1.85 GBq, 50 mCi, 0.61 μmol) was added a solution of 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one (Building B) (417 μg, 1.25 μmol, 2.0 equiv.) in THF (120 μL). A 0.5 M solution of sodium tert-butoxide (6.3 μL, 3.1 μmol, 5 equiv.) in THF was then added and the reaction mixture was stirred at room temperature for 130 min. The reaction was quenched by the addition of water (20 μl). The solvent was evaporated under a stream of argon and the remaining solid was dissolved in MeCN / H2O 1:1 (160 μL). The crude product was purified by HPLC (Sunfire C18 OBD, 4.6 × 250 mm, MeCN[A] / HO+5% MeCN[B], gradient: 1–18 min 10:90 to 90:10 [A]:[B], 18.0–18.1 min 90:10 to 95:5, 21.0–21.1 min 95:5 to 10:90, run time 24 min, flow rate 1 mL / min, 236 nm, oven temperature 40 °C; 5 injections). Pure fractions were combined, frozen, and lyophilized under vacuum for 2 h. The pure tritium-labeled compound (1040 MBq, 28.1 mCi) was dissolved and stored in ethanol (10 mL) until further use. Radiochemical purity >99% was determined by radio-HPLC.

[0076] b) 6-chloro-5-(2-fluoro-5-hydroxy-phenyl)-7-methyl-1-([ 3 H3]methyl)-3H-1,4-benzodiazepin-2-one 6-Chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1-([ 3 A solution of [H3]methyl)-3H-1,4-benzodiazepin-2-one (124 μg, 0.351 μmol, 1 equiv, 28.1 mCi) was concentrated to dryness on a Rotavap at 40 °C. The residue was dissolved in dichloromethane (0.5 mL) and transferred to a 1 mL Alltech tube. The solvent was evaporated under a stream of argon. This was repeated three times with a total of 1.5 mL of dichloromethane. The residue was dissolved in dichloromethane (extra dried, over molecular sieves, 0.15 mL) and treated with a 1 M solution of boron tribromide (3.5 μL, 3.5 μmol, 10 equiv). The tube was closed with a Teflon sealed plastic cap. The solution turned yellow and was stirred at 40 °C (oil bath temperature) for 3 h. Radio-HPLC analysis revealed almost complete consumption of the starting material. The reaction was quenched by adding water (20 μL) at room temperature. The solvent was evaporated under a stream of argon and the remaining solid was dissolved in MeCN / HO 1:1 (200 μL). The crude product was purified by HPLC (Sunfire C18 OBD, 4.6 × 250 mm, MeCN[A] / HO+5%MeCN[B], gradient: 1–18 min 10:90 to 90:10[A]:[B], 18.0–18.1 min 90:10 to 95:5, 21.0–21.1 min 95:5 to 10:90, run time 24 min, flow rate 1 mL / min, 236 nm, oven temperature 40 °C; 5 injections). Pure fractions were combined, frozen, and lyophilized under vacuum for 2 h. The pure tritium-labeled compound (410.7 MBq, 11.1 mCi) was dissolved in ethanol (10 mL) and stored. Radiochemical purity of 97% was determined by radio-HPLC and specific activity of 3.1 TBq / mmol (83 Ci / mmol) was determined by mass spectrometry (MS). The identity of the labeled compound was confirmed by HPLC (by co-injecting an unlabeled reference standard) and MS. MS: m / z=333.1 [M(H)+H]+ (2%), 335.1 [M( 3 H)+H] + (0%), 337.1 [M( 3 H2)+H] + (7%), 339.1 [M( 3 H3)+H] + (91%).

[0077] Example 11 C]1 [ 11 C]6,7-dichloro-5-(2-fluoro-5-hydroxyphenyl)-1-methyl-3H-1,4-benzodiazepin-2-one [ka] The radiochemical synthesis of this tracer was performed using the same procedure as described above, except that the preparative mobile phase flow rate was 15 mL / min. 11 C] 6-chloro-5-(2-fluoro-5-hydroxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one (Example [ 11 The fraction proceeded as [C]2). The preparative retention time of the radiotracer was 6.1 min. Quality control of this radiotracer was performed identically to that described below, except that the mobile phase was 35:65 acetonitrile (MeCN) / TEA buffer (pH 7.2) and the UV wavelength monitored was 254 nm. Comparable radiochemical and chemical purity, specific activity (molar activity), and chemical identity results were obtained.

[0078] Example 11 C]2 [ 11 C]6-Chloro-5-(2-fluoro-5-hydroxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one [ka] The standard gas carbon dioxide target of the General Electric (GE) Medical Systems (GEMS, Uppsala, Sweden) PETTrace cyclotron was filled with high-purity nitrogen containing 0.5% oxygen. The target was irradiated with a proton beam at 60 μA for 25 min to obtain approximately 2–3 Ci (74–111 GBq) of [ 11 C] carbon dioxide was produced. The radioactive gas was released in about 10 minutes. 11 Transfer the CH3 to a GE FXMeI module for synthesis and then for radiosynthesis 11 CH3I was transferred to a suitable hot cell by helium gas. The precursor 5-[5-[tert-butyl(dimethyl)silyl]oxy-2-fluoro-phenyl]-6-chloro-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one (1 ± 0.3 mg) was dissolved in 200 μL of dimethylformamide (DMF) and added to a vial containing potassium carbonate (1 ± 0.3 mg) and then sealed. Prior to the end of bombardment (EOB), the vial was placed into a lead-lined synthesis cell. 11 After trapping CH3I, the vial was heated (80°C) for 3 min. Hydrochloric acid (1 mL) was added to the reaction mixture and the vial was heated (80°C) for 1 min. The reaction solution was diluted with 1 mL of 30% acetonitrile:70% aqueous buffer (57 mM TEA adjusted to pH 7.2 with o-phosphoric acid) and injected onto a semi-preparative HPLC column (XBridge C-18, 10 μm, 10 mm x 150 mm) and eluted with 30% acetonitrile:70% aqueous buffer (57 mM TEA adjusted to pH 7.2 with o-phosphoric acid) at 10 mL / min, and the radioactive content and UV (254 nm) of the eluate were monitored. The product peak (t R= 6.7 min, k' = 5.7) was collected in 50 mL of water. The product solution was eluted onto a conditioned Waters C18 SepPak Plus (Waters Corp.) and the SepPak was washed with HPLC water (10 mL). The radiotracer product was filtered from the SepPak through a 0.2 μm sterile Millipore FG filter (25 mm) with absolute ethanol (1 mL) followed by sterile saline (10 mL) into a sterile product vial prefilled with sterile saline (4 mL). An aliquot was removed from the final product vial for quality control analysis.

[0079] Analytical HPLC was performed to determine radiochemical and chemical purity, specific activity (molar activity), and chemical identity using an XBridge C-18 column (3.5 μm, 4.6 mm×100 mm) eluted with 30:70 acetonitrile (MeCN) / TEA buffer (pH 7.2) at 2 mL / min and monitored at 236 nm. The final radiotracer product exhibited greater than 95% radiochemical purity, with an average yield of product greater than 100 mCi, which co-eluted with an authentic cold reference. The average specific activity (molar activity) was greater than 10 Ci / micromole (370 GBq / micromole).

[0080] Example 3 - Assay Procedure γ1-containing GABA A Membrane preparation and binding assays for subtypes GABA A The affinity of the compounds of formula (I) and (II) at γ1 subunit-containing receptors (see Examples 1 and 2 above) was determined by binding of [ 3 H]RO7239181 (67.3 Ci / mmol; Roche; described, for example, in WO 2021198124) binding. For better protein expression of α2 subunit-containing receptors, human GABA AThe 28 amino acid long signal peptide (Met1~Ala28) of the α2 subunit is expressed in human GABA A It was replaced with a 31 amino acid long signal peptide (Met1~Ser31) of the α5 subunit.

[0081] Different GABA APellets collected from HEK293F cells expressing receptor subtypes were resuspended in mannitol buffer pH 7.2-7.4 (mannitol 0.29 M, triethylamine 10 mM, acetic acid 10 mM, EDTA 1 mM + protease inhibitors (20 tablets of Complete per liter, Roche Diagnostics, Cat. No. 05 056 489 001)), washed twice and then resuspended at a dilution of 1:10-1:15 in the same buffer. Cell disruption was performed by agitating the suspension for 15 min at 435 psi in a Parr vessel #4637, then the suspension was centrifuged at 1000 x g for 15 min at 4 °C (Beckman Avanti J-HC; rotor JS-4.2). The supernatant (S1) was transferred to a 2 l Schott flask and the pellet (P1) was resuspended to 175 ml with mannitol buffer. The resuspended pellet was transferred to a 250 ml Corning centrifuge beaker and centrifuged at 1500×g for 10 min at 4°C (Beckman Avanti J-HC; rotor JS-4.2). The supernatant (S1) was then transferred to a 2 liter Schott flask and the pellet was discarded. The supernatant (S1) was centrifuged in a 500 ml Beckman polypropylene centrifuge beaker at 15'000×g for 30 min at 4°C (Beckman Avanti J-20 XP; rotor JLA-10.500). The pellet (P2) was resuspended in mannitol buffer 1:1 and frozen at -80°C. The supernatant (S2) was centrifuged in a 100 ml Beckman polypropylene centrifuge tube at 48000×g for 50 min at 4°C (Beckman Avanti J-20 XP; rotor JA-18). The supernatant (S3) was discarded and the pellet (P3) was resuspended in 1:1 mannitol buffer. P2 and P3 protein concentrations were determined using the BIORAD Standard assay method with bovine serum albumin as standard and measured on a NANO-Drop1000. The membrane suspension was aliquoted (500 μl / tube) and stored at -80°C until required.

[0082] Membrane homogenates were resuspended and polytronized (Polytron PT1200E Kinematica AG) in 10 mM potassium phosphate, 100 mM KCl binding buffer, pH 7.4, to final assay concentrations determined in previous experiments.

[0083] Radioligand binding assays were performed using 100 μL of cell membranes, 1.5 nM (α5β2γ1) or 20–30 nM (α1β2γ1, α2β2γ1) of [ 3 H]RO7239181, and [0.3–10000] × 10 -9 The experiments were carried out in a volume of 200 μL (96-well plate) containing test compounds in the range of M. Non-specific binding was determined to be 10×10 -6 (α5β2γ1) and 30×10 -6 M was defined by RO7239181 and typically represented less than 5% (α5β2γ1) and less than 20% (α1β2γ1, α2β2γ1) of total binding. Assays were incubated to equilibrium at 4°C for 1 h, then membranes were filtered onto unifilters (96-well white microplates with bound GF / C filters preincubated for 20-50 min in 0.3% polyethyleneimine) using a Filtermate 196 harvester (Packard BioScience) and washed four times with cold potassium phosphate 10 mM pH 7.4, KCl 100 mM binding buffer. After dehydration, filter-retained radioactivity was detected by liquid scintillation counting. K i Values ​​were calculated using Excel-Fit (Microsoft) and are the average of duplicate determinations.

[0084] The compounds of the accompanying examples were tested in the above assay, and preferred compounds had a GABA receptor activity of 100 nM or less. A [from γ1 subunit-containing receptors (e.g., α5β2γ1, α2β2γ1, α1β2γ1) 3 H]K for displacement of RO7239181 iValues ​​were found to be 0.01 to 0.01. Compounds with Ki (nM) < 50 are most preferred. Representative test results obtained by the above assay measuring binding affinity to HEK293 cells expressing the human (h) receptor are shown in Table 1.

[0085] γ2-containing GABA A Membrane preparation and binding assays for subtypes GABA A The affinity of compounds at γ2 subunit-containing receptors was determined by their affinity to HEK293F cells expressing human (transiently transfected) receptors of the composition α1β3γ2. 3 H]flumazenil (81.1 Ci / mmol; Roche) binding was measured by competition.

[0086] Different GABA A Pellets collected from HEK293F cells expressing the γ2 receptor subtype were resuspended in mannitol buffer pH 7.2–7.4 and incubated with GABA A Cells expressing γ1 subunit-containing receptors were treated as above.

[0087] Radioligand binding assays were performed using 100 μL of cell membranes and 1 nM of [ 3 H]flumazenil and [0.1 10 -3 -10]×10 -6 The assay was performed in a volume of 200 μL (96-well plate) containing test compounds in the range of M. Non-specific binding was determined by 10 -5 M diazepam was defined as binding and was typically less than 5% of total binding. Assays were incubated at equilibrium for 1 h at 4°C and harvested onto GF / C unifilters (Packard) by filtration using a Packard harvester and washing with ice-cold wash buffer (50 mM Tris, pH 7.5). After dehydration, filter-retained radioactivity was detected by liquid scintillation counting. i Values ​​were calculated using Excel-Fit (Microsoft) and are the average of duplicate determinations.

[0088] The compounds of the accompanying examples were tested in the above assay, and preferred compounds exhibited a human GABA receptor activity of 100 nM or greater. A [From the α1β3γ2 subtype of receptor 3 Large K for the substitution of [H]flumazenil i It was found that the value of K i Most preferred are compounds having α1β3γ2 (nM)>300. In a preferred embodiment, the compounds of the present invention are γ2 subunit-containing GABA receptor antagonists. A Compared with γ1 subunit-containing GABA receptors, A In particular, the compounds of the present invention selectively bind to the receptor with a 10-fold or greater "K i α1β3γ2(nM) / K i γ2 / γ1 selectivity ratio defined as “α2β2γ1 (nM)” or “Log[K i α1β3γ2(nM) / K i Representative test results obtained by the above assay measuring binding affinity to HEK293 cells expressing the human (h) receptor are shown in Table 1 below. [Table 1]

[0089] GABA A Functional expression of receptors; Preparation of Xenopus oocytes Xenopus laevis oocytes at maturation stages V-VI were treated with GABA A Oocytes prepared for RNA microinjection were purchased from Ecocyte, Kastrup-Rauxel, Germany, and kept at 20°C in modified Barth's medium (composition in mM: NaCl 88, KCl 1, NaHCO3 2.4, HEPES 10, MgSO4 0.82, CaNO3 0.33, CaCl2 0.33, pH = 7.5) until the experiment.

[0090] Xenopus oocyte microinjection Oocytes were plated in 96-well plates for microinjection using the Roboinject automated device (MultiChannelSystems, Reutlingen, Germany). A Approximately 50 nL of an aqueous solution containing RNA transcripts of the subunits of the receptor subtype was injected into each oocyte. RNA concentrations ranged between 20 and 200 pg / μL / subunit and were adjusted in pilot experiments to determine the RNA concentration of GABA receptor subtypes. A GABA responses of appropriate size and maximal effect were obtained for the reference benzodiazepine positive allosteric modulators (PAMs) flunitrazepam, triazolam and midazolam at the receptor benzodiazepine (BZD) binding site. Oocytes were kept in modified Barth's medium (composition in mM: NaCl 88, KCl 1, NaHCO3 4, HEPES 10, MgSO4 0.82, CaNO3 0.33, CaCl2 0.33, pH = 7.5) at 20 °C until the experiment.

[0091] Electrophysiology Electrophysiological experiments were performed 3–5 days after microinjection of mRNA using a Robocyte instrument (MultiChannelSystems, Reutlingen, Germany). During the experiments, oocytes were always superfused in a solution containing (in mM): NaCl 90, KCl 1, HEPES 5, MgCl2 1, CaCl2 1 (pH 7.4). Oocytes were filled with a solution containing KCl 1 M + K-acetate 1.5 M and impaled by two glass microelectrodes (resistance: 0.5–0.8 MΩ) voltage-clamped at −80 mV. Recordings were performed at room temperature using a Robocyte two-electrode voltage-clamp system (Multichannelsystem). After an initial equilibration period of 1.5 min GABA, the maximum current response (EC 20 ) for 1.5 min. After another rest interval of 2.5 min, GABA was added again to elicit responses of similar amplitude and shape. 0.5 min after the start of this second GABA application, while GABA was still present, its K iTest compounds were added at a concentration approximately 30-fold greater than α2β2γ1.Current traces were recorded at a digitization rate of 10 Hz during, immediately prior to, and after GABA application.

[0092] Each compound and concentration was tested in at least three oocytes. Different oocytes were used for different compound concentrations. The reference PAMs, flunitrazepam, triazolam and midazolam, inhibited the α2β2γ1 GABA receptors. A It enhanced GABA-induced currents in oocytes expressing the receptor subtype by approximately 60%.

[0093] Data analysis For analysis, the digitized current traces of the first and second GABA responses were superimposed and, if necessary, rescaled to equal maximal amplitude. The ratio between the two responses during the time interval of the test compound experiment was calculated point-by-point. The extreme value of the resulting "ratio trace" was designated the "GABA EC 20 Compound efficacy ("fold increase") was expressed as "% modulation of IL-1 expression" (100*(fold increase-1)).

[0094] The results are shown in Table 2. [Table 2]

[0095] Example 4 - Reference Compound Benzodiazepine reference compounds (classical commercially available benzodiazepines) and their structural analogues listed below were compared with GABA A Receptor α1β2γ1 and α2β2γ1 subtypes and GABA A Their affinity for the receptor subtype α1β3γ2 was tested, and the results are shown in Table 3. [ka]

[0096] [Table 3]

[0097] Example 5 - In vitro autoradiography Using coronal brain sections from GABAA γ1 receptor knockout mice (C57BL / 6NTac-Gabrg1) and wild-type controls, 3 H]-(I) and [ 3 Autoradiographic analysis of the β-H]-(II) was performed. Tissue sections (10 μm) were cut on a cryostat, thaw-mounted onto glass microscope slides, and incubated with 0.3 nM of the radioligand ([ 3 H]-(II) and [ 3 The sections were incubated at room temperature for 30 min in incubation buffer (50 mM Tris-HCl, pH 7.4) containing 10 mM EDTA and 10 mM EDTA (molar activities of 83 Ci / mmol and 81 Ci / mmol for [H]-(I), respectively. After incubation, all sections were rinsed three times for 10 min in ice-cold washing buffer (50 mM Tris-HCl, pH 7.4) and immersed three times in distilled water at 4°C. Slide-mounted brain sections were dried for at least 2 h in a ventilated refrigerator and exposed to Fuji Imaging Plates for 5 days. Imaging plates were scanned at 25 μm resolution with a Fujifilm high-resolution plate scanner. Visualization and quantification of autoradiography was performed by the MCID™ image analysis program.

[0098] result Autoradiograms revealed a distributed binding pattern consistent with the expected enriched expression of the GABAA γ1 receptor subtype in limbic regions such as the amygdala (Figure 1). Radioligand binding was clearly reduced in brain slices from GABAA γ1 receptor knockout mice. 3 H]-(II) showed 97% specific binding in the amygdala, whereas [ 3 H]-(I) revealed 65% specific binding.

[0099] Example 6 - In vivo PET scan In vivo 11C]-(I) and [ 11 PET imaging experiments were performed in male papio anubis olive brown baboons to characterize [C]-(II). The final radiotracer product was radiochemically pure (>95%) up to 40 min after the end of synthesis, with a mean final calculated specific activity of >555 GBq / μmol. The PET camera used was a Siemens HRRT with an in-plane field-of-view (FOV) of 30 cm and an axial FOV of 24 cm. Each dynamic PET scan was initiated with an intravenous bolus injection of the radiotracer (~700 MBq) and lasted for 90 min in 3D list mode. A set of volumes of interest (VOIs) of 16 brain regions was defined on the MRI of each individual animal with reference to a standard VOI template. Coregistration parameters were used to transfer the VOIs into PET space, and time-activity curves (TACs) of the brain regions were generated.

[0100] result When injected intravenously in baboons, both PET tracers showed rapid initial brain uptake (peaking at or near 10 min in most regions, but later peaks in lower peak regions) and gradual washout, as seen in Figure 2. 11 C]-(II) is generally 11 C]-(I), suggesting faster entry into the brain. 11 The slower clearance of [C]-(I) suggested slower dissociation or more likely nonspecific binding for this tracer. Overall, both tracer candidates showed good transport across the blood-brain barrier, low nonspecific retention, and appropriate clearance kinetics. Taken together, these properties make both radiotracers promising PET imaging agents for visualization of the GABAA γ1 receptor subtype.

Claims

1. Formula (I) or (II): 【Chemistry 1】 A compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, wherein the compound includes a radiolabel.

2. The aforementioned radioactive label is １１ C and ３ A compound of formula (I) or (II) according to claim 1, selected from H. 【Request Item 3】 【Chemistry 2】 A compound of formula (I) or (II) according to claim 2, selected from the group consisting of a pharmaceutically acceptable salt thereof. 【Request Item 4】 【Chemistry 3】 A compound of formula (I) or (II) according to claim 3, or a pharmaceutically acceptable salt thereof. 【Request Item 5】 【Chemistry 4】 A compound of formula (I) or (II) according to claim 3, or a pharmaceutically acceptable salt thereof. 【Request Item 6】 【Chemistry 5】 A compound of formula (I) or (II) according to claim 3, or a pharmaceutically acceptable salt thereof. 【Request Item 7】 【Transformation 6】 A compound of formula (I) or (II) according to claim 3, or a pharmaceutically acceptable salt thereof.

8. GABA in mammals Ａ A pharmaceutical product comprising a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, for use in a method of imaging diagnosis of γ1, wherein the method is (a) administering a detectable amount of the compound to the mammal, (b) GABA Ａ To detect the compound when it is associated with γ1, Pharmaceuticals, including those listed above.

9. The pharmaceutical product for use according to claim 8, wherein the detection is performed via autoradiography and / or positron emission tomography (PET).

10. GABA Ａ A pharmaceutical product comprising a radiolabeled compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, for use in γ1 occupational research.

11. GABA in the mammalian brain Ａ Use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical for imaging diagnostics of γ1.

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