Pyrazole ring compounds, preparation methods thereof, compositions and applications

Abstract

The present invention provides a compound represented by the general formula (I), its cis-trans isomers, enantiomers, diastereoisomers, racemates, solvates, hydrates, or pharmaceutically acceptable salts and esters thereof, a preparation method thereof, a pharmaceutical composition containing the compound, and the use of the compound as an α5-GABA A receptor modulator, wherein R1, R2, X1, X2, A are as defined in the specification,

Description

Technical Field

[0001] The present invention relates to pyrazole ring compounds having a regulatory function on α5-GABA A receptors, methods for their preparation, pharmaceutical compositions containing them, and their use as drugs. Background Art

[0002] γ-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the mammalian central nervous system. There are two types of GABA A receptors existing in nature. One type is the GABA B receptor, which is a member of the ligand-gated ion channel superfamily, and the other type is the GABA A receptor, which is a member of the G-protein coupled receptor superfamily. The GABA A receptor subunits found in mammals include α1-6, β1-4, γ1-3, δ, ε, θ, and ρ1-3 subunits, etc. Among them, the α subunit, β subunit, and γ subunit are essential for forming a complete functional GABA A receptor, and the α subunit is crucial for the binding of benzodiazepine to the GABA A receptor.

[0003] The GABA A receptor containing α5 (α5-GABA A receptor) accounts for less than 5% of the GABA A receptors in the mammalian brain, has a very low expression level in the cerebral cortex, but accounts for more than 20% of the GABA A receptors in the brain hippocampal tissue, and is hardly expressed in other brain regions. Considering the specific distribution and functional research of the α5-GABA A receptor in the brain hippocampal tissue, many pharmaceutical companies including Roche, Merck, etc. are engaged in the research of α5-GABA A receptor ligands, and a large number of compounds have been synthesized one after another, especially inverse agonists of the GABA A receptor containing the α5 subunit for the brain hippocampal tissue. Among them, α5IA and MRK-016 have shown good therapeutic effects on cognitive diseases in animal disease models. It is generally believed that inverse agonists of the GABA A receptor of the α5 subunit can be used to treat cognitive diseases, especially Alzheimer's disease. Patent application US20110224278 discloses that inverse agonists of the GABA A receptor containing the α5 subunit can be used to treat multi-infarct dementia and stroke-related diseases.

[0004] As the GABA of the α5 subunit AWhen inverse agonists of receptors are used to treat related diseases such as Alzheimer's disease, multi-infarct dementia, and stroke, the compound needs to cross the blood-brain barrier and enter the brain to exert its pharmacological effects. It is reported in the literature (Jones et al., Bioorg Med Chem Lett. 2006, 16(4). 872-875) that the binding of the compound to inhibit ( 3 H)RO-15-1788 (a specific inverse agonist labeled with α5-GABA A receptor) in the brain can be detected. MRK016 can effectively inhibit ( 3 H)RO-15-1788 binding in the central nervous system, while MRK016-M3 can hardly significantly inhibit ( 3 H)RO-15-1788 binding in the central nervous system. MRK016-M3 is considered unable to exert an inverse agonistic function on the GABA A receptor of the α5 subunit in the brain.

[0005] In 2002, the laboratory of Zhang Xu reported that the α5-GABA A receptor is also mainly expressed in small neurons and its expression increases in the nerve transection model (Xiao HS et al., Proc Natl Acad Sci U SA. 2002, 99(12), 8360-8365). Patent application CN103239720 discloses that the α5-GABA A receptor is expressed in the peripheral nervous system and its expression increases significantly in the partial nerve injury model. Moreover, the inverse agonist of the α5-GABA A receptor can exert the effect of inhibiting various pains by selectively binding to the α5-GABA A receptor in the peripheral nervous system. Animal experiment model data show that the stronger the inverse agonistic effect of the inverse agonist, the better its pain inhibitory effect.

