Difluoromethyl-pyridin-2-yl triazoles

Abstract

The present invention relates to difluoromethyl-pyridin-2-yl triazoles of general formula (I), which are modulators of the GABAA receptor containing the alpha5 subunit, useful in the treatment of central nervous system diseases and other diseases. In addition, the invention relates to a method for preparing a pharmaceutical composition and to a method for producing the compounds according to the invention.

Description

[0001] This application is a divisional application of Chinese patent application No. 202180066477.8, entitled "Difluoromethyl-pyridin-2-yltriazole", filed on August 4, 2021 (PCT application No. PCT / EP2021 / 071755). Technical Field

[0002] This invention relates to difluoromethyl-pyridin-2-yltriazole of general formula (I), which is a GABA containing an α5 subunit. A Receptor modulators. These compounds can be used to treat central nervous system and other diseases. Furthermore, this invention relates to methods for preparing pharmaceutical compositions and methods for manufacturing compounds according to the invention. Background Technology

[0003] It has been shown that GABA A The α5 subunit represents a therapeutic target for treating various diseases and disorders of the central nervous system. GABA has been established as a therapeutic target. A The α5 subunit is linked to various neurological disorders, circadian rhythm disorders, and pain syndromes. There is particular interest in modulating GABA containing the α5 subunit. A Compounds of the receptor are particularly useful candidates for the treatment of cognitive impairment, Alzheimer's disease, schizophrenia, positive, negative and / or cognitive symptoms associated with schizophrenia, cognitive impairment associated with schizophrenia, and cognitive deficits associated with Down syndrome, autism, neurofibromatosis type I, or post-stroke.

[0004] WO 2012 / 062687 A1 (corresponding to EP-2638029-A1) discloses triazole derivatives for the treatment of neurological disorders.

[0005] WO 2020 / 016443 A1 discloses difluoromethyl- Phenyl Triazoles, used as GABA receptor modulators, are employed to treat neurological disorders. The compound contains a difluoromethyl group attached to a phenyl group linked to the triazole ring.

[0006] This invention describes difluoromethyl- Pyridine -2-pyridyltriazole. The compound contains a difluoromethyl group attached to a 2-pyridine ring. Compared to similar compounds described in WO 2020 / 016443, the combination of the 2-pyridyl group and the attached difluoromethyl group surprisingly results in a compound containing an α5 subunit of GABA. AA significantly more effective modulator of the receptor. The modulatory potency of the compound can be measured as the Ki value of the compound in assay A described below. In the case of the present invention, the combination of pyridyl (instead of the phenyl used in phenyltriazole of WO 2020 / 016443) with difluoromethyl at position 4 unexpectedly increases the potency of the claimed compound by at least about 10 times or more overall.

[0007] Efficacy and GABA A The 5R binding constant Ki is related, where the increased potency translates into a lower effective dose of the corresponding compound for disease treatment.

[0008] Purpose of the invention

[0009] Surprisingly, pyridin-2-yl-triazole of general formula (I) was discovered.

[0010]

[0011] in:

[0012] ·Xa and Xb are distinct from each other and represent C or N, and

[0013] R1 is a substituted phenyl group or a 5- or 6-membered heterocyclic ring containing 1, 2, or 3 heteroatoms.

[0014] It is GABA A Excellent negative regulator of 5R (i.e., GABA) A (A negative regulator of the α5 subunit), which has a similar effect to GABA. A The improved properties associated with the 5R binding characteristics mean lower compound doses for disease treatment and minimized side effects. Furthermore, the compounds of this invention exhibit excellent CNS permeability, a low efflux rate from the ventricles, which is essential for drugs to have the desired effect in the CNS, and high metabolic stability.

[0015] Therefore, one aspect of the present invention relates to compounds according to formula (I) or salts thereof, which are GABA. A The negative regulator of 5R.

[0016] Another aspect of the invention relates to compounds according to formula (I) or pharmaceutically acceptable salts thereof, which are GABA. A 5R is a negative regulator that has high GABA content. A 5R combined characteristics.

[0017] Another aspect of the invention relates to compounds according to formula (I) or pharmaceutically acceptable salts thereof, which are GABA. A 5R is a negative regulator that has high GABA content. AIt possesses 5R binding properties, excellent CNS permeability, a low efflux rate from the ventricles, and high metabolic stability.

[0018] In another aspect, the present invention relates to pharmaceutical compositions comprising at least one compound according to formula (I) or a pharmaceutically acceptable salt thereof, optionally and one or more inert carriers and / or diluents.

[0019] Another aspect of the present invention relates to a method for manufacturing the compounds of the present invention.

[0020] In particular, the present invention relates to compounds according to formula (I) or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising compounds according to formula (I) or pharmaceutically acceptable salts thereof, for the prevention and / or treatment of possible GABA-induced irritation. A Diseases or conditions negatively influenced by the 5Rs include, but are not limited to, acute neurological disorders, chronic neurological disorders, cognitive impairment, Alzheimer's disease, memory deficits, schizophrenia, positive, negative, and / or cognitive symptoms associated with schizophrenia, cognitive impairment associated with schizophrenia, bipolar disorder, autism, Down syndrome, neurofibromatosis type I, postoperative cognitive decline, sleep disorders, circadian rhythm disorders, amyotrophic lateral sclerosis (ALS), AIDS-related dementia, psychotic disorders, substance-induced psychotic disorders, anxiety disorders, generalized anxiety disorder, panic disorder, paranoid disorder, obsessive-compulsive disorder, acute stress disorder, drug addiction, movement disorders, Parkinson's disease, restless legs syndrome, cognitive deficits, multi-infarct dementia, mood disorders, and depression. Major depressive disorder, neuropsychiatric disorders, psychosis, attention deficit hyperactivity disorder, neuropathic pain, stroke, attention deficit disorder, eating disorders, anorexia, anorexia nervosa, cachexia, weight loss, muscle atrophy, pain disorders, chronic pain, nociceptive pain, postoperative pain, osteoarthritis pain, rheumatoid arthritis pain, musculoskeletal pain, burn pain, eye pain, pain due to inflammation, pain due to fracture, hyperalgesia, neuropathic pain, herpes-related pain, HIV-related neuropathic pain, traumatic nerve injury, recovery after traumatic brain injury, post-stroke pain, post-ischemic pain, fibromyalgia, chronic headache, migraine, tension headache, diabetic neuropathic pain, phantom limb pain, visceral pain, and skin pain.

[0021] Other results or conclusions of the invention will become apparent to those skilled in the art directly from the foregoing and the following comments. Detailed Implementation

[0022] This invention relates to compounds of general formula (I).

[0023]

[0024] or its salt, wherein

[0025] Xa and Xb are not the same as each other and represent C or N, and R1 is a substituted phenyl or a 5- or 6-membered substituted heterocyclic ring containing 1, 2 or 3 heteroatoms.

[0026] Specifically, Xa or Xb is C.

[0027] R1 is preferably selected from

[0028] • Phenyl groups substituted with carbamoyl groups;

[0029] • Unsubstituted 2-pyridone or 2-pyridone substituted with halogens such as fluorine or substituted with methyl or ethyl groups on nitrogen;

[0030] • 2, 3 or 4-pyridyl groups substituted with cyano-(NC-) or methylthioyl-, amino-, methyl-amino-, methanesulfonyl- or halogen;

[0031] • Unsubstituted pyrimidinyl group - or C 1-6 -alkyl-, amino-, -hydroxymethyl-substituted pyrimidinyl-; or pyrazinyl- or pyridazinyl;

[0032] • Be C 1-6 -alkyl- or NC-CH2-CH2-substituted pyrrole- and C-substituted 1-6 -alkyl-, C 3-5 -cycloalkyl-, NC-CH2-CH2-, amino-, methyl-amino- or halogen-substituted pyrazolyl-;

[0033] • Be C 1-6 -alkyl-, carbamoyl-, NC-CH2-CH2-, amino- or -methylamino-substituted imidazolyl-;

[0034] • Unsubstituted triazole group - or C 1-3 -alkyl-such as methyl-substituted triazolyl-;

[0035] • Oxazolyl- substituted with methyl group and thiophene- substituted with NC-CH2-CH2- group.

[0036] Unless otherwise stated, groups, residues, and substituents (especially R1) are as defined above and below. If a residue, substituent, or group appears multiple times in a compound, they may have the same or different meanings. Some preferred meanings of groups and substituents in compounds according to the invention will be given below.

[0037] In yet another embodiment of the invention, R1 is selected from...

[0038] • Phenyl groups substituted with carbamoyl groups, such as

[0039]

[0040] as well as

[0041] • Unsubstituted 2-pyridones or 2-pyridones substituted with halogens such as fluorine or substituted with methyl or ethyl groups on nitrogen, such as

[0042]

[0043] In yet another embodiment of the invention, R1 is

[0044] • 2, 3, or 4-pyridyl groups substituted with NC- or -methylthioyl, amino-, methyl-amino-, or -methylsulfonyl or halogen, such as

[0045]

[0046]

[0047] In yet another embodiment of the invention, R1 is selected from...

[0048] In yet another embodiment of the invention, R1 is

[0049] • Unsubstituted pyrimidinyl group - or C 1-6 -alkyl-, amino-, -hydroxymethyl-substituted pyrimidinyl-; or pyrazinyl- or pyridazinyl, such as

[0050]

[0051] In yet another embodiment of the invention, R1 is

[0052] • Be C 1-6 -alkyl- or NC-CH2-CH2-substituted pyrrole- and C-substituted 1-6 -alkyl-, C 3-5 -cycloalkyl-, NC-CH2-CH2-, -amino, -methyl-amino or -halogen-substituted pyrazolyl-, such as

[0053]

[0054]

[0055] In yet another embodiment of the invention, R1 is

[0056] • Be C 1-6 -alkyl-, carbamoyl-, NC-CH2-CH2-, amino- or -methylamino-substituted imidazolyl-, such as

[0057]

[0058]

[0059] In yet another embodiment of the invention, R1 is

[0060] • Unsubstituted triazole group - or C 1-3 -alkyl-substituted triazole-, such as

[0061]

[0062] In yet another embodiment of the invention, R1 is

[0063] • Oxazolyl groups substituted with methyl groups or thiophene groups substituted with NC-CH2-CH2-, such as

[0064]

[0065] Further preferred are the following compounds listed in Table 1:

[0066] Table 1 Compounds (C#) 1 to 56

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] Some of the terms used above and below to describe the compounds according to the invention will now be defined more precisely.

[0088] Terms not explicitly defined herein shall be given the meanings that a person skilled in the art would interpret based on this disclosure and the context. However, as used herein, unless otherwise specified, the following terms shall have the meanings indicated and shall be governed by the following conventions.

[0089] In the following definitions of groups or radicals, the number of carbon atoms is usually specified before the group, for example, C 1-6 -alkyl refers to an alkyl group or radical having 1 to 6 carbon atoms. Typically, in groups such as HO-, H2N-, (O)S-, (O)2S-, NC- (cyano), HOOC-, and F3C-, a skilled craftsman can identify the attachment point to one or more groups in the molecule from the free valence of the group itself. For composite groups containing two or more daughter groups, the last named daughter group is the group attachment point; for example, the substituent "aryl-C" 1-3 -alkyl- means related to C 1-3 -alkyl-bonded aryl, the C 1-3 -Alkyl groups are attached to the core or to the group to which the substituent is attached.

[0090] Generally speaking, the attachment site of a given residue to another group should be variable, that is, any capable atom in the residue with the hydrogen to be replaced can be the attachment site to the attached group, unless otherwise stated.

[0091] When the compounds of this invention are described by chemical name or as structural formula, in any case of inconsistency, the structural formula shall prevail.

[0092] Unless expressly indicated, throughout the specification and appended claims, the given chemical formula or name shall include tautomers and all stereoisomers, optical isomers and geometric isomers (e.g., enantiomers, diastereomers, E / Z isomers, etc.) and their racemates, as well as mixtures of individual enantiomers in different proportions, mixtures of diastereomers, or any mixture of the foregoing forms, wherein such isomers and enantiomers are present, and salts, including pharmaceutically acceptable salts and their solvates, such as hydrates, including solvates of free compounds or solvates of salts of compounds.

[0093] The phrases “pharmaceutically acceptable” or “physiologically acceptable” are used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, to the extent of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio.

[0094] As used herein, a “pharmaceutically acceptable salt” refers to a derivative of the disclosed compound, wherein the parent compound is modified by preparing its acidic or basic salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral salts or organic salts of basic residues such as amines; and basic salts or organic salts of acidic residues such as carboxylic acids. For example, such salts include those derived from benzenesulfonic acid, benzoic acid, citric acid, ethanesulfonic acid, fumaric acid, gentian acid, hydrobromic acid, hydrochloric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, 4-methylbenzenesulfonic acid, phosphoric acid, salicylic acid, succinic acid, sulfuric acid, and tartaric acid.

[0095] Other pharmaceutically acceptable salts can be formed using cations from ammonia, L-arginine, calcium, 2,2'-iminodiethanol, L-lysine, magnesium, N-methyl-D-glucosamine, potassium, sodium, and tris(hydroxymethyl)-aminomethane.

[0096] Pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety using conventional chemical methods. Typically, such salts are prepared by reacting the free acidic or basic form of these compounds with a sufficient amount of a suitable base or acid in water or in an organic diluent (such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, or mixtures thereof). Salts of other acids besides those mentioned above (such as trifluoroacetates, which can be used to purify or isolate the compounds of the present invention) also constitute a part of the present invention.

[0097] As used herein, the term “substituted” means that any one or more hydrogen atoms on a specified atom are selectively substituted from the indicated group, provided that the substitution does not exceed the feasible valence of the specified atom, and that the substitution produces a stable compound.

[0098] As used herein, the term "partially unsaturated" means the presence of one, two, or more, preferably one or two, double bonds in a specified group or portion. Preferably, as used herein, the term "partially unsaturated" does not include groups or portions that are completely unsaturated.

[0099] The term "halogen" usually refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0100] The term "C" alone or in combination with another free radical 1-n -alkyl (where n is an integer from 2 to n) represents an acyclic, saturated, branched, or straight-chain hydrocarbon radical having 1 to n carbon atoms. For example, the term C 1-5 -Alkyl groups include H3C-, H3C-CH2-, H3C-CH2-CH2-, H3C-CH(CH3)-, H3C-CH2-CH2-CH2-, H3C-CH2-CH(CH3)-, H3C-CH(CH3)-CH2-, H3C-C(CH3)2-, H3C-CH2-CH2-CH2-CH2-, H3C-CH2-CH2-CH(CH3)-, H3C-CH2-CH(CH3)-CH2-, H3C-CH(CH3)-CH2-CH2-, H3C-CH2-C(CH3)2-, H3C-C(CH3)2-CH2-, H3C-CH(CH3)-CH(CH3)- and H3C-CH2-CH(CH2CH3)-.

[0101] The term "C" alone or in combination with another free radical 3-n -cycloalkyl (where n is an integer from 4 to n) represents a cyclic, saturated, unbranched hydrocarbon radical having 3 to n carbon atoms. For example, the term C 3-7 -Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0102] Many of the terms given above can be reused in the definition of a formula or group, and in each case have one of the meanings given above independently of each other.

[0103] The compounds according to the invention can be obtained using synthetic methods known in principle. Preferably, the compounds are obtained by the method according to the invention, which will be described in more detail below.

[0104] The general chemical synthetic route used for the compounds disclosed herein is

[0105] Route 1:

[0106]

[0107]

[0108] Route 2:

[0109]

[0110]

[0111] abbreviation:

[0112]

[0113]

[0114] Analytical HPLC methods

[0115] Method A

[0116]

[0117] Analytical column: Sunfire C18 (Waters) 2.5μm; 3.0 x 30mm; column temperature: 60℃

[0118] Method B

[0119]

[0120] Analytical column: Xbridge BEH C18, 2.1 x 30 mm, 1.7 μm; column temperature: 60 °C

[0121] Method C

[0122]

[0123] Analytical column: Sunfire (Waters) 2.5μm; 3.0 x 30mm; column temperature: 60℃

[0124] Method D

[0125]

[0126] Analytical column: XBridge C18 (Waters) 2.5μm; 3.0 x 30mm; column temperature: 60℃

[0127] Method E

[0128]

[0129] Analytical column: Sunfire C18 (Waters) 2.5μm; 3.0 x 30mm; column temperature: 60℃

[0130] Method F

[0131]

[0132] Analytical column: Sunfire C18 (Waters) 2.5μm; 3.0 x 30mm; column temperature: 60℃

[0133] Method G

[0134]

[0135] Analytical column: XBridge C18 (Waters) 2.5μm; 3.0 x 30mm; column temperature: 60℃

[0136] Method H

[0137]

[0138] Analytical column: XBridge C18 (Waters) 2.5 μm; 3.0 x 30 mm; column temperature: 60℃ Method I

[0139]

[0140] Analytical column: XBridge BEH C18 2.1 x 30 mm 1.7 μm; Column temperature: 60℃

[0141] Method J

[0142]

[0143] Analytical column: Sunfire C18 (Waters) 2.5μm; 3.0 x 30mm; column temperature: 60℃

[0144] Method K

[0145]

[0146] Analytical column: Sunfire C18 (Waters) 2.5μm; 3.0 x 30mm; column temperature: 60℃

[0147] Method L

[0148]

[0149] Analytical column: Hypercarb 3.0 x 30 mm 3 μm; Column temperature: 60℃

[0150] Method M

[0151]

[0152] Analytical column: Acquity UPLC 1.8μm C18 (2.1 x 50 mm), Column temperature: 25℃

[0153] Method N

[0154]

[0155] Analytical column: Kinetex XB-C18 2.6μm (4.6 x 50 mm), Column temperature: 25℃

[0156] Preparation of starting compounds I to XVIII

[0157] Example I [3-(5-difluoromethyl-pyridin-2-yl)-5-methyl-3H-[1,2,3]triazol-4-yl]-methanol

[0158]

[0159] 2-Azide-5-difluoromethyl-pyridine (25.7 g, 151 mmol) was stirred in 50 mL of but-2-alkyl-1-ol at 120 °C for 5 days. Excess alcohol was evaporated under vacuum as much as possible. 9.00 g of 3-(5-difluoromethyl-pyridin-2-yl)-5-methyl-3H-[1,2,3]triazol-4-yl]-methanol as a white solid was obtained by column chromatography on silica gel using DCM / acetone (10:1) as eluent.

[0160] 1 H NMR (400MHz, DMSO-d6) δppm 2.32-2.43 (m, 3H) 4.86 (s, 2H) 7.26 (t, J = 56Hz 1H) 8.11 (d, J = 8.59Hz, 1H) 8.31 (s, 1H) 8.84 (d, J = 0.76Hz, 1H).

[0161] Example II 2-Bromo-5-(difluoromethyl)pyridine

[0162]

[0163] Diethylaminosulfur trifluoride (185 mL; 1.40 mol) was added dropwise to a solution of 6-bromopyridine-3-carboxaldehyde (186 g, 1.00 mol) in 1.86 L of dichloromethane at 0 °C. The mixture was stirred at room temperature for 18 h. The reaction mixture was poured into ice and saturated NaHCO3. The aqueous phase was extracted three times with DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (silica gel, hexane / EE (4 / 1)) to give 170 g of product.

[0164] C6H4 BrF2N (M = 208.0 g / mol)

[0165] ESI-MS: 208 [M+H] +

[0166] 1 H NMR (DMSO-d6, 400MHz): δ = 8.65 (d, J = 1.3Hz, 1H), 7.91-8.08 (m, 1H), 7.76-7.92ppm (m, 1H), 7.17 (t, J = 56Hz, 1H).

[0167] Example III: 5-(difluoromethyl)-2-[2-(trimethylsilyl)ethynyl]pyridine

[0168]

[0169] Ethynyltrimethylsilane (160 mL, 1.15 mol) was added dropwise to a mixture of Example II (120 g, 0.58 mol), bis(triphenylphosphine)palladium chloride (II) (20.2 g, 0.03 mol), copper iodide (I) (5.49 g, 0.03 mol), and triethylamine (250 mL, 1.73 mol) in 600 mL of tetrahydrofuran at 0 °C. The resulting mixture was stirred at room temperature for 18 h. The mixture was filtered through diatomaceous earth. The filter cake was washed with EtOAc. The filtrate was washed with water, dried over Na2SO4, filtered, and concentrated. Purification by column chromatography (silica gel, hexane / EE(19 / 1)) yielded 117 g of product.

[0170] C 11 H 13 F2Nsi (M = 225.3 g / mol)

[0171] ESI-MS: 226 [M+H] +

[0172] 1H NMR (DMSO-d6, 400MHz): δ = 8.76 (d, J = 0.8Hz, 1H), 8.01 (dt, J = 8.1, 0.9Hz, 1H), 7.69 (d, J = 8.1Hz, 1H), 7.16 (t, J = 56Hz, 1H) 0.23-0.30ppm (m, 9H).

[0173] Example IV: 5-Difluoromethyl-2-ethynyl-pyridine

[0174]

[0175] Water (15.0 mL, 834 mmol) was added to the solution of Example III (100.0 g, 0.42 mol) in 800 mL of tetrahydrofuran. The resulting solution was cooled to 0 °C, and then 1.0 M tetrabutylammonium fluoride (143 mL; 0.50 mol) in THF was added dropwise. After 1 h, TLC indicated that the reaction was complete. Water was added, and the aqueous layer was extracted three times with diethyl ether. The combined organic layers were dried, filtered, and carefully concentrated. The residue was purified by column chromatography (silica gel, hexane / DCM (1 / 1 to 0 / 1)) to give the product.

[0176] C8H5F2N (M = 153.1 g / mol)

[0177] ESI-MS: 154 [M+H] +

[0178] 1 H NMR (DMSO-d6, 400MHz): δ = 8.77 (d, J = 0.8Hz, 1H), 8.03 (br d, J = 8.1Hz, 1H), 7.72 (d, J = 8.1Hz, 1H), 7.17 (t, J = 56Hz, 1H) 4.50ppm (s, 1H).

[0179] Example V: 5-Difluoromethyl-2-(1-trimethylsilylmethyl-1H-[1,2,3]triazol-4-yl)-pyridine

[0180]

[0181] Copper iodide (I) (10.9 g, 0.06 mol) and N,N-diisopropylethylamine (50.360 mL, 0.29 mol) were added to a solution of Example IV (50.0 g, 0.30 mol) in 1.5 L DMF, followed by dropwise addition of trimethylsilylmethyl azide (50.6 mL, 0.34 mol). The resulting mixture was stirred at room temperature for 24 h. The reaction was quenched by adding water / salt water, followed by the addition of EtOAc, and the mixture was filtered through diatomaceous earth. The filtrate was extracted three times with EtOAc, dried over Na2SO4, filtered, and concentrated. Purification was performed by column chromatography (silica gel, hexane / EE (3 / 1)), followed by grinding the solid with pentane and drying to give 68.0 g of product.

[0182] C 12 H 16 F2N4Si (M = 282.4 g / mol)

[0183] ESI-MS: 283 [M+H] +

[0184] R t (HPLC): 3.52 min (Method M)

[0185] 1 H NMR (DMSO-d6, 400MHz): δ = 8.78 (s, 1H), 8.52 (s, 1H), 8.13-8.20 (m, 1H), 8.05-8.12 (m, 1H), 7.17 (t, J = 56Hz, 1H), 4.10 (s, 2H), 0.11ppm (s, 9H).

[0186] Example VI 5-Difluoromethyl-2-(1-methyl-1H-[1,2,3]triazol-4-yl)-pyridine

[0187]

[0188] Water (11.9 mL, 0.66 mol) was added to a solution of Example V (93.0 g, 0.33 mol) in 1.86 L of tetrahydrofuran. The resulting solution was cooled to 0 °C, and tetrabutylammonium fluoride (395.2 mL, 0.40 mol) was added dropwise. The reaction mixture was stirred at 0 °C for 1.5 h. Water was added and THF was evaporated. The formed precipitate was filtered, washed with water, and dried to give 51.0 g of product.

[0189] C9H8F2N4 (M = 210.1 g / mol)

[0190] ESI-MS: 211 [M+H] +

[0191] 1 H NMR (DMSO-d6, 400MHz): δ = 8.80 (d, J = 1.0Hz, 1H), 8.65 (s, 1H), 8.14-8.19 (m, 1H), 8.03-8.13 (m, 1H), 7.17 (t, J = 56Hz, 1H), 4.13ppm (s, 3H).

[0192] Example VII: 5-(5-difluoromethyl-pyridin-2-yl)-3-methyl-3H-[1,2,3]triazol-4-carboxaldehyde

[0193]

[0194] A 2.5 M solution of n-butyllithium (114.2 mL, 0.29 mol) in hexane was added dropwise to a solution of Example VI (40.0 g, 0.19 mol) in 1.2 L of tetrahydrofuran at -65 °C. The resulting mixture was stirred at this temperature for 1.5 h. Then, N,N-dimethylformamide (147.4 mL, 1.90 mol) was added dropwise, and the reaction mixture was stirred at 0 °C for 30 min. The reaction was quenched by the slow addition of an aqueous solution of NH4Cl. The aqueous layer was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated. Purification was performed by column chromatography (silica gel, DCM / EE (7 / 3 to 5 / 5)), followed by grinding with pentane to give 19.9 g of product.

[0195] C9H8F2N4 (M = 238.2 g / mol)

[0196] ESI-MS: 239 [M+H] +

[0197] R t (HPLC): 2.68 min (Method M)

[0198] 1 H NMR (DMSO-d6, 400MHz): δ = 10.68 (s, 1H), 8.92 (d, J = 0.8Hz, 1H), 8.34 (d, J = 8.3Hz, 1H), 8.22 (dd, J = 8.1, 1.0Hz, 1H), 7.23 (t, J = 56Hz, 1H), 4.29ppm (s, 3H).

[0199] Example VIII 5-(5-difluoromethyl-pyridin-2-yl)-3-methyl-3H-[1,2,3]triazol-4-carboxaldehyde

[0200]

[0201] The product of Example VII (19.9 g, 0.08 mol) was dissolved in 199 mL of methanol and 99.5 mL of tetrahydrofuran. The resulting solution was cooled to 0 °C. Sodium borohydride (6.32 g, 0.17 mol) was then added in portions, and the reaction mixture was stirred at this temperature for 2 h. The reaction was quenched by adding water. MeOH was evaporated, and the resulting precipitate was collected by filtration, washed with water, and dried. The solid was ground with pentane to give 19.4 g of product.

[0202] C9H8F2N4 (M = 240.2 g / mol)

[0203] ESI-MS: 241 [M+H] +

[0204] R t (HPLC): 3.52 min (Method N)

[0205] 1 H NMR (DMSO-d6, 400MHz): δ = 8.83 (s, 1H), 8.22 (d, J = 8.1Hz, 1H), 8.11 (br d, J = 8.1Hz, 1H), 7.18 (t, J = 56Hz, 1H), 5.53 (s, 1H), 5.09 (d, J = 3.8Hz, 2H), 4.11ppm (s, 3H).

[0206] Example IX.1 3-[3-(5-difluoromethyl-pyridin-2-yl)-5-methyl-3H-[1,2,3]triazol-4-ylmethoxy]-6-iodopyridazine

[0207]

[0208] Sodium hydride (1.10 g, 25.0 mmol) and 3,6-diiodopyridazine (5.50 g, 17.0 mmol) were added to Example I (4.00 g, 16.6 mmol) in 50 mL THF. The reaction mixture was stirred overnight at 80 °C. The reaction mixture was evaporated. The residue was quenched with water, and the product was extracted with DCM. The organic phases were combined, dried over MgSO4, and evaporated. The crude product was purified by column chromatography (silica gel, CH / EE(6 / 4)) to give 6.30 g of product.

[0209] C 14 H 11 F 2I N6O (M = 444.2 g / mol)

[0210] ESI-MS: 445 [M+H] +

[0211] R t (HPLC): 0.57 min (Method A)

[0212] 1 H NMR (DMSO-d6, 400MHz): δ = 8.53-8.80 (m, 1H), 8.33 (dt, J = 8.5, 1.1Hz, 1H), 7.96 (d, J = 9.1 Hz, 1H), 7.22 (t, J = 52Hz, 1H), 6.95 (d, J = 9.1Hz, 1H), 5.95 (s, 2H), 2.36-2.45ppm (m, 3H).

[0213] The compounds mentioned in Examples IX.2 to IX.4 below were prepared according to the general procedure of Example IX.1 above.

[0214] Table of compounds in Examples IX.2 to IX.4 :

[0215]

[0216]

[0217] The reaction conditions mentioned in the table below were used in Examples IX.2 to IX.4.

[0218] surface The reaction conditions used in Examples IX.2 to IX.4 above.

[0219]

[0220]

[0221] Example X.1: 1-(6-Chlorpyridazin-3-yl)-1H-pyrazole-4-carboxynitrile

[0222]

[0223] Add 3,6-dichloropyridazine (500 mg, 3.40 mmol) and potassium carbonate (1.40 g, 10.1 mmol) to 5 mL of 4-cyanopyrazole (312 mg, 3.40 mmol) in DMF, and stir the reaction mixture overnight at room temperature. Quench the mixture with ice water and filter the precipitate. Wash the residue with water and dry the separated solid in a vacuum drying oven to give 524 mg of product.

[0224] C8H4ClN5 (M = 205.6 g / mol)

[0225] ESI-MS: 206 [M+H] +

[0226] R t (HPLC): 0.80 min (Method C)

[0227] The following compounds were prepared according to the general procedure described in Example X.1 above:

[0228]

[0229]

[0230] Example XI: 3-Chloro-6-(5-methyl-1H-1,2,4-triazol-1-yl)pyridazine

[0231]

[0232] 16.4 g (113 mmol) of 6-chloro-pyridazin-3-yl)-hydrazine and 15.5 g (136 mmol) of N-[1-dimethylamino-methyl-(E)-ylidene]-acetamide were dissolved in 164 mL of acetic acid. The mixture was placed in a preheated oil bath at 80 °C. The reaction was stirred at this temperature for 30 min (monitored by TLC), and the acetic acid was evaporated under vacuum. The residue was dissolved in EtOAc, and the organic layer was slowly neutralized with NaHCO3 (concentrated aqueous solution). The organic layer was dried over Na2SO4 and the solvent was evaporated. The product was purified on silica gel using EtOAc / hexane (3:1) as the eluent. 10.2 g of 3-chloro-6-(5-methyl-[1,2,4]triazol-1-yl)-pyridazine as a creamy solid was obtained.

[0233] Example XII: methyl 1-(6-chloropyridazine-3-yl)-1H-imidazol-4-carboxylate

[0234]

[0235] 1H-imidazolium-4-carboxylate (5.00 g, 39.6 mmol) was added to sodium hydride (1.74 g, 43.6 mmol) in 50 mL DMF at 0 °C. The reaction mixture was stirred for 30 min. A solution of 3,6-dichloropyridazine (5.90 g, 39.6 mmol) in 30 mL DMF was added to the reaction mixture at 0 °C, and the mixture was stirred for 20 h to reach room temperature. The reaction mixture was quenched with water under ice cooling, and the precipitate was filtered, washed, and dried to give 3.90 g of product.

[0236] C9H7ClN4O2 (M=238.6g / mol)

[0237] ESI-MS: 239 [M+H] +

[0238] 1 H NMR (400MHz, DMSO-d6) δ = 8.74 (d, J = 1.3Hz, 1H), 8.72 (d, J = 1.0Hz, 1H), 8.43 (d, J = 9.3Hz, 1H), 8.25 (d, J = 9.3Hz, 1H), 3.82 (s, 3H).

[0239] Example XIII: 1-(6-Chlorpyridazine-3-yl)-1H-imidazol-4-carboxylic acid

[0240]

[0241] 1 M NaOH (16.0 mL, 16.0 mmol) was added to 100 mL of 1,4-dioxane in Example XII (3.80 g, 15.8 mmol) and the mixture was stirred at room temperature for 18 h. The reaction mixture was quenched with ice and 1 M HCl (16.0 mL, 16.0 mmol). The precipitate was filtered, washed, and dried to yield 3.30 g of product.

[0242] C8H5ClN4O2 (M=224.6g / mol)ESI-MS: 225[M+H] +

[0243] R t (HPLC): 0.58 min (Method C)

[0244] Example XIV 1-(6-Chlorpyridazin-3-yl)-1H-imidazol-4-carboxamide

[0245]

[0246] DIPEA (2.30 mL, 13.4 mmol) and TBTU (1.40 g, 4.50 mmol) were added to 10 mL of DMF in Example VI (1.00 g, 4.50 mmol), and the mixture was stirred at room temperature for 10 min. Ammonium bicarbonate (1.10 g, 13.4 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. The mixture was quenched with ice, and the precipitate was filtered, washed, and dried to give 0.80 g of product.

[0247] C8H6ClN5O (M=223.6g / mol)

[0248] ESI-MS: 224 [M+H] +

[0249] R t (HPLC): 0, 54 min (Method C)

[0250] Example XV: 1-[6-(4-cyano-1H-imidazol-1-yl)pyridazin-3-yl]-1H-imidazol-4-carboxynitrile

[0251]

[0252] Example III (20.5 g, 99.9 mmol) and potassium carbonate (41.4 g, 299 mmol) were added to 100 mL of DMF containing 9.30 g, 99.9 mmol, and the reaction mixture was stirred at 50 °C for 18 h. Then, 5.00 g, 53.7 mmol of 1H-imidazolium-4-carboxynitrile was added, and the mixture was stirred at 50 °C for 3 days.

[0253] The mixture was quenched with water, and the precipitate was filtered and dried to give 25.8 g of product.

[0254] C 12 H6N8 (M=262.2g / mol)ESI-MS: 263[M+H] +

[0255] R t (HPLC): 0.74 min (Method C)

[0256] Example XVI.1: 3-Chloro-6-(4-Chloro-1H-pyrazol-1-yl)pyridazine

[0257]

[0258] 3,6-Dichloropyridazine (500 mg, 3.36 mmol) and cesium carbonate (2.40 g, 7.38 mmol) were added to 5 mL of 4-chloro-1H-pyrazole (688 mg, 7.00 mmol), and the reaction mixture was stirred overnight at room temperature. The mixture was quenched with water, and the precipitate was filtered to give 686 mg of product.

[0259] C8H6N8 (M = 215.0 g / mol)

[0260] ESI-MS: 216 [M+H] +

[0261] R t (HPLC): 0.51 min (Method A)

[0262] The following compounds were prepared according to the general procedure described above (Example XVI.1):

[0263]

[0264]

[0265]

[0266] Example XVII: 6-(pyrazin-2-yl)-2,3-dihydropyridazin-3-one

[0267]

[0268] Acetylpyrazine (3.00 g, 24.6 mmol) was added to 2-oxoacetic acid hydrate (2.26 g, 25.0 mmol) in an aqueous K₂CO₃ solution (6.79 g, 49.0 mmol in 30 mL of water). The mixture was stirred at room temperature for 6 h. Then, acetic acid (12.9 mL, 221 mmol) and hydrazine hydrate (1.42 mL, 29.0 mmol) were added, and the reaction mixture was refluxed for 2 h. The solution was cooled to room temperature and basicized to pH 7 with K₂CO₃. The precipitate was filtered and dried in an oven at 40 °C to obtain 1.29 g of product.

[0269] C8H6N4O (M = 174.1 g / mol)

[0270] ESI-MS: 175 [M+H] +

[0271] R t (HPLC): 0.23 min (Method A)

[0272] Example XVIII: 3-Chloro-6-(pyrazin-2-yl)pyridazine

[0273]

[0274] Example XVII (1.50 g, 6.03 mmol) in POCl3 (5.00 mL, 53.6 mmol) was stirred at 100 °C for 1 h. The reaction mixture was evaporated, and the residue was diluted with DCM on cooling. 10 mL of saturated NaHCO3 solution was added, and this solution was added dropwise to ice-cold saturated NaHCO3 solution with stirring until the solution was neutral. After 30 min, the solution was filtered through diatomaceous earth and extracted with DCM. The organic layer was collected, dried, and the solvent was evaporated. The product was purified by column chromatography (silica gel, CH / EE(1 / 1)) to give 570 mg of product.

[0275] C8H6ClN4 (M = 192.6 g / mol)

[0276] ESI-MS: 193 [M+H] +

[0277] R t (HPLC): 0.33 min (Method A)

[0278] Preparation of the final compound

[0279] Example 1: 5-[6-({1-[5-(difluoromethyl)pyridin-2-yl]-4-methyl-1H-1,2,3-triazol-5-yl}methoxy)pyridazin-3-yl]-1-methyl-1,2-dihydropyridin-2-one]

[0280]

[0281] Under argon atmosphere, 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxane-2-yl)-1,2-dihydropyridin-2-one (159 mg, 0.70 mmol) was added to 1.5 mL of methanol in Example IX.1 (250 mg, 0.60 mmol), 3 mL of 1,4-dioxane, 2 M sodium carbonate aqueous solution (0.60 mL, 1.10 mmol), and Pd-PEPPSI (9.50 mg, 0.01 mmol), and the reaction mixture was stirred at 100 °C for 3 h. The mixture was purified by preparative HPLC to obtain 96.1 mg of product.

[0282] C 20 H 17 F2N7O2 (M = 425.4 g / mol)

[0283] ESI-MS: 426 [M+H] +

[0284] R t (HPLC): 0.58 min (Method D)

[0285] 1 H NMR(400MHz, DMSO-d6)δppm 2.41-2.46(m,3H)3.48-3.57(m,3H)5.96-6.08(m,2H)6.47-6.58(m,1H)7.21(t,J=56Hz 1H)7.23(s,1H)7.95-8.06(m,1H)8.13-8.22(m,2H)8.29-8.38(m,1H)8.51(d,J=2.66Hz,1H)8.65-8.71(m,1H).

[0286] The following compounds were prepared according to the general procedure described in Example 1 above:

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300] For the compounds in the examples, the reaction conditions in Table 2 were used.

[0301] Table 2: Reaction conditions of Examples 1-35

[0302]

[0303]

[0304] Example 36

[0305]

[0306]

[0307] At 0°C, a solution of 2 mol / L sodium tert-amyloxide in Me-THF (83.9 μL, 0.20 mmol) was added dropwise to 2 mL DMSO of Example I (50.0 mg, 0.20 mmol) and Example XIV (46.5 mg, 0.20 mmol). The reaction mixture was stirred overnight at room temperature, then overnight at 50°C, and then stirred at 75°C for 3 days. The mixture was purified by preparative HPLC to obtain 3.20 mg of product.

[0308] C 18 H 15 F2N9O2 (M = 427.4 g / mol)

[0309] ESI-MS: 428 [M+H] +

[0310] R t (HPLC): 0.77 min (Method C)

[0311] The following compounds were prepared according to the general procedure described in Example 36 above:

[0312]

[0313]

[0314] For compounds 37-41 in Examples, the reaction conditions in the table below were used.

[0315] Reaction conditions of compounds 37-41 in Examples surface

[0316]

[0317]

[0318] Example 42: 1-[6-({1-[5-(difluoromethyl)pyridin-2-yl]-4-methyl-1H-1,2,3-triazol-5-yl}methoxy)pyridazin-3-yl]-1H-imidazol-4-carboxynitrile

[0319]

[0320] Cesium carbonate (814 mg, 2.00 mmol) and Example XV (273 mg, 1.00 mol) were added to 5 mL of ACN in Example I (200 mg, 1.00 mmol), and the mixture was stirred overnight at 90 °C. The reaction mixture was quenched with water, and the precipitate was filtered to obtain the crude product. The crude product was purified by silica gel column chromatography (CH / EE) and finally recrystallized in MeOH / EE / ether to give 151 mg of product.

[0321] C 18 H 13 F2N9O (M = 409.3 g / mol)

[0322] ESI-MS: 410 [M+H] +

[0323] R t (HPLC): 0.50 min (Method A)

[0324] 1 H NMR(400MHz,DMSO-d6)δppm 2.34-2.48(m,3H)5.97-6.14(m,2H)7.22(t,J=56Hz,1H)7.50-7.64(m,1H)8.16-8.19(m,1H)8.1 9-8.21(m,1H)8.31-8.43(m,1H)8.67(d,J=1.14Hz,1H)8.73(d,J=1.27Hz,1H)8.90-8.95(m,1H).

[0325] The following compounds 43-47 in the table below were prepared according to the general procedure described in Example 42 above:

[0326] Compounds 43-47 surface

[0327]

[0328]

[0329]

[0330] For compounds 43-47 in Examples, the reaction conditions in the table below were used. The reaction conditions for compounds 43-47 in Examples... surface

[0331]

[0332] Example 48:3-({4-[5-(difluoromethyl)pyridin-2-yl]-1-methyl-1H-1,2,3-triazol-5-yl}methoxy)-6-(5-methyl-1H-1,2,4-triazol-1-yl)pyridazine

[0333]

[0334] Sodium tert-amyl oxide (101 mg, 0.92 mmol) was added to 10 mL of dioxane in Examples VIII (200 mg, 0.83 mmol) and XI (163 mg, 0.83 mmol). The reaction mixture was stirred overnight at 90 °C, and then stirred over the weekend at room temperature. The reaction mixture was diluted with water and the filtrate was precipitated. The crude solid was recrystallized in MeOH to obtain 212 mg of product.

[0335] C 17 H 15 F2N9O (M = 399.4 g / mol)

[0336] ESI-MS: 400 [M+H] +

[0337] R t (HPLC): 0.46 min (Method A)

[0338] The following compounds 49 and 50 were prepared according to the general procedure of Example 48 above:

[0339]

[0340]

[0341] For compounds 49-50 in Examples, the reaction conditions in the table below were used.

[0342] Reaction conditions of compounds 49-50 in Examples surface

[0343]

[0344] Example 51: 3-(4-chloro-1H-pyrazol-1-yl)-6-({4-[5-(difluoromethyl)pyridin-2-yl]-1-methyl-1H-1,2,3-triazol-5-yl}methoxy)pyridazine

[0345]

[0346] Sodium hydride (21.8 mg, 0.50 mmol) was added to 2 mL of DCM containing Example VIII (100 mg, 0.42 mmol) and Example XVI.1 (89.5 mg, 0.42 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was then purified directly by preparative HPLC to obtain 41.6 mg of product.

[0347] C 17 H 13 ClF₂N₈O (M = 418.8 g / mol)

[0348] ESI-MS: 397 [M+H] +

[0349] R t (HPLC): 0.86 min (Method F)

[0350] The following compounds were prepared according to the general procedure described above (Example 51):

[0351]

[0352]

[0353] For compounds 52-53 in Examples, the reaction conditions in the table below were used.

[0354] Reaction conditions of compounds 52-53 in Examples surface

[0355]

[0356] Example 54: 3-({1-[5-(difluoromethyl)pyridin-2-yl]-4-methyl-1H-1,2,3-triazol-5-yl}methoxy)-6-(4-fluoro-1H-pyrazol-1-yl)pyridazine

[0357]

[0358] Under argon atmosphere, 30.0 mg (0.10 mmol) of Example IX.1, copper iodide (I) (5.10 mg, 0.03 mmol), potassium phosphate (57.3 mg, 0.30 mmol), and (1R,2R)-N,N'-dimethyl-1,2-cyclohexanediamine (8.50 μL, 0.05 mmol) were added to 1 mL of dioxane containing 4-fluoro-1H-pyrazole (6.00 mg, 0.10 mmol). Then 100 μL of 25% ammonia was added, and the mixture was stirred for another 15 min. The reaction mixture was filtered through an Alox column and an SPE-thiol column, and then purified by preparative HPLC to obtain 20.2 mg of product.

[0359] C 17 H 13 F3N8O (M = 402.3 g / mol)

[0360] ESI-MS: 403 [M+H] +

[0361] R t (HPLC): 0.84 min (Method F)

[0362] The following compound 55 was prepared according to the general procedure of Example 54 above:

[0363]

[0364] For compound 55 of example, the following reaction conditions were used.

[0365]

[0366] Example 56: 3-({1-[5-(difluoromethyl)pyridin-2-yl]-4-methyl-1H-1,2,3-triazol-5-yl}methoxy)-6-(pyrazin-2-yl)pyridazine

[0367]

[0368] Example IX.2 (53.0 mg, 0.15 mmol), cesium fluoride (46.0 mg, 0.30 mmol), and xphos (26.0 mg, 0.03 mmol) were added to 2 mL of dioxane under argon atmosphere. The reaction mixture was purified by preparative HPLC to obtain 17.0 mg of product.

[0369] C 18 H 14 F2N8O (M = 396.3 g / mol)

[0370] ESI-MS: 397 [M+H] +

[0371] R t (HPLC): 0, 92 min (Method G)

[0372] 1 H NMR(400MHz,DMSO-d6)δppm 2.47(s,3H)6.11(s,2H)7.22(t,J=56Hz,1H)7.35(d,J=9.25Hz,1H)8.20(d,J=8.49Hz,1H)8.32 -8.37(m,1H)8.39(d,J=9.25Hz,1H)8.67(d,J=1.14Hz,1H)8.74-8.84(m,2H)9.55-9.66(m,1H).

[0373] Biological Examples

[0374] Assay A: In vitro inhibition 3 H-Flumazenil ( 3 H-Ro15-1788) combines with human GABA expression A α5β3γ 2s HEK receptor cell

[0375] The benzodiazepine modifier unit can be selectively replaced with an antagonist. 3 H-Flumazenil labeling.

[0376] According to reports, 3 H-flumazenil's affinity for different subunit combinations is shown for α1β2γ2; α2β2γ2; α3β2γ2 and α5β2γ2. 2s The receptors were 1.0 nM, 1.1 nM, 1.5 nM and 0.4 nM, and 107 nM and 90 nM for α4β2γ2 and α6β2γ2 receptors, respectively (see Sieghart; Pharmacol. Rev. 1995 47 181-234).

[0377] Regarding the binding of 3H-flumazenil, the mutation of α5β3γ 2s GABA A The pharmacology of the receptor is similar to that of the wild-type receptor.

[0378] Cell culture and membrane preparation

[0379] Stable expression of recombinant human GABA A α5β3γ 2s HEK-293 cell lines containing the receptor (plasmid H46 / E9 / B10) were seeded in T175 polystyrene flasks or roller flasks (1700 cm²). 2Fisher Scientific CCI-431191) and in Durbecco's Modified Eagle Medium (DMEM) (containing GlutaMAX) TM The cells were cultured at 37°C, 5% CO2, supplemented with 10% fetal bovine serum and one or both of the following antibiotics: hygromycin B (50 pg / ml; γ2 subunit) or G418 (0.5 mg / ml; Ω5 subunit).

[0380] Once the culture reaches confluence, remove the DMEM and wash the cells once in Durbeco phosphate-buffered saline (DPBS) (10 ml for T175 flasks; 50 ml for roller flasks). After approximately 5 min, add DPBS to the culture (10 ml for T175 flasks; 100 ml for roller flasks), and gently shake or tap the flask to detach the cells easily from the surface. Transfer the cell suspension to Falcon tubes and centrifuge at 23,500 x g for 10 min at 2°C. Using an Ultra-Turrax homogenizer, wash the precipitate once in 15 ml Tris-citrate buffer (50 mM, pH 7.1) and centrifuge at 27,000 x g for 10 min at 2°C. Resuspend the washed precipitate in 15 ml Tris-citrate buffer and freeze at -80°C until the day of the binding experiment.

[0381] Measurement

[0382] On the day of the experiment, the cell membrane preparation was thawed and centrifuged at 27,000 x g for 10 min at 2 °C. The precipitate was resuspended in Tris-citrate buffer to 15–50 pg protein / assay using an UltraTurrax homogenizer and then used for binding assays.

[0383] Add 500 μl of cell suspension to 25 μl of test compound solution and 25 μl of... 3 Mix in H-flumazenil (1 nM, final concentration) and incubate at 2 °C for 40 min. Nonspecific binding was determined using clonazepam (1 μM, final concentration).

[0384] All dilutions and assays of the test compound were performed in glass vials / 96-inch plates. The test compound and... 3The solution of H-flumazenil was prepared to 22x the desired final concentration. The compound was dissolved in 100% DMSO (10 mM stock solution), diluted in 48% ethanol-water, and tested in triplicate at consecutive 1:3 or 1:10 dilutions. When screening a large number of compounds, only one concentration of each compound was tested in a single well. Reference compounds were routinely not included, but for each experiment performed, the total binding and non-specific binding were compared with data obtained during assay validation.

[0385] The union may be terminated in the following ways:

[0386] 1) Use a Brandel cell harvester to quickly filter the cells onto a Whatman GF / C glass fiber filter, then wash 5 times with 1 ml of ice-cold buffer; or 2) Use a Tomtec cell harvester to quickly filter the cells onto a UniFilter GF / C glass fiber filter plate, then wash with approximately 5 ml of ice-cold buffer.

[0387] The amount of radiation on the filter is determined using the following method through conventional liquid scintillation counting:

[0388] 1) Tri-Garb TM Counters (PerkinElmer Life and Analytical Sciences), used for individual large filters, or

[0389] 2) Topcount TM Counter (PerkinElmer Life and Analytical Sciences), used for 96-well filter plates. Specific binding is total binding minus non-specific binding.

[0390] calculate

[0391] In calculating IC 50 (make 3 Before H-flumazenil can achieve 50% specific binding inhibition at the test compound concentration (μM), 25%–75% specific binding inhibition must be obtained.

[0392] IC testing of compounds 50 The value is determined based on the following equation:

[0393] B = 100 - (100 * C) n / (IC 50 n +C n ))

[0394] Where B is the percentage of total specific binding; c is the concentration of the test compound; and n is the Hill coefficient. For screening purposes, n is set to 1. The IC50 was calculated from the concentration response curve using a nonlinear regression method with the curve fitting program GraphPad Prism. 50 value.

[0395] The equations of Cheng and Prusoff can be used to obtain IC 50 Calculate the Ki value of the test compound:

[0396] K = IC 50 / (1+L / K d )

[0397] in 3 H-Flumazenil K d It is 0.36 nM, and L is the inhibition assay. 3 The measured concentration of H-flumazenil.

[0398] result

[0399] The potency observed in assay A for compounds 1, 13, 10 and 28 of example WO 2020 / 016433 and compounds 42, 56, 5 and 55 of example of the present invention is shown in the table below.

[0400]

[0401]

[0402]

[0403]

[0404] Determination of B: α5β2γ2GABA A In vitro evaluation of receptor regulation.

[0405] The regulatory efficacy of the compound of formula (I) was determined by electrophysiological recordings in oocytes using a two-electrode voltage-clamp (TEVC) technique. Human GABA was injected into oocytes at a ratio of 3:1:3. A cRNAs of receptor subunits α5, β2, and γ2, and via interaction with the second-maximal EC 5-20GABA concentration (0.5 μM) (referred to as the GABA control) was used in combination to evaluate regulatory efficacy. As a standard, starting from the lowest concentration, the compound was tested at five concentrations (3.16, 0.316, 0.0316, 0.00316, and 0.000316 μM) on each oocyte. The peak current amplitude, after background subtraction, was normalized to the corresponding GABA control current, converted to a percentage change, and the + / - SEM was plotted as a function of increasing compound concentration. The plotted data points were fitted to the empirical Hill equation using nonlinear regression. From this fitting procedure, the maximum efficacy (bottom) and potency (Log EC) were derived. 50 The 95% confidence interval of ).

[0406]

[0407]

[0408] These data show that the compounds of the present invention exhibit strong negative regulation of target binding and GABA receptor function. The data also show that the compounds, particularly compared to compounds known from WO 2020 / 016433, exhibit stronger GABA binding. A The 5R binding aspect exhibits improved properties, implying that the effective dose of compounds used for disease treatment is lower (see also: Ballard, TM et al. (2009). RO4938581, a novel cognitive enhancer acting at GABA). A α5subunit-containing receptors. Psychopharmacology (2009) 202:207-223; J. Pharmacol. Exp. Ther. (2006) 316:1335-1345).

[0409] Efflux was evaluated in Madin-Darby canine kidney (MDCK) cells transfected with the human MDR1 gene to assess brain penetration. (Drug Metabolism and Disposition February 2008,36(2)268-275; DOI: https: / / doi.org / 10.1124 / dmd.107.017434)

[0410] The apparent permeability (PE) of compounds across the MDCK-MDR1 cell monolayer was measured in both apex-to-basal (AB) and base-to-apex (BA) transport directions (pH 7.4, 37°C). AB permeability (PEAB) represents drug absorption from the blood to the brain, and BA permeability (PEBA) represents drug efflux from the brain back into the blood via both passive permeability and active transport mechanisms mediated by efflux and uptake transporters expressed on MDCK-MDR1 cells, primarily by overexpression of human MDR1 P-gp. Compounds were classified into permeability / absorption categories by comparing their AB permeability to that of reference compounds with known in vitro permeability and oral absorption in humans. Permeability that is the same or similar in both transport directions indicates passive permeability, while vector permeability points to an additional active transport mechanism. A PEBA higher than PEAB indicates the involvement of active efflux mediated by MDR1 P-gp. Active transport was concentration-dependent and saturated.

[0411] MDCK-MDR1 cells (1-2 x 10e5 cells / cm²) were seeded onto filter inserts (Costartranswell polycarbonate or PET filters, 0.4 μm pore size) and cultured (DMEM) for 7 days. MDR1 expression was then enhanced by culturing cells in complete medium with 5 mM sodium butyrate for 2 days. The compounds were dissolved in a suitable solvent (e.g., DMSO, 1-20 mM stock solution). The stock solution was diluted with HTP-4 buffer (128.13 mM NaCl, 5.36 mM KCl, 1 mM MgSO4, 1.8 mM CaCl2, 4.17 mM NaHCO3, 1.19 mM Na2HPO4 x 7H2O, 0.41 mM NaH2PO4 xH2O, 15 mM HEPES, 20 mM glucose, 0.25% BSA, pH 7.4) to prepare transport solutions (0.1–300 μM compound, final DMSO <= 0.5%). The transport solutions (TL) were applied to the donor side (top or base side) to measure AB or BA permeability (3 filters replicate). The receiver side contained the same buffer as the donor side. Samples were collected from the donor side at the beginning and end of the experiment, and from the receiver side at different time intervals for up to 2 hours for concentration measurements by HPLC-MS / MS or scintillation counting. The sampled receiver volume was replaced with fresh receiver solution.

[0412]

[0413] These data show that the compounds of this invention possess excellent brain penetration properties and a low efflux rate from the ventricles. Metabolic stability in human liver microsomes (human MSTs) was evaluated.

[0414] The metabolic stability of the compounds according to the present invention can be studied as follows:

[0415] Metabolic degradation of the test compound was determined using pooled human liver microsomes at 37 °C. A final incubation volume / time point of 100 μL contained TRIS buffer pH 7.6 (at room temperature) (0.1 M), MgCl2 (5 mM), microsomal protein (1 mg / mL), and the test compound at a final concentration of 1 μM. The reaction was initiated by adding the reduced form of β-nicotinamide adenine dinucleotide phosphate (NADPH, 1 mM) after a short pre-incubation period at 37 °C and terminated by transferring aliquots to the solvent at different time points. The amount of the parent compound in the aliquots of the supernatant was determined by LCMS / MS after centrifugation (10000 g, 5 min). The half-life (t1 / 2) was determined by the slope of the semi-logarithmic plot of the concentration-time curve.

[0416] Example Human MST t1 / 2[min] Example Human MST t1 / 2>[min] 1 >130 28 >130 3 >130 29 97 4 40 30 >130 5 83 31 127 6 116 32 78 7 >130 33 98 8 >130 35 >130 9 101 37 129 17 >130 39 >130 18 >130 48 >130 20 >130 49 >130 22 >130 50 130 23 >130 52 >130 24 62 53 >130 25 >130 56 >130

[0417] Given its regulation of GABA containing the α5 subunit AThe ability to activate receptors and their favorable pharmacokinetic properties, compounds of general formula (I) according to the invention, or physiologically acceptable salts thereof, are suitable for the treatment and / or prophylactic treatment of all diseases or conditions that may be affected by the regulation of GABAA receptors containing α5 subunits. Therefore, compounds according to the invention, including their physiologically acceptable salts, are particularly suitable for the prevention or treatment of diseases, especially acute neurological disorders, chronic neurological disorders, cognitive impairment, Alzheimer's disease, memory deficits, schizophrenia, positive, negative and / or cognitive symptoms associated with schizophrenia, cognitive impairment associated with schizophrenia, bipolar disorder, autism, Down syndrome, neurofibromatosis type I, postoperative cognitive decline, sleep disorders, circadian rhythm disorders, amyotrophic lateral sclerosis (ALS), AIDS-related dementia, psychotic disorders, substance-induced psychotic disorders, anxiety disorders, generalized anxiety disorder, panic disorder, paranoid disorder, obsessive-compulsive disorder, acute stress disorder, drug addiction, movement disorders, Parkinson's disease, restless legs syndrome, cognitive impairment, and multiple infarctions. Dementia, mood disorders, depression, major depressive disorder, neuropsychiatric disorders, psychosis, attention deficit hyperactivity disorder, neuropathic pain, stroke, attention deficit disorder, eating disorders, anorexia, anorexia nervosa, cachexia, weight loss, muscle atrophy, pain disorders, chronic pain, nociceptive pain, postoperative pain, osteoarthritis pain, rheumatoid arthritis pain, musculoskeletal pain, burn pain, eye pain, pain due to inflammation, pain due to fracture, hyperalgesia, neuropathic pain, herpes-related pain, HIV-related neuropathic pain, traumatic nerve injury, recovery after traumatic brain injury, post-stroke pain, post-ischemic pain, fibromyalgia, chronic headache, migraine, tension headache, diabetic neuropathic pain, phantom limb pain, visceral pain, and skin pain.

[0418] The compounds according to the invention, including their physiologically acceptable salts, are even more suitable for treating, in particular, cognitive impairment, postoperative cognitive decline, Alzheimer's disease, schizophrenia, positive, negative and / or cognitive symptoms associated with schizophrenia, cognitive impairment associated with schizophrenia, cognitive deficits associated with Down syndrome, cognitive deficits associated with autism, cognitive deficits associated with neurofibromatosis type I, or post-stroke cognitive deficits.

[0419] In another aspect of the invention, the invention relates to a method for treating or preventing the aforementioned diseases and symptoms, the method comprising administering to a human an effective amount of a compound of general formula (I) or a pharmaceutically acceptable salt thereof.

[0420] The daily dose range of compounds of general formula (I) that can be applied is generally from 0.1 to 1000 mg, preferably from 1 to 500 mg, administered orally 1 to 4 times daily in each case.

[0421] Each dosage unit can conveniently contain from 0.1 to 500 mg, preferably 1 to 100 mg.

[0422] The actual pharmaceutically effective dose or therapeutic dose will, of course, depend on factors known to those skilled in the art, such as the patient's age and weight, route of administration, and severity of disease. In any case, the dosage and manner of administration of the combination will allow for the delivery of a pharmaceutically effective dose based on the patient's unique condition.

[0423] Suitable formulations for use with the compound of formula (I) (including its pharmaceutically acceptable salts) will be apparent to those skilled in the art, and include, for example, tablets, pills, capsules, suppositories, lozenges, sugar tablets, solutions, syrups, elixirs, capsules, injections, inhalers, powders, etc. The content of one or more pharmaceutically active compounds should vary from 0.1 to 95 wt.%, preferably 5.0 to 90 wt.%, of the composition as a whole.

[0424] Suitable tablets can be obtained, for example, by mixing one or more compounds according to Formula I with known excipients, such as inert diluents, carriers, disintegrants, adjuvants, surfactants, binders, and / or lubricants. Tablets may also consist of several layers.

[0425] For this purpose, compounds of formula I prepared according to the present invention can be formulated, optionally together with other active substances, with one or more inert conventional carriers and / or diluents, such as corn starch, lactose, glucose, microcrystalline cellulose, magnesium stearate, citric acid, tartaric acid, water, polyvinylpyrrolidone, water / ethanol, water / glycerol, water / sorbitol, water / polyethylene glycol, propylene glycol, cetearyl alcohol, carboxymethyl cellulose or fatty substances such as stearates or suitable mixtures thereof.

[0426] The compounds according to the invention can also be used in combination with other active substances, particularly for the treatment and / or prevention of the aforementioned diseases and conditions. The examples are listed as follows: donepezil, memantine, acetazolamide, carbamazepine, escricazepine acetate, ethosuximide, gabapentin, lacosamide, lamotrigine, levetiracetam, brivaracetam, nitrazepam, oxcarbazepine, piracetam, phenobarbital, phenytoin, pregabalin, primidone, rufenamide, sodium valproate, staefenol, tiagabine, topiramate, vigabatrin, zonisamide, levodopa, carbidopa, haloperidol, loxapine, thioridazine, morpholinone, meperidine, hydroxypromethazine, mesoridazine, trifluoperazine, perphenazine, chlorpromazine, aripiprazole, asenapine maleate, clozapine, ipraridone, lurasidone, olanzapine, paliperidone, quetiapine, risperidone, ziprasidone, and zolpidem.

[0427] The dosage of the above-mentioned combination of partners is usually 1 / 5 to 1 / 1 of the normally recommended minimum dose.

[0428] Therefore, in another aspect, the present invention relates to the use of a compound according to the invention, or a pharmaceutically acceptable salt thereof, in combination with at least one of the above-described active substances as a combined partner, for the preparation of a pharmaceutical composition suitable for treating or preventing the above-described diseases or conditions.

[0429] The use of compounds according to the invention in combination with another active substance can be performed simultaneously or at staggered times, but particularly within short time intervals. If they are administered simultaneously, both active substances are given to the patient together; while if they are used at staggered times, both active substances are given to the patient over a period of less than or equal to 12 hours, particularly less than or equal to 6 hours.

[0430] Therefore, in another aspect, the present invention relates to a pharmaceutical composition comprising a compound according to the invention or a pharmaceutically acceptable salt thereof and at least one of the above-described active substances as a combination partner, optionally together with one or more inert carriers and / or diluents.

[0431] The compounds according to the invention can be present together in a formulation, such as tablets or capsules, or separately in two identical or different formulations, such as in the form of a so-called kit.

Claims

1. A compound having formula (I) or a salt thereof. in: Xa and Xb are distinct from each other and represent C or N, and R1 is C 1-6 -alkyl- or NC-CH2-CH2-substituted pyrrole-; C 1-6 -alkyl-, C 3-5 -cycloalkyl-, NC-CH2-CH2-, amino-, methyl-amino-, or -halogen-substituted pyrazolyl-; or 2. The compound or a salt thereof according to claim 1, wherein R1 is reacted with C 1-6 -alkyl- or NC-CH2-CH2-substituted pyrrole-.

3. The compound or a salt thereof according to claim 1, wherein R1 is 4. The compound or a salt thereof according to claim 1, wherein R1 is reacted with C 1-6 -alkyl-, C 3-5 -cycloalkyl-, NC-CH2-CH2-, amino-, methyl-amino-, or -halogen-substituted pyrazolyl-.

5. The compound or a salt thereof according to claim 1, wherein R1 is 6. The compound according to claim 1 or a salt thereof, selected from:

7. A salt of the compound according to any one of claims 1-6, which is used as a pharmaceutical agent.

8. A pharmaceutical preparation made from a compound or a salt thereof according to any one of claims 1 to 6.

9. A pharmaceutical composition comprising at least one compound according to one or more of claims 1 to 6 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is used to treat or prevent acute neurological disorders, chronic neurological disorders, cognitive impairment, Alzheimer's disease, memory deficits, schizophrenia, positive, negative and / or cognitive symptoms associated with schizophrenia, cognitive impairment associated with schizophrenia, bipolar disorder, autism, Down syndrome, neurofibromatosis type I, postoperative cognitive decline, sleep disorders, circadian rhythm disorders, amyotrophic lateral sclerosis (ALS), AIDS-related dementia, psychotic disorders, substance-induced psychotic disorders, anxiety disorders, generalized anxiety disorder, panic disorder, paranoid disorder, obsessive-compulsive disorder, acute stress disorder, drug addiction, movement disorders, Parkinson's disease, restless legs syndrome, cognitive deficits, and multi-infarct dementia. Mood disorders, depression, major depressive disorder, neuropsychiatric disorders, psychosis, attention deficit hyperactivity disorder, neuropathic pain, stroke, attention deficit disorder, eating disorders, anorexia, anorexia nervosa, cachexia, weight loss, muscle atrophy, pain disorders, chronic pain, nociceptive pain, postoperative pain, osteoarthritis pain, rheumatoid arthritis pain, musculoskeletal pain, burn pain, eye pain, pain due to inflammation, pain due to fracture, hyperalgesia, neuropathic pain, herpes-related pain, HIV-related neuropathic pain, traumatic nerve injury, recovery after traumatic brain injury, post-stroke pain, post-ischemic pain, fibromyalgia, chronic headache, migraine, tension headache, diabetic neuropathic pain, phantom limb pain, visceral pain, and skin pain.

11. The pharmaceutical composition for use according to claim 10, wherein the pharmaceutical composition comprises 0.1 to 1000 mg, preferably 1 to 500 mg, of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in a therapeutically effective amount.

12. The compound of any one or more of claims 1 to 6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 9, for the prevention or treatment of cognitive impairment, postoperative cognitive decline, Alzheimer's disease, schizophrenia, positive, negative and / or cognitive symptoms associated with schizophrenia, cognitive impairment associated with schizophrenia, cognitive deficits associated with Down syndrome, cognitive deficits associated with autism, cognitive deficits associated with neurofibromatosis type I, or post-stroke cognitive deficits.

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