PET TRACERS FOR VISUALIZING GABAA GAMMA1 RECEPTORS

Abstract

The present invention provides radiolabeled GABA.sub.A 1 positive allosteric modulators (PAM) that are useful for medical imaging.

Claims

1. A compound of formula (I) or (II) ##STR00019## or a pharmaceutically acceptable salt thereof, wherein said compound comprises a radiolabel.

2. The compound of formula (I) or (II) according to claim 1, wherein said radiolabel is selected from .sup.11C and .sup.3H.

3. The compound of formula (I) or (II) according to claim 2, selected from the group consisting of ##STR00020## or a pharmaceutically acceptable salt thereof.

4. The compound of formula (I) or (II) according to claim 3, which is ##STR00021## or a pharmaceutically acceptable salt thereof.

5. The compound of formula (I) or (II) according to claim 3, which is ##STR00022## or a pharmaceutically acceptable salt thereof.

6. The compound of formula (I) or (II) according to claim 3, which is ##STR00023## or a pharmaceutically acceptable salt thereof.

7. The compound of formula (I) or (II) according to claim 3, which is ##STR00024## or a pharmaceutically acceptable salt thereof.

8. A method of diagnostic imaging of GABA.sub.A 1 in a mammal, comprising: (a) administering to the mammal a detectable quantity of a radiolabeled compound according to acclaim 1, or of a pharmaceutically acceptable salt thereof, and (b) detecting the radiolabeled compound when associated with GABA.sub.A 1.

9. The method according to claim 8, wherein said detecting is done via autoradiography and/or positron-emission tomography (PET).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 shows in vitro autoradiograms of [.sup.3H]-(I) and [.sup.3H]-(II) in coronal mouse brain sections (refer to Example 5 for details).

[0012] FIG. 2 shows line plots of regional time-activity curves (TACs) of [.sup.11C]-(I) and [.sup.11C]-(II) in baboons (refer to Example 6 for details).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0013] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0014] The term pharmaceutically acceptable salt refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, in particular 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-acetylcystein and the like. In addition, these salts may be prepared by addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, 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.

[0015] The term mammal includes humans, non-human primates such as chimpanzees and other apes and monkey species, farm animals such as cattle, horses, sheep, goats, and swine, domestic animals such as rabbits, dogs, and cats, laboratory animals including rodents, such as rats, mice, and guinea pigs. In certain embodiments, a mammal is a human. The term mammal does not denote a particular age or sex.

[0016] The term radiolabel refers to radioactive isotopes of, e.g., hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine. In some embodiments, the radiolabels used in the context of the invention are useful for PET imaging and/or autoradiography. Examples of isotopes that can be incorporated into the compounds of formula (I) and (II) as radiolabels include .sup.3H, .sup.11C, .sup.14C, .sup.13N, .sup.15O, .sup.18F, and .sup.36Cl, respectively. Preferred radiolabels are .sup.3H, .sup.11C, .sup.13N, .sup.15O, and .sup.18F. Further preferred radiolabels are .sup.3H, .sup.11C and .sup.18F. Particularly preferred radiolabels are .sup.3H and .sup.11C.

Compounds of the Invention

[0017] In a first aspect, the present invention provides a compound of formula (I) or (II)

##STR00002##

or a pharmaceutically acceptable salt thereof, wherein said compound comprises a radiolabel.

[0018] The compounds of formula (I) and (II) of the invention are radiolabeled (i.e., isotopically-labeled) by having one or more atoms therein replaced by isotopes 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, such as, but not limited to .sup.3H, .sup.11C, .sup.14C, .sup.13N, .sup.15O, .sup.18F, and .sup.36Cl, respectively. 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 isotopes tritium, i.e., .sup.3H, carbon-11, i.e., .sup.11C and fluorine-18, i.e., .sup.18F are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. For example, a compound of formula (I) can be enriched with 1, 2, 5, 10, 25, 50, 75, 90, 95, or 99 percent of a given isotope.

[0019] Substitution with positron emitting isotopes, such as .sup.11C, .sup.18F, .sup.15O and .sup.13N, can be useful in Positron Emission Topography (PET) studies, e.g. for examining substrate receptor occupancy.

[0020] Substitution with beta minus radiation emitting isotopes, such as .sup.3H, can be useful in autoradiography studies, e.g. for examining substrate receptor occupancy.

[0021] In one embodiment, said radiolabel is selected from .sup.3H, .sup.11C, .sup.13N, .sup.15O, .sup.18F, and .sup.36Cl.

[0022] In a preferred embodiment, said radiolabel is selected from .sup.11C, .sup.18F and .sup.3H.

[0023] In a preferred embodiment, said radiolabel is selected from .sup.11C and .sup.3H.

[0024] In a particularly preferred embodiment, said radiolabel is .sup.11C.

[0025] In a particularly preferred embodiment, said radiolabel is .sup.3H.

[0026] In a particularly preferred embodiment, said radiolabel is .sup.18F.

[0027] In one embodiment, the compound of formula (I) or (II) of the invention is selected from the group consisting of

##STR00003## [0028] or a pharmaceutically acceptable salt thereof.

[0029] In one embodiment, the compound of formula (I) or (II) of the invention is

##STR00004## [0030] or a pharmaceutically acceptable salt thereof.

[0031] In one embodiment, the compound of formula (I) or (II) of the invention is

##STR00005## [0032] or a pharmaceutically acceptable salt thereof.

[0033] In one embodiment, the compound of formula (I) or (II) of the invention is

##STR00006## [0034] or a pharmaceutically acceptable salt thereof.

[0035] In one embodiment, the compound of formula (I) or (II) of the invention is

##STR00007##

or a pharmaceutically acceptable salt thereof.

Using the Compounds of the Invention

[0036] The radiolabeled compounds of the present invention are potent GABA.sub.A 1 positive allosteric modulators (PAM) that may be used, for example, as PET tracers for the GABA.sub.A 1 receptor to validate target engagement of therapeutic GABA.sub.A 1 modulators, as well as to investigate the function of the GABA.sub.A 1 receptor under normal and disease conditions.

[0037] Thus, in one aspect, the present invention provides a method of diagnostic imaging of GABA.sub.A 1 in a mammal, comprising: [0038] (a) administering to the mammal a detectable quantity of a radiolabeled compound described herein, or of a pharmaceutically acceptable salt thereof; and [0039] (b) detecting the radiolabeled compound when associated with GABA.sub.A 1.

[0040] In a preferred embodiment, said diagnostic imaging is diagnostic imaging of the brain.

[0041] In a preferred embodiment, said detecting is detecting via autoradiography and/or PET.

[0042] In a preferred embodiment, said detecting is detecting via autoradiography.

[0043] In a preferred embodiment, said detecting is detecting via PET.

[0044] In a further aspect, the present invention provides a radiolabeled compound as described herein, or a pharmaceutically acceptable salt thereof, for use in a method of diagnostic imaging described herein.

[0045] In a further aspect, the present invention provides the use of a radiolabeled compound as described herein, or a pharmaceutically acceptable salt thereof, in a method of diagnostic imaging described herein.

[0046] In a further aspect, the present invention provides a radiolabeled compound as described herein, or a pharmaceutically acceptable salt thereof, for use in GABA.sub.A 1 occupancy studies. Such occupancy studies may be conducted, for example, as described in Scientific Reports (2021), 11(1), 7700.

[0047] In a further aspect, the present invention provides the use of the radiolabeled compound disclosed herein in GABA.sub.A 1 occupancy studies.

[0048] In one embodiment, said GABA.sub.A 1 occupancy studies comprise contacting GABA.sub.A 1 with a radiolabeled compound disclosed herein, or a pharmaceutically acceptable salt thereof.

[0049] In a further aspect, the present invention provides a method for studying the occupancy of GABA.sub.A 1 receptors, said method comprising contacting said GABA.sub.A 1 receptors with a radiolabeled compound described herein.

[0050] In one embodiment, said GABA.sub.A 1 occupancy studies are in vitro occupancy studies.

EXAMPLES

[0051] The invention will be more fully understood by reference to the following examples. The claims should not, however, be construed as limited to the scope of the examples.

[0052] All reaction examples and intermediates were prepared under an argon atmosphere if not specified otherwise.

Building Block Syntheses

[0053] The building blocks can be produced according to the following synthetic procedures.

Building Block A

6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one

##STR00008##

a) 5-chloro-2-methyl-3,1-benzoxazin-4-one

[0054] 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 h. The reaction solution was concentrated in vacuo. The residue was suspended in acetonitrile, the solid was filtered and the filter cake was dried in in vacuo to afford the title compound (11.3 g, 66%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 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).

b) N-[3-chloro-2-(2-fluoro-5-methoxy-benzoyl)phenyl]acetamide

[0055] 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 aqueous saturated NH.sub.4Cl and extracted with ethyl acetate. The organic layer was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The residue was purified by preparative HPLC (Phenomenex luna C.sub.18, 10 m, 25050 mm, 0.05% HCl in water/acetonitrile) to afford the title compound (3.63 g, 42%) as a light yellow solid. MS: 322.1 ([{.sup.35Cl}M+H].sup.+), 324.1 ([{.sup.37Cl}M+H].sup.+), ESI pos.

c) (2-amino-6-chloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone

[0056] 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 with saturated aqueous NaHCO.sub.3 and water successively. The organic layer was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo to afford the title compound (2.87 g, 83%) as an off-white solid. MS: 280.0 ([{.sup.35Cl}M+H].sup.+), 282.0 ([{.sup.37Cl}M+H].sup.+), ESI pos.

d) (6-amino-2,3-dichloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone

[0057] 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 (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The residue was purified by preparative HPLC (Phenomenex Synergi C18, 10 m, 15025 mm, 0.1% trifluoroacetic acid in water/acetonitrile) to afford the title compound (367 mg, 33%) as a light yellow solid. MS: 313.9 ([{.sup.35Cl, .sup.35Cl}M+H].sup.+), 315.9 ([{.sup.35Cl, .sup.37Cl}M+H].sup.+), ESI pos.

e) 6.7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one

[0058] 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 m, 15025 mm, 0.1% trifluoroacetic acid in water/acetonitrile) to afford the title compound (280 mg, 38%) as a yellow solid. MS: 353.0 ([{.sup.35Cl, .sup.35Cl}M+H].sup.+), 355.0 ([{.sup.35Cl, .sup.37Cl}M+H].sup.+), ESI pos.

Building Block B

6-chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one

##STR00009##

a) (6-amino-3-bromo-2-chloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone

[0059] In analogy to experiment of Building block A d, (2-amino-6-chloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone (Building block A c) using N-bromosuccinimide instead of N-chlorosuccinimide was converted into the title compound (1.63 g, 64%) which was obtained as a light yellow solid. MS: 357.9 ([{.sup.79Br, .sup.35Cl}M+H].sup.+), 359.9 ([{.sup.81Br, .sup.35Cl or .sup.79Br, .sup.37Cl}M+H].sup.+), ESI pos.

b) 7-bromo-6-chloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one

[0060] In analogy to experiment of Building block A e, (6-amino-3-bromo-2-chloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone was converted into the title compound (1.63 g, 64%) as a light yellow solid (860 mg, 38%) which was obtained as a light yellow solid. MS: 396.9 ([{.sup.79Br, .sup.35Cl}M+H].sup.+), 398.9 ([{.sup.81Br, .sup.35Cl or .sup.79Br, .sup.37Cl}M+H].sup.+), ESI pos.

c) 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one

[0061] 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. for 6 h under nitrogen. The reaction was diluted with methanol, filtered through a plug of Celite and the filtrate was concentrated in vacuo. The residue was treated with water and extracted with ethyl acetate. The organic layer was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The residue was purified by preparative HPLC (Phenomenex Synergi C18, 10 m, 15025 mm, 0.1% trifluoroacetic acid in water/acetonitrile) to afford the title compound (230 mg, 45%) as a light brown solid. MS: 333.1 ([{.sup.79Br, .sup.35Cl}M+H].sup.+), 335.1 ([{.sup.81Br, .sup.35Cl or .sup.79Br, .sup.37Cl }M+H].sup.+), ESI pos.

.SUP.11.C Radiolabeling Precursor 1

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

##STR00010##

a) tert-butyl N-[3,4-dichloro-2-[(2-fluoro-5-methoxy-phenyl)-hydroxy-methyl]phenyl]carbamate

[0062] To a solution of tert-butyl N-(3,4-dichlorophenyl)carbamate (5.82 g, 22.2 mmol) in THF (64 mL) was added dropwise from a dry-ice-cooled dropping-funnel at 90 C. tert-butyllithium, 1.7 M in pentane (28.7 ml, 48.8 mmol) and the resulting mixture was stirred at 85 C. for an additional 0.5 h. Then was added dropwise from a dry-ice-cooled dropping-funnel at 85 to 90 C. a solution of 2-fluoro-5-methoxybenzaldehyde (3.76 g, 24.4 mmol) in THF (16 ml). After stirring at 90 to 85 C. for an additional 0.5 h the mixture was allowed to warm to 65 C. and was then quenched by dropwise addition of saturated aqueous NH.sub.4Cl. 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, the solid was filtered and dried in high vacuum to afford the title compound (4.29 g, 46%) as a white solid. MS: 414.2 ([{.sup.35Cl, .sup.35Cl}MH].sup.), 416.2 ([{.sup.35Cl, .sup.37Cl}MH].sup.), ESI neg.

b) tert-butyl (3,4-dichloro-2-(2-fluoro-5-methoxybenzoyl)phenyl)carbamate

[0063] 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 at 22 C. water (95 ml), potassium bromide (383 mg, 3.22 mmol) and sodium bicarbonate (773 mg, 9.21 mmol) to give two layers that were cooled to 0 C. Then TEMPO (75.5 mg, 483 mol) was added and sodium hypochlorite (21.4 g, 17.8 ml, 34.5 mmol) was added under vigorous stirring dropwise (over 1 h), while internal temperature was kept below 2 C. The mixture was allowed to warm to 22 C. and extracted with DCM (3100 ml). The organic layers were washed with water (1100 ml) and half-sat NaCl (1100 ml), dried over Na.sub.2SO.sub.4, filtered and evaporated. The residue (light brown foam) was treated with EtOAc (50 ml) to give precipitation, the suspension was stirred for 30 minutes and the solid was filtered off, washed with EtOAc (220 ml) and dried to give product (5.602 g, 59%) as white solid. The filtrate was concentrated, adsobed on Isolute sorbent and purified by flash chromatography (silica gel, 330 g, adsorbed on Isolute HM-N, EtOAc in heptane 5% to 10%) to give additional product (3.00 g, 31%) as white solid. 412.2 ([{.sup.35Cl, .sup.35Cl}MH].sup.), 414.2 ([{.sup.35Cl, .sup.37Cl}MH].sup.), ESI neg.

c) (6-amino-2,3-dichloro-phenyl)-(2-fluoro-5-methoxy-phenyl)methanone

[0064] To a solution of tert-butyl N-[3,4-dichloro-2-[(2-fluoro-5-methoxy-phenyl)-hydroxy-methyl]phenyl]carbamate (8.60 g, 20.8 mmol) in DCM (200 mL) was added at 22 C. 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 batch17.9 mmol-scale) was treated with saturated aqueous NaHCO.sub.3 and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo to afford the title compound (11.0 g, 90%) as a yellow solid. MS: 314.0 ([{.sup.35Cl, .sup.35Cl}M+H].sup.+), 316.0 ([{.sup.35Cl, .sup.37Cl}M+H].sup.+), ESI pos.

d) 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one

Building Block A, Alternative Synthesis

[0065] 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 further 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 NaHCO.sub.3 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 afford the title compound (5.47 g, 58%) as a yellow solid. MS: 353.0 ([{.sup.35Cl, .sup.35Cl}M+H].sup.+), 355.0 ([{.sup.35Cl, .sup.37Cl}M+H].sup.+), ESI pos.

e) 6,7-dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one

[0066] To a light 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 dropwise at 65 C. boron tribromide (1.77 g, 7.08 mmol). The mixture was allowed to warm to 20 C. and stirred for 0.5 h. The mixture was quenched with half-saturated aqueous NaHCO.sub.3 and extracted with DCM. The organic layer was washed with half-saturated aqueous NaHCO.sub.3, dried over sodium sulfate, filtered and concentrated in vacuo. The aqueous layer was again extracted with ethyl acetate, 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 afford the title compound (220 mg, 46%) as a light yellow solid. MS: 339.0 ([{.sup.35Cl, .sup.35Cl}M+H].sup.+), 341.0 ([{.sup.35Cl, .sup.37Cl}M+H].sup.+), ESI pos.

f) 5-[5-[tert-butyl(dimethyl)silyl]oxy-2-fluoro-phenyl]-6,7-dichloro-1,3-dihydro-1,4-benzodiazepin-2-one

[0067] To a solution of 6,7-dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one (175 mg, 0.516 mmol) in DMF (1.75 mL) was added at 22 C. imidazole (77.3 mg. 1.14 mmol) 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 with aqueous NaOH (0.1 M) and brine successively, 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 afford the title compound (122 mg, 52%) as a white solid. MS: 453.2 ([{.sup.35Cl, .sup.35Cl}M+H].sup.+), 455.1 ([{.sup.35Cl, .sup.37Cl}M+H].sup.+), ESI pos.

.SUP.11.C Radiolabeling Precursor 2

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

##STR00011##

a) 6-chloro-5-(2-fluoro-5-hydroxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one

[0068] 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) was added at 65 C. 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 NaHCO.sub.3 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 afford the title compound (177 mg, 37%) as a light yellow solid. MS: 319.1 ([{.sup.79Br, .sup.35Cl}M+H].sup.+), 321.1 ([{.sup.81Br, .sup.35Cl or .sup.79Br, .sup.37Cl}M+H].sup.+), ESI pos.

b) 5-[5-[tert-Butyl(dimethyl)silyl]oxy-2-fluoro-phenyl]-6-chloro-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one

[0069] To a solution of 6-chloro-5-(2-fluoro-5-hydroxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one (184 mg, 0.577 mol) in DMF (1.8 mL) was added at 22 C. 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 afford the title compound (110 mg, 44%) as a white solid. MS: 433.2 ([{.sup.79Br, .sup.35Cl}M+H].sup.+), 435.2 ([{.sup.81Br, .sup.35Cl or .sup.79Br, .sup.37Cl}M+H].sup.+), ESI pos.

EXAMPLES

Example 1

6,7-dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1-methyl-3H-1,4-benzodiazepin-2-one (I)

##STR00012##

a) 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1-methyl-3H-1,4-benzodiazepin-2-one

[0070] A solution of 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one (building block 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 m, 15025 mm, 0.1% trifluoroacetic acid in water/acetonitrile) to afford the title compound (114 mg, 91%) as a white solid. MS: 367.1 ([{.sup.35Cl, .sup.35Cl}M+H].sup.+), 369.1 ([{.sup.35Cl, .sup.37Cl}M+H].sup.+), ESI pos.

b) 6,7-dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1-methyl-3H-1,4-benzodiazepin-2-one

[0071] 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 dropwise boron tribromide (273 mg, 1.09 mmol). The reaction mixture was stirred at 0 C. for 1 h, allowed to warm 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 m, 15025 mm, 0.225% formic acid in water/acetonitrile) to afford the title compound (59 mg, 77%) as a white solid. MS: 353.1 ([{.sup.35Cl, .sup.35Cl}M+H].sup.+), 355.1 ([{.sup.35Cl, .sup.37Cl}M+H].sup.+), ESI pos.

Example 2

6-chloro-5-(2-fluoro-5-hydroxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one (II)

##STR00013##

a) 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one

[0072] In analogy to experiment of example 1 a, 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one (building block B) was converted into the title compound (76 mg, 36%) which was obtained as a yellow solid. MS: 347.1 ([{.sup.35Cl}M+H].sup.+), 349.1 ([{.sup.37Cl}M+H].sup.+), ESI pos.

b) 6-chloro-5-(2-fluoro-5-hydroxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one

[0073] In analogy to experiment of example 1 b, 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one was converted into the title compound (72 mg, 83%) which was obtained as an off-white solid. MS: 333.0 ([{.sup.35Cl}M+H].sup.+), 335.0 ([{.sup.37Cl}M+H].sup.+), ESI pos.

Example [.SUP.3.H]1

6,7-Dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1-([.SUP.3.H.SUB.3.]methyl)-3H-1,4-benzodiazepin-2-one

##STR00014##

a) 6,7-Dichloro-5-(2-fluoro-5-methoxy-phenyl)-1-([.SUP.3.H.SUB.3.]methyl)-3H-1,4-benzodiazepin-2-one

[0074] To [.sup.3H.sub.3]methyl nosylate (1.85 GBq, 50 mCi, 0.61 mol) was added a solution of 6,7-dichloro-5-(2-fluoro-5-methoxy-phenyl)-1,3-dihydro-1,4-benzodiazepin-2-one (building block A) (443 g, 1.25 mol, 2.0 equiv.) in THF (120 L). Then a 0.5 M solution of sodium tert-butoxide (6.3 L, 3.1 mol, 5.0 equiv.) in THF was added, and the reaction mixture was stirred for 2 h at room temperature. The reaction was quenched by the addition of water (20 L). The solvent was evaporated under a stream of argon, and the remaining solid was dissolved in MeCN/H.sub.2O 1:1 (180 L). The crude product was purified by HPLC (Sunfire C18 OBD, 4.6250 mm, MeCN [A]/H.sub.2O+5% MeCN [B], gradient: 1-18 min 10:90 to 90:10 [A]:[B], 18.0-18.1 90:10 to 95:5, 21.0 to 21.1 95:5 to 10:90, run time 24 min, flow rate 1 mL/min, 236 nm, oven temperature 40 C.; 5 injections). The 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.

b) 6,7-Dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1-([.SUP.3.H.SUB.3.]methyl)-3H-1,4-benzodiazepin-2-one

[0075] 6,7-Dichloro-5-(2-fluoro-5-methoxy-phenyl)-1-([.sup.3H.sub.3]methyl)-3H-1,4-benzodiazepin-2-one (expected from previous experiment: 131 g, 0.35 mol, 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 in total 1.5 mL of dichloromethane. The residue was dissolved in dichloromethane (extra dry, over molecular sieves, 0.15 mL) and treated with a IM solution of borontribromide (5.3 L, 5.3 mol, 15 equiv.). The tube was closed with a teflon-sealed plastic cap. The solution turned yellow and was stirred for 4 h at 40 C. (oil bath temperature. Radio-HPLC analysis revealed almost complete consumption of the starting material. At room temperature, the reaction was quenched by the addition of water (20 L). The solvent was evaporated under a stream of Argon, and the remaining solid was dissolved in MeCN/H.sub.2O 1:1 (150 L). The crude product was purified by HPLC (Sunfire C18 OBD, 4.6250 mm, MeCN [A]/H.sub.2O+5% MeCN [B], gradient: 1-18 min 10:90 to 90:10 [A]:[B], 18.0-18.1 90:10 to 95:5, 21.0 to 21.1 95:5 to 10:90, run time 24 min, flow rate 1 mL/min, 236 nm, oven temperature 40 C.; 5 injections). The pure fractions were combined, frozen and lyophilized under vacuum for 2 h. The pure tritium-labeled compound (337 MBq, 9.1 mCi) was dissolved and stored in ethanol (10 mL). The radiochemical purity of 96% was determined by radio-HPLC and the specific activity of 3.0 TBq/mmol (81 Ci/mmol) by mass spectrometry (MS). The identity of the labeled compound was confirmed by HPLC (by co-injecting the unlabeled reference standard) and by MS. MS: m/z=353.0 [M(H)+H].sup.+ (3%), 355.0 [M(.sup.3H)+H].sup.+ (0%), 357.0 [M(.sup.3H.sub.2)+H].sup.+ (6%), 359.0.1 [M(.sup.3H.sub.3)+H].sup.+ (90%).

Example [.SUP.3.H]2

6-Chloro-5-(2-fluoro-5-hydroxy-phenyl)-7-methyl-1-([.SUP.3.H.SUB.3.]methyl)-3H-1,4-benzodiazepin-2-one

##STR00015##

a) 6-Chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1-([.SUP.3.H.SUB.3.]methyl)-3H-1,4-benzodiazepin-2-one

[0076] To [.sup.3H.sub.3] methyl nosylate (1.85 GBq, 50 mCi, 0.61 mol) was added a solution of 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one (building block B) (417 g, 1.25 mol, 2.0 equiv.) in THF (120 L). Then a 0.5 M solution of sodium tert-butoxide (6.3 L, 3.1 mol, 5 equiv.) in THF was added, and the reaction mixture was stirred for 130 min at room temperature. The reaction was quenched by the addition of water (20 l). The solvent was evaporated under a stream of argon, and the remaining solid was dissolved in MeCN/H.sub.2O 1:1 (160 L). The crude product was purified by HPLC (Sunfire C18 OBD, 4.6250 mm, MeCN [A]/H.sub.2O+5% MeCN [B], gradient: 1-18 min 10:90 to 90:10 [A]:[B], 18.0-18.1 90:10 to 95:5, 21.0 to 21.1 95:5 to 10:90, run time 24 min, flow rate 1 mL/min, 236 nm, oven temperature 40 C.; 5 injections) The 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. The radiochemical purity of >99% was determined by radio-HPLC.

b) 6-Chloro-5-(2-fluoro-5-hydroxy-phenyl)-7-methyl-1-([.SUP.3.H.SUB.3.]methyl)-3H-1,4-benzodiazepin-2-one

[0077] The solution of 6-chloro-5-(2-fluoro-5-methoxy-phenyl)-7-methyl-1-([.sup.3H.sub.3]methyl)-3H-1,4-benzodiazepin-2-one (124 g, 0.351 mol, 1 equiv., 28.1 mCi) in ethanol (10 mL) was concentrated to dryness at the 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 in total 1.5 mL of dichloromethane. The residue was dissolved in dichloromethane (extra dry, over molecular sieves, 0.15 mL) and treated with a 1M solution of borontribromide (3.5 L, 3.5 mol, 10 equiv.). The tube was closed with a teflon-sealed plastic cap. The solution turned yellow and was stirred for 3 h at 40 C. (oil bath temperature). Radio-HPLC analysis revealed almost complete consumption of the starting material. At room temperature, the reaction was quenched by the addition of water (20 L). The solvent was evaporated under a stream of Argon and the remaining solid dissolved in MeCN/H.sub.2O 1:1 (200 L). The crude product was purified by HPLC (Sunfire C18 OBD, 4.6250 mm, MeCN [A]/H.sub.2O+5% MeCN [B], gradient: 1-18 min 10:90 to 90:10 [A]:[B], 18.0-18.1 90:10 to 95:5, 21.0 to 21.1 95:5 to 10:90, run time 24 min, flow rate 1 mL/min, 236 nm, oven temperature 40 C.; 5 injections) The 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 and stored in ethanol (10 mL). The radiochemical purity of 97% was determined by radio-HPLC and the specific activity of 3.1 TBq/mmol (83 Ci/mmol) by mass spectrometry (MS). The identity of the labeled compound was confirmed by HPLC (by co-injecting the unlabeled reference standard) and by MS. MS: m/z=333.1 [M(H)+H].sup.+ (2%), 335.1 [M(.sup.3H)+H].sup.+ (0%), 337.1 [M(.sup.3H.sub.2)+H].sup.+ (7%), 339.1 [M(.sup.3H.sub.3)+H].sup.+ (91%).

Example [.SUP.11.C]1

[.SUP.11.C]6,7-Dichloro-5-(2-fluoro-5-hydroxy-phenyl)-1-methyl-3H-1,4-benzodiazepin-2-one

##STR00016##

[0078] The radiochemical synthesis of this tracer proceeded as [.sup.11C]6-chloro-5-(2-fluoro-5-hydroxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one (Example [.sup.11C]2) except the preparative mobile phase flow rate was 15 mL/min. The preparative retention time of the radiotracer was 6.1 min. Quality control of this radiotracer was done the same as below except 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.

Example [.SUP.11.C]2

[.SUP.11.C]6-Chloro-5-(2-fluoro-5-hydroxy-phenyl)-1,7-dimethyl-3H-1,4-benzodiazepin-2-one

##STR00017##

[0079] A standard gas carbon dioxide target of a 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 A for 25 min to produce approximately 2-3 Ci (74-111 GBq) of [.sup.11C]carbon dioxide. The radioactive gas was transferred to a GE FXMeI module that synthesizes .sup.11CH.sub.3I in approximately 10 min, after which the .sup.11CH.sub.3I was transferred by helium gas to an appropriate hot cell for radiosynthesis. The precursor 5-[5-[tert-butyl(dimethyl)silyl]oxy-2-fluoro-phenyl]-6-chloro-7-methyl-1,3-dihydro-1,4-benzodiazepin-2-one (10.3 mg) was dissolved in 200 L of dimethylformamide (DMF) and added to a vial containing potassium carbonate (10.3 mg) that was subsequently sealed. Prior to the end of bombardment (EOB), the vial was placed in a lead-lined synthesis cell. After trapping of .sup.11CH.sub.3I, 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 the semipreparative HPLC column (XBridge C-18, 10 m, 10 mm150 mm), eluting with 30% acetonitrile:70% aqueous buffer (57 mM TEA adjusted to pH 7.2 with o-phosphoric acid) at 10 mL/min with the effluent monitored for radioactivity content and UV (254 nm). The product peak (t.sub.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 water HPLC water (10 mL). The radiotracer product was eluted from the SepPak with absolute ethanol (1 mL) followed by sterile saline (10 mL) through a 0.2 m sterile Millipore FG filter (25 mm) into a sterile product vial preloaded with sterile saline (4 mL). Aliquots were removed from the final product vial for quality control analysis.

[0080] 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 m, 4.6 mm100 mm) eluted with 30:70 acetonitrile (MeCN)/TEA buffer (pH 7.2), eluted at 2 mL/min, and monitored at 236 nm. The final radiotracer product demonstrated a radiochemical purity of greater than 95% with an average yield over 100 mCi of product which co-eluted with authentic cold reference. The average specific activity (molar activity) was greater than 10 Ci per micromole (370 GBq per micromole).

Example 3Assay Procedures

Membrane Preparation and Binding Assay for 1-Containing GABA.SUB.A .Subtypes

[0081] The affinity of compounds of formula (I) and (II) (see Examples 1 and 2 above) at GABA.sub.A 1 subunit-containing receptors was measured by competition for [.sup.3H]RO7239181 (67.3 Ci/mmol; Roche; described, e.g., in WO2021198124) binding to membranes from HEK293F cells (ThermoFisher R79007) expressing human (transiently transfected) receptors of composition 521, 221, 121. For better protein expression of the 2 subunit-containing receptors, the 28 amino acid long signal peptide (Met1 to Ala28) of the human GABA.sub.A 2 subunit was substituted by the 31 amino acid long signal peptide (Met1 to Ser31) of human GABA.sub.A 5 subunit.

[0082] Harvested pellets from HEK293F cells expressing the different GABA.sub.A receptor subtypes were resuspended in Mannitol Buffer pH 7.2-7.4 (Mannitol 0.29M, Triethylamine 10 mM, Acetic acid 10 mM, EDTA 1 mM plus protease inhibitors (20 tablets Complete, Roche Diagnostics Cat. No. 05 056 489 001 per liter)), washed two times and then resuspended at 1:10 to 1:15 dilution in the same buffer. Cell disruption was performed by stirring the suspension in a Parr vessel #4637 at 435 psi for 15 minutes, and then the suspensions were centrifuged at 1000g for 15 minutes at 4 C. (Beckman Avanti J-HC; rotor JS-4.2). The supernatant (S1) was transferred in a 2 l Schott flask and the pellet (P1) was resuspended with Mannitol Buffer up to 175 ml. The resuspended pellet was transferred into a 250 ml Corning centrifugal beaker and centrifuged at 1500g for 10 minutes at 4 C. (Beckman Avanti J-HC; rotor JS-4.2). The supernatant (S1) was then transferred in the 2 l Schott flask and the pellet was discarded. The supernatants (S1) were centrifuged in 500 ml Beckman polypropylene centrifugal beaker at 15000g for 30 minutes at 4 C. (Beckman Avanti J-20 XP; rotor JLA-10.500). The pellet (P2) was resuspended with Mannitol Buffer 1:1 and frozen at 80 C. The supernatant (S2) was centrifuged in 100 ml Beckman polypropylene centrifugal tubes at 48000g for 50 minutes at 4 C. (Beckman Avanti J-20 XP; rotor JA-18). The supernatant (S3) was discarded and the pellet (P3) was resuspended with 1:1 Mannitol Buffer. The P2 and P3 protein concentration was determined with the BIORAD Standard assay method with bovine serum albumin as standard and measured on the NANO-Drop 1000. The membrane suspension was aliquots (500 l per tube) and stored at 80 C. until required.

[0083] Membrane homogenates were resuspended and polytronised (Polytron PT1200E Kinematica AG) in Potassium Phosphate 10 mM, KCl 100 mM binding buffer at pH 7.4 to a final assay concentration determined with a previous experiment.

[0084] Radioligand binding assays were carried out in a volume of 200 L (96-well plates) which contained 100 L of cell membranes, [.sup.3H]RO7239181 at a concentration of 1.5 nM (521) or 20-30 nM (121, 221) and the test compound in the range of [0.3-10000]10.sup.9 M. Nonspecific binding was defined by 1010.sup.6 (521) and 3010.sup.6 M RO7239181 and typically represented less than 5% (521) and less than 20% (121, 221) of the total binding. Assays were incubated to equilibrium for 1 hour at 4 C. and then, membranes were filtered onto unifilter (96-well white microplate with bonded GF/C filters preincubated 20-50 minutes in 0.3% Polyethylenimine) with a Filtermate 196 harvester (Packard BioScience) and washed 4 times with cold Potassium Phosphate 10 mM pH 7.4, KCl 100 mM binding buffer. After anhydrousing, filter-retained radioactivity was detected by liquid scintillation counting. K.sub.i values were calculated using Excel-Fit (Microsoft) and are the means of two determinations.

[0085] The compounds of the accompanying examples were tested in the above described assays, and the preferred compounds were found to possess a K.sub.i value for the displacement of [.sup.3H]RO7239181 from GABA.sub.A 1 subunit-containing receptors (e.g. 521, 221, 121) of 100 nM or less. Most preferred are compounds with a K.sub.i (nM)<50. Representative test results, obtained by the above described assay measuring binding affinity to HEK293 cells expressing human (h) receptors, are shown in the Table 1.

Membrane Preparation and Binding Assay for 2-Containing GABA.SUB.A .Subtypes

[0086] The affinity of compounds at GABA.sub.A 2 subunit-containing receptors was measured by competition for [.sup.3H]Flumazenil (81.1 Ci/mmol; Roche) binding to HEK293F cells expressing human (transiently transfected) receptors of composition 132.

[0087] Harvested pellets from HEK293F cells expressing the different GABA.sub.A 2 receptor subtypes were resuspended in Mannitol Buffer pH 7.2-7.4 and processed as described above for the cells expressing the GABA.sub.A 1 subunit-containing receptors.

[0088] Radioligand binding assays were carried out in a volume of 200 L (96-well plates) which contained 100 L of cell membranes, [.sup.3H]Flumazenil at a concentration of 1 nM and the test compound in the range of [0.1.Math.10.sup.310]10.sup.6 M. Nonspecific binding was defined by 10.sup.5 M Diazepam and typically represented less than 5% of the total binding. Assays were incubated to equilibrium for 1 hour at 4 C. and harvested onto GF/C uni-filters (Packard) by filtration using a Packard harvester and washing with ice-cold wash buffer (50 mM Tris; pH 7.5). After anhydrousing, filter-retained radioactivity was detected by liquid scintillation counting. K.sub.i values were calculated using Excel-Fit (Microsoft) and are the means of two determinations.

[0089] The compounds of the accompanying examples were tested in the above described assay, and the preferred compounds were found to possess large K.sub.i value for displacement of [.sup.3H]Flumazenil from the 132 subtype of the human GABA.sub.A receptor of 100 nM or above. Most preferred are compounds with a K.sub.i 132 (nM)>300. In a preferred embodiment the compounds of the invention are binding selectively for the 1 subunit-containing GABA.sub.A receptors relative to 2 subunit-containing GABA.sub.A receptors. In particular, compounds of the present invention have 2/1 selectivity ratio defined as K.sub.i 132 (nM)/K.sub.i 221 (nM) above 10-fold, or LogSel defined as Log[K.sub.i 132 (nM)/K.sub.i 221 (nM)] above 1. Representative test results, obtained by the above described assay measuring binding affinity to HEK293 cells expressing human (h) receptors, are shown in the Table 1 below.

TABLE-US-00001 TABLE 1 Ki h- Ki h- Ki h- Ki h- 2/1 GABA.sub.A GABA.sub.A GABA.sub.A GABA.sub.A Selec- Exam- 521 221 121 132 tivity ple (nM) (nM) (nM) (nM) Ratio LogSel 1 0.34 2.27 2.90 28.7 12.7 1.10 2 0.53 1.49 3.49 58.2 39.0 1.59

Functional Expression of GABA.SUB.A .Receptors

Xenopus Oocytes Preparation

[0090] Xenopus laevis oocytes at maturation stages V-VI were used for the expression of cloned mRNA encoding GABA.sub.A receptor subunits. Oocytes ready for RNA micro-injection were bought from Ecocyte, Castrop-Rauxel, Germany and stored in modified Barth's medium (composition in mM: NaCl 88, KCl 1, NaHCO.sub.3 2.4, HEPES 10, MgSO.sub.4 0.82, CaNO.sub.3 0.33, CaCl.sub.2 0.33, pH=7.5) at 20 C. until the experiment.

Xenopus Oocytes Microinjection

[0091] Oocytes were plated in 96-well plates for microinjection using the Roboinject automated instrument (MultiChannelSystems, Reutlingen, Germany). Approximately 50 nL of an aqueous solution containing the RNA transcripts for the subunits of the desired GABA.sub.A receptor subtype was injected into each oocyte. RNA concentrations ranged between 20 and 200 pg/L/subunit and were adjusted in pilot experiments to obtain GABA responses of a suitable size and a maximal effect of Flunitrazepam, Triazolam and Midazolam, reference benzodiazepine positive allosteric modulators (PAM) at the GABA.sub.A receptor benzodiazepine (BZD) binding site. Oocytes were kept in modified Barth's medium (composition in mM: NaCl 88, KCl 1, NaHCO.sub.3 4, HEPES 10, MgSO.sub.4 0.82, CaNO.sub.3 0.33, CaCl.sub.2 0.33, pH=7.5) at 20 C. until the experiment.

Electrophysiology

[0092] Electrophysiological experiments were performed using the Roboocyte instrument (MultiChannelSystems, Reutlingen, Germany) on days 3 to 5 after the micro-injection of mRNA. During the experiment the oocytes were constantly superfused by a solution containing (in mM) NaCl 90, KCl 1, HEPES 5, MgCl.sub.2 1, CaCl.sub.2 1 (pH 7.4). Oocytes were impaled by two glass microelectrodes (resistance: 0.5-0.8 M) which were filled with a solution containing KCl 1M+K-acetate 1.5 M and voltage-clamped to 80 mV. The recordings were performed at room temperature using the Roboocyte two-electrode voltage clamp system (Multichannelsystem). After an initial equilibration period of 1.5 min GABA was added for 1.5 min at a concentration evoking approximately 20% of a maximal current response (EC.sub.20). After another rest interval of 2.5 min GABA was again added evoking a response of similar amplitude and shape. 0.5 min after the onset of this second GABA application the test compound, at a concentration corresponding to approximatively 30-fold its K.sub.i 221, was added while GABA was still present. Current traces were recorded at a digitization rate of 10 Hz during and shortly before and after the GABA application.

[0093] Each compound and concentration was tested on at least 3 oocytes. Different oocytes were used for different compound concentrations. The reference PAMs, Flunitrazepam, Triazolam and Midazolam, potentiated the GABA-induced current in 221 GABA.sub.A receptor subtype expressing oocytes by approximatively 60%.

Data Analysis

[0094] For the analysis, the digitized current traces of the first and second GABA response were superimposed and, if necessary, rescaled to equal maximal amplitudes. The ratio between the two responses during the time interval of test compound application was calculated point by point. The extremum of the resulting ratio trace was taken as the efficacy (Fold increase) of the compound expressed as % modulation of GABA EC.sub.20 (100*(Fold increase1)).

[0095] The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Ki h-GABA.sub.A Fold increase h-GABA-A Efficacy Example 221 (nM) 221 oocyte @ 30-fold Ki (GABA)% 1 2.27 1.90 90 2 1.49 1.70 70

Example 4Reference Compounds

[0096] Benzodiazepines reference compounds (classical marketed benzodiazepines) and their structural analogues listed below were tested for their affinity towards the GABA.sub.A receptor 121 and 221 subtypes as well as in the GABA.sub.A receptor 132 subtype. The results are shown in Table 3.

##STR00018##

TABLE-US-00003 TABLE 3 Ki Ki Ki h-GABA.sub.A h-GABA.sub.A h-GABA.sub.A 2/1 121 221 132 Selectivity Example (nM) (nM) (nM) Ratio LogSel Alprazolam 5923 3945 19.6 0.0050 2.3 Triazolam 44.2 46.2 1.5 0.032 1.5 Estazolam ND 3182 28.4 0.0089 2.0 Midazolam 1153.2 737.7 5.0 0.0068 2.2 RE-A 6.84 2.79 1.5 0.54 0.27 Example 1 2.90 2.27 28.7 12.7 1.1

Example 5In Vitro Autoradiography

[0097] Autoradiographical analyses of [.sup.3H]-(I) and [.sup.3H]-(II) were performed with coronal brain sections from GABA.sub.A 1 receptor knockout mice (C57BL/6NTac-Gabrg1) and wild-type controls. Tissue sections (10 m) were cut in a cryostat, thaw-mounted on microscope glass slides and incubated in incubation buffer (50 mM Tris-HCl, pH 7.4) containing 0.3 nM radioligand (molar activity 83 Ci/mmol and 81 Ci/mmol for [.sup.3H]-(II) and [.sup.3H]-(I), respectively) at room temperature for 30 minutes. After incubation, all sections were rinsed three times in ice-cold wash buffer (50 mM Tris-HCl, pH 7.4) for 10 minutes and dipped three times in distilled water at 4 C. Slide-mounted brain sections were dried in a ventilated fridge for at least 2 hours and exposed to a Fuji Imaging Plate for 5 days. The imaging plate was scanned at a resolution of 25 m in a Fujifilm high-resolution plate scanner. Visualization and quantification of autoradiographies was performed by the MCID image analysis program.

Results

[0098] Autoradiograms revealed a distribution binding pattern in line with the expected enriched expression of the GABA.sub.A 1 receptor subtype in limbic brain regions such as the amygdala (FIG. 1). Radioligand binding was clearly reduced in brain sections from GABA.sub.A 1 receptor knockout mice. [.sup.3H]-(II) showed 97% specific binding in the amygdala, while [.sup.3H]-(I) revealed 65% specific binding.

Example 6In Vivo PET Scans

[0099] PET imaging experiments were performed in male papio anubis olive brown baboons to examine the characteristics of [.sup.11C]-(I) and [.sup.11C]-(II) in vivo. The final radiotracer product was radiochemically pure (>95%) up to 40 min post its end of synthesis with an average final calculated specific activity of >555 GBq/mol. The PET camera used was the Siemens HRRT with an in-plane field-of-view (FOV) of 30 cm and an axial FOV of 24 cm. Each dynamic PET scan started with an intravenous bolus injection of the radiotracer (approx. 700 MBq) and continued for 90 min in a 3D list mode. A set of volumes of interest (VOIs) for 16 brain regions were defined on MRIs of individual animals referring to a standard VOI template. VOIs were transferred to PET space using the coregistration parameters to generate time-activity curves (TACs) of brain regions.

Results

[0100] Upon intravenous injection in baboons, both PET tracers showed rapid initial brain uptake (peaks before or around 10 min in most regions, but slower peaks in lower peak regions) and gradual washout as visible in FIG. 2. [.sup.11C]-(II) had earlier peaks than [.sup.11C]-(I) in general, suggestive of faster entrance to the brain. Slower clearance of [.sup.11C]-(I) suggested slower dissociation or more likely higher non-specific binding for this tracer. Altogether, both tracer candidates demonstrated good transport across the blood-brain barrier, low non-specific retention, and appropriate clearance kinetics. Collectively, these properties render both radiotracers a promising PET imaging agent for the visualization of the GABA.sub.A 1 receptor subtype.