[0006] For different disease types, it is necessary to select whether the inverse agonist of the GABA A receptor corresponding to the α5 subunit has blood-brain barrier permeability; the inverse agonist that binds to the α5-GABA A receptor in the peripheral nervous system can exert the effect of inhibiting various pains and avoid side effects on the central nervous system; while the inverse agonist that binds to the α5-GABA A receptor in the central nervous system is used to treat cognitive diseases.

[0007] By AModify the compound structure of the inverse agonist of the receptor to adjust the physicochemical properties of the compound (such as log D, PSA, etc.), so that the efflux transporter (such as Pgp transporter) located at the blood-brain barrier exerts an effect on the compound, control its efflux ratio, and obtain properties such as good blood-brain barrier permeability or inability to pass through the blood-brain barrier for the compound, which can exert effects on different disease types.

[0008] Currently, there are many reports on the inverse agonist of the GABA A receptor of the α5 subunit. Among them, isoxazole compounds represented by RG1662 have high binding selectivity and functional selectivity; patent reports such as WO2009071476, etc.; in addition, phenyltriazole compounds can also act as inverse agonists for the α5-GABA A receptor. Patent reports include WO2012062623, WO2012062687, WO2012076590, WO2014001278, WO2014001279, WO2014001280, WO2014001281, WO2014001282, WO2020016443. Summary of the Invention

[0009] The present invention provides a pyrazole ring compound, a preparation method thereof, a pharmaceutical composition and uses thereof. This type of compound has strong inverse agonist activity, high binding selectivity and good solubility and other pharmaceutical properties for the α5-GABA A receptor.

[0010] The present invention provides a pyrazole ring compound having the following general formula I, its solvate, its hydrate or its pharmaceutically acceptable salt or its precursor compound,

[0011]

[0012] wherein,

[0013] R1 and R2 are each independently selected from hydrogen, halogen, cyano, C 1-6 alkyl, C 3-6 cycloalkyl, and the C 1-6 alkyl and C 3-6 cycloalkyl are optionally substituted by 1-3 R';

[0014] R' are each independently selected from hydrogen, halogen or C 1-3 alkyl;

[0015] X1 and X2 are each independently selected from N or CH;

[0016] A is selected from

[0017] -C(O)NR3R4;

[0018] a 5- or 6-membered heteroaryl;

[0019] a 3- to 6-membered heterocycloalkyl;

[0020] said 5- or 6-membered heteroaryl and 3- to 6-membered heterocycloalkyl may each independently be optionally substituted with 1 to 3 R;

[0021] R is independently selected from hydrogen, oxo, halogen, cyano, hydroxy, amino, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylamino, C 1-6 alkylamino-C(O)-, C 3-6 cycloalkylamino-C(O)-, 3- to 6-membered heterocycloalkylamino-C(O)-, C 3-6 cycloalkyl or 3- to 6-membered heterocycloalkyl, said C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylamino, C 1-6 alkylamino-C(O)-, C 3-6 cycloalkyl, 3- to 6-membered heterocycloalkyl is optionally substituted with 1 to 3 R';

[0022] R3 and / or R4 are independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, 3- to 6-membered heterocycloalkyl, 5- to 10-membered heteroaryl or 5- to 10-membered heteroaryl(C 1-6 )alkyl, which may each independently be optionally substituted with 1 to 3 R; said 5- to 10-membered heteroaryl and 3- to 6-membered heterocycloalkyl may include 1, 2 or 3 heteroatoms selected from N, O or S as ring atoms;

[0023] R3 and R4 together with the attached N atom may form a 4- to 7-membered heterocycle, said 4- to 7-membered heterocycle may include 1, 2 or 3 heteroatoms selected from N, O or S as ring atoms, said 4- to 7-membered heterocycle may each independently be optionally substituted with 1 to 3 R.

[0024] Said R1 is selected from F or -CHF2.

[0025] Said R2 is selected from -Me, Cl or CN.

[0026] Said R3 and / or R4 are independently selected from H, Me, Et, i-Pr or Said R3 and R4 together with the attached N atom may form a morpholine ring or a piperazine ring; which may each independently be optionally substituted with 1 to 3 R.

[0027] The above-mentioned 5- or 6-membered heteroaryl group is selected from imidazolyl, pyrazolyl, pyridyl and pyrimidinyl; the imidazolyl, pyrazolyl, pyridyl and pyrimidinyl groups may each optionally be substituted by 1-3 R groups.

[0028] The above-mentioned 3- to 6-membered heterocycloalkyl group is selected from piperazinyl; the piperazinyl group may each optionally be substituted by 1-3 R groups.

[0029] The above-mentioned R groups are each independently selected from hydrogen, oxo, halogen, cyano, hydroxy, amino, C 1-4 alkyl, C 1-4 alkoxy, C 1-4 alkylamino, C 1-4 alkylamino-C(O)-, C 3-6 cycloalkylamino-C(O)-, 3- to 6-membered oxygen-containing heterocycloalkylamino-C(O)-, C 3-6 nitrogen-containing cycloalkyl or nitrogen-containing fused cycloalkyl, and the 3- to 6-membered nitrogen-containing heterocycloalkylamino-C(O)-, C 3-6 nitrogen-containing cycloalkyl, or nitrogen-containing fused cycloalkyl may optionally be substituted by 1-3 R' groups. In a specific embodiment, R is preferably 5- or 6-membered nitrogen-containing heterocycloalkylamino-C(O)-, C 5-6 nitrogen-containing cycloalkyl or nitrogen-containing fused cycloalkyl or C 5-6 nitrogen-containing heteroaryl.

[0030] The above-mentioned A is selected from

[0031] The above-mentioned pyrazole ring compounds are selected from any of the following compounds:

[0032]

[0033]

[0034]

[0035] The second aspect of the present invention provides a composition comprising any one of the above-mentioned compounds.

[0036] The third aspect of the present invention provides a pharmaceutical composition comprising any one of the above-mentioned compounds and one or more of pharmaceutically acceptable carriers, diluents or excipients.

[0037] The fourth aspect of the present invention provides the use of the above-mentioned compound, or the above-mentioned composition, or the above-mentioned pharmaceutical composition in the preparation of an α5-GABA A receptor modulator.

[0038] The fifth aspect of the present invention provides the use of the above-mentioned compound or the above-mentioned composition in the preparation of a medicament for treating or preventing a disease related to α5-GABA AUse in drugs for receptor-related diseases.

[0039] The sixth aspect of the present invention provides a method for treating or preventing a disease related to the α5-GABA A receptor, that is, administering an effective dose of any of the above-mentioned compounds or the above-mentioned composition or the above-mentioned pharmaceutical composition to a patient.

[0040] The seventh aspect of the present invention provides the use of the above-mentioned compound or the above-mentioned composition in the preparation of drugs for treating or preventing the following diseases: pain, Alzheimer's disease, multi-infarct dementia, and stroke.

[0041] The eighth aspect of the present invention provides a method for treating or preventing pain, Alzheimer's disease, multi-infarct dementia, and stroke, that is, administering an effective dose of the above-mentioned compound or the composition as above or the above-mentioned pharmaceutical composition to a patient.

[0042] The above-mentioned pain is neuropathic pain, inflammatory pain, and cancer pain.

[0043] In a specific implementation, the above-mentioned pain is selected from: headache, facial pain, neck pain, shoulder pain, back pain, chest pain, abdominal pain, back pain, lumbar pain, lower limb pain, muscle and bone pain, vascular pain, gout, arthritis pain, visceral pain, pain caused by infectious diseases (such as AIDS and postherpetic neuralgia), polyostotic pain, pain related to sickle cell anemia, autoimmune diseases, multiple sclerosis or inflammation, chronic pain caused by injury or surgery, nociceptive pain, painful diabetes, trigeminal neuralgia, lumbar or cervical radicular pain, glossopharyngeal neuralgia, autonomic reflex pain, reflex sympathetic dystrophy, nerve root avulsion, cancer, chemical injury, toxin, nutritional deficiency, viral or bacterial infection, pain related to degenerative osteoarthropathy.

[0044] The pain caused by the above-mentioned infectious diseases is AIDS or postherpetic neuralgia.

[0045] The ninth aspect of the present invention provides a method for preparing the above-mentioned compound, and the preparation method is selected from any of the following groups of methods:

[0046]

[0047] R1, R2, X1, X2, A are as defined above; (1)

[0049] a) Generally, compound I-1 and compound I-2 form compound I-3 through a Chan-Lam reaction in a halogenated alkane or ether solvent;

[0050] b) Compound I-3 is reduced in an ether or alcohol solvent to obtain compound I-4;

[0051] c) Compound I-4 reacts with Compound I-6 to form Compound I; or (2)

[0053] a) Generally, Compound I-1 and Compound I-2 form Compound I-3 through the Chan-Lam reaction in a halogenated alkane or ether solvent;

[0054] b) Compound I-3 is reduced in an ether or alcohol solvent to obtain Compound I-4;

[0055] d) Compound I-4 reacts with Compound I-7 to obtain I-5;

[0056] e) I-5 reacts with Compound AH to form Compound I. Detailed implementation manners

[0057] The present application will be described in detail below through examples, but this does not mean any adverse limitations to the present application. The present application has been described in detail herein, and its specific implementation manners have also been disclosed. For those skilled in the art, various changes and improvements made to the specific implementation manners of the present application without departing from the spirit and scope of the present application will be obvious.

[0058] The experimental materials and reagents used in the following examples can be obtained from commercial channels without special instructions. The present invention uses the following abbreviations: ACN represents acetonitrile, CuCN represents cuprous cyanide, Cu(OAc)2 represents copper acetate, DCM represents dichloromethane, DIBAL-H represents diisobutylaluminum hydride, DIEA represents diisopropylethylamine, DMAP represents 4-dimethylaminopyridine, DMF represents N,N-dimethylformamide, DMSO represents dimethyl sulfoxide, EtOAc represents ethyl acetate, HATU represents 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, HPLC represents high performance liquid chromatography, Pd(dppf)Cl2 represents 1,1'-bis(diphenylphosphino)ferrocene dichloropalladium, pyr. represents pyridine, RuPhos represents 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl, Ruphos-Pd-G3 represents palladium(II) (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl) methanesulfonate, THF represents tetrahydrofuran, TEA represents triethylamine, TLC represents thin layer chromatography, LCMS represents liquid chromatography-mass spectrometry, tol. represents toluene, h represents hour, and min represents minute.

[0059] Definition

[0060] Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following definitions. A particular term or phrase should not be considered indefinite or unclear merely because it is not specifically defined, but should be construed according to its ordinary meaning. When a trade name appears in this text, it is intended to refer to the corresponding product or active ingredient.

[0061] As used herein, the term "pharmaceutically acceptable" pertains to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0062] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared from compounds having specific substituents found in the present invention with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with an adequate amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with an adequate amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, where the inorganic acids include, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc., and organic acid salts, where the organic acids include, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid, etc.; also salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can thus be converted into either base or acid addition salts.

[0063] The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid or base groups. Generally, the preparation method of such salts is to react these compounds in the form of free acids or bases with a stoichiometric amount of the appropriate base or acid in water, an organic solvent, or a mixture of both.

[0064] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomer- or diastereoisomer-enriched mixtures, all of which mixtures are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and their mixtures are included within the scope of the present invention.

[0065] Unless otherwise specified, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of each other.

[0066] Unless otherwise specified, the terms "cis-trans isomers" or "geometric isomers" are caused by the inability of double bonds or single bonds of ring carbon atoms to rotate freely.

[0067] Unless otherwise specified, the term "diastereoisomer" refers to stereoisomers that have two or more chiral centers and are not mirror images of each other between molecules.

[0068] Unless otherwise specified, "(D)" or "(+)" indicates dextrorotation, "(L)" or "(-)" indicates levorotation, and "(DL)" or "(±)" indicates racemic.

[0069] Unless otherwise specified, the solid wedge bond and the dashed wedge bond represent the absolute configuration of a stereocenter, the straight solid bond and the straight dashed bond represent the relative configuration of a stereocenter, and the wavy line represents the solid wedge bond or the dashed wedge bond or the wavy line represents the straight solid bond and the straight dashed bond

[0070] The compounds of the present invention may exist in specific forms. Unless otherwise specified, the term "tautomer" or "tautomeric form" refers to different functional group isomers that are in dynamic equilibrium at room temperature and can rapidly interconvert. If tautomers are possible (such as in solution), a chemical equilibrium of tautomers may be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions that occur through proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions that occur through the reorganization of some bonding units. A specific example of keto-enol tautomerization is the interconversion between the two tautomers of pentane-2,4-dione and 4-hydroxy-3-en-2-one.

[0071] The solvates of the present invention are formed by the dissolution of a substance in a solvent, where the solvent molecules combine with the solute molecules or ions, etc., causing the solute to change its original state to form a solvate. The hydrates of the present invention are compounds containing water and are the most common type of solvate in small molecule drugs. Here, water can be connected to other parts of the drug through a coordination bond, such as a hydrated metal ion, or through a covalent bond, such as chloral hydrate, or through a hydrogen bond, such as a hydrated drug crystal, etc.

[0072] The term "prodrug compound" generally refers to a functional group derivatization of the compound of general formula (I), and its derivative can be easily converted into the compound of general formula (I) in vivo. Generally, the selection and preparation of suitable prodrugs can be referred to, for example, as described in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.

[0073] The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound. These isotopes have the same number of atoms, but their atomic mass or mass number is different from the atomic mass or mass number that predominantly exists in nature. For example, a compound can be labeled with a radioactive isotope, such as deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention. Isotope variants may confer certain therapeutic advantages. For example, deuterium can replace hydrogen to form deuterated drugs. The bond formed by deuterium and carbon is stronger than that formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxicity and side effects, increased drug stability, enhanced efficacy, and extended drug biological half-life, or can provide standard compounds that can be used for the characterization of biological samples. By conventional techniques well-known to those skilled in the art, or by methods similar to those described in the routes and examples of the present invention, using appropriate isotope-enriched reagents and / or intermediates, isotope-enriched compounds within the general formula (I) can be prepared without undue experimentation.

[0074] "Optional" or "optionally" means that the subsequent described event or condition may but does not necessarily occur, and this description includes the cases where the described event or condition occurs and the cases where the described event or condition does not occur.

[0075] The nomenclature rules used in the present invention are based on the IUPAC system of nomenclature.

[0076] The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by substituents, which may include deuterium and variants of hydrogen, as long as the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are replaced, and oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted. Unless otherwise specified, the type and number of substituents can be arbitrary based on what is chemically achievable.

[0077] When any variable (such as R) appears more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted by 0 - 2 Rs, the group may optionally be substituted by up to two Rs, and each R has independent options in each case. In addition, combinations of substituents and / or their variants are only permitted if such combinations result in stable compounds.

[0078] When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0079] When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when L in A - L - Z represents a single bond, it means that the structure is actually A - Z.

[0080] When the listed linking groups do not specify their connection directions, their connection directions are arbitrary. For example, The linking group L in At this time the benzene ring and the cyclopentyl group can be connected in the same direction as the reading order from left to right to form or the phenyl group and the cyclopentyl group can be connected in the direction opposite to the reading order from left to right to form The combination of the linking group, the substituent and / or its variant is only allowed if such a combination results in a stable compound.

[0081] Unless otherwise specified, the number of atoms in a ring is usually defined as the ring member count. For example, a "3-7 membered ring" refers to a "ring" composed of 3-7 atoms arranged in a ring.

[0082] Unless otherwise specified, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0083] Unless otherwise specified, the term "C 1-6 alkyl" is used to represent a straight-chain or branched saturated hydrocarbon group composed of 1 to 6 carbon atoms. The C 1-6 alkyl includes C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-4 、C6 and C5 alkyls, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of C 1-6 alkyl include but are not limited to methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.

[0084] Unless otherwise specified, the term "C 1-6 alkoxy" represents those alkyl groups containing 1 to 6 carbon atoms that are connected to the rest of the molecule through an oxygen atom. The C 1-6 alkyl includes C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-4 、C6、C5、C4 and C3 alkoxys, etc.; Examples of C 1-6 alkoxy include but are not limited to methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy, s-butoxy and t-butoxy), pentoxy (including n-pentoxy, isopentoxy and neopentoxy), hexoxy, etc.

[0085] Unless otherwise specified, the term "C 1-6"Alkylamino" refers to those alkyl groups containing 1 to 6 carbon atoms that are attached to the rest of the molecule through an amino group. The C 1-6 alkyl includes C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-4 、C6, C5, C4, C3, and C2 alkylamino, etc.; examples of C 1-6 alkylamino include, but are not limited to, -NHCH3, -N(CH3)2, -NHCH2CH3, -N(CH3)CH2CH3, -N(CH2CH3)2, -NHCH2CH2CH3, -NHCH(CH3)2, -NHCH2CH2CH2CH3, etc.

[0086] Unless otherwise specified, the term "C 3-6 cycloalkyl" refers to a saturated cyclic hydrocarbon group consisting of 3 to 6 carbon atoms, which is a monocyclic and bicyclic system. The C 3-6 cycloalkyl includes C 3-5 、C 4-5 and C 5-6 cycloalkyl, etc.; it can be monovalent, divalent, or polyvalent. Examples of C 3-6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0087] Unless otherwise specified, the term "3-6 membered heterocycloalkyl" alone or in combination with other terms separately represents a saturated monocyclic group consisting of 3 to 6 ring atoms, where 1, 2, 3, or 4 of the ring atoms are heteroatoms independently selected from O, S, and N, and the remaining are carbon atoms, where the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., NO and S(O) z , z is 1 or 2). In addition, for this "3-6 membered heterocycloalkyl", the heteroatom can occupy the position where the heterocycloalkyl is connected to the rest of the molecule. The 3-6 membered heterocycloalkyl includes 4-6 membered, 5-6 membered, 4 membered, 5 membered, and 6 membered heterocycloalkyl, etc. Examples of 3-6 membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl, tetrahydrofuryl (including tetrahydrofuran-2-yl), piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, etc.

[0088] Unless otherwise specified, the terms "5- or 6-membered heteroaryl ring" and "5- or 6-membered heteroaryl group" of the present invention can be used interchangeably. The term "5- or 6-membered heteroaryl group" refers to a cyclic group having a conjugated π-electron system composed of 5 to 6 ring atoms, wherein 1, 2, 3, or 4 ring atoms are heteroatoms independently selected from O, S, and N, and the rest are carbon atoms. The nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms can be optionally oxidized (i.e., NO and S(O) z , z is 1 or 2). The 5- or 6-membered heteroaryl group can be connected to the rest of the molecule through a heteroatom or a carbon atom, and the 5- or 6-membered heteroaryl group includes 5-membered and 6-membered heteroaryl groups, etc. Examples of the 5- or 6-membered heteroaryl group include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl, 3-pyrazolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl, 4H-1,2,4-triazolyl), tetrazolyl, isoxazolyl (including 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, etc.), furyl (including 2-furyl, 3-furyl, etc.), thienyl (including 2-thienyl, 3-thienyl, etc.), pyridyl (including 2-pyridyl, 3-pyridyl, 4-pyridyl, etc.), pyrazinyl, pyrimidinyl (including 2-pyrimidinyl, 4-pyrimidinyl, etc.), etc.

[0089] Unless otherwise specified, C n-n+m or C n -C n+m includes any specific case of n to n + m carbons, such as C 1-7 includes C1, C2, C3, C4, C5, C6, and C7, and also includes any range from n to n + m, such as C 1-7 includes C 1-3 , C 1-6 , C 3-6 , C 4-7 and C 5-7 etc.; similarly, n-membered to n + m-membered means that the number of atoms in the ring is from n to n + m, for example, 3- to 7-membered rings include 3-membered, 4-membered, 5-membered, 6-membered, and 7-membered rings, and also include any range from n to n + m, such as 3- to 7-membered rings include 3- to 6-membered rings, 4- to 7-membered rings, 5- to 7-membered rings, and 6- to 7-membered rings, etc.

[0090] The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (such as a nucleophilic substitution reaction). For example, representative leaving groups include trifluoromethanesulfonate; chlorine, bromine, iodine; sulfonate groups such as methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-toluenesulfonate, etc.; acyloxy groups such as acetoxy, trifluoroacetoxy, and the like.

[0091] The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group", or "mercapto protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: formyl; acyl groups such as alkanoyl groups (such as acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl groups such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups such as benzyl (Bn), trityl (Tr), 1,1-bis-(4'-methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing side reactions of the hydroxyl group. Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl, and tert-butyl; acyl groups such as alkanoyl groups (such as acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like.

[0092] As used in the present invention, the term "treatment" refers to administering one or more pharmaceutical substances, particularly the compound of formula (I) and / or its pharmaceutically acceptable salts as described in the present invention, to an individual suffering from a disease or having symptoms of the disease, for the purpose of curing, alleviating, reducing, modifying, treating, improving, ameliorating, or affecting the disease or the symptoms of the disease. The term "prevention" as used in the present invention refers to administering one or more pharmaceutical substances, particularly the compound of formula (I) and / or its pharmaceutically acceptable salts as described in the present invention, to an individual having a constitution predisposed to the disease, for the purpose of preventing the individual from contracting the disease. When referring to a chemical reaction, the terms "treatment", "contact", and "reaction" refer to adding or mixing two or more reagents under appropriate conditions to produce the indicated and / or desired product. It should be understood that the reaction producing the indicated and / or desired product may not necessarily directly result from the combination of the two initially added reagents, i.e., one or more intermediates may be formed in the mixture, and these intermediates ultimately lead to the formation of the indicated and / or desired product.

[0093] As used in the present invention, "patient" is defined as any warm-blooded animal, such as, but not limited to, mice, guinea pigs, dogs, horses, or humans, and preferably the patient is a human.

[0094] As used herein, the term "effective amount" generally refers to an amount sufficient to produce a beneficial effect on an individual. The effective amount of the compounds of the present invention can be determined by conventional methods (such as modeling, dose escalation studies or clinical trials) in combination with conventional influencing factors (such as the mode of administration, the pharmacokinetics of the compound, the severity and course of the disease, the medical history of the individual, the health status of the individual, the degree of response of the individual to the drug, etc.).

[0095] As described above, the novel compounds of the present invention, their pharmaceutically acceptable salts and prodrugs have important pharmacological properties and are inverse agonists of the α5-GABA A receptor. Therefore, the compounds of the present invention can be used alone or in combination with other drugs for the treatment or prevention of diseases mediated by GABA A receptor ligands containing the α5 subunit. These diseases include, but are not limited to, pain, Alzheimer's disease, multi-infarct dementia and stroke.

[0096] Accordingly, the present invention also relates to a pharmaceutical composition comprising a compound as defined above and a pharmaceutically acceptable carrier and / or adjuvant.

[0097] Likewise, the present invention also includes the compounds as described above for use in the manufacture of a medicament for the treatment or prevention of diseases related to the α5-GABA A receptor, especially for the treatment or prevention of the following diseases: pain, Alzheimer's disease, multi-infarct dementia and stroke.

[0098] The technical and scientific terms not specifically defined herein have the meanings commonly understood by those skilled in the art to which the present invention pertains.

[0099] Preparation method

[0100] The present invention also relates to a method for producing the compound of general formula (I) as defined above, and the compound synthesis method is shown as follows:

[0101] Synthesis method:

[0102]

[0103]

[0104] Usually, compound I-1 and compound I-2 are provided by commercial raw materials. Compound I-1 is reacted with compound I-2 in halogenated alkane or ether solvents, such as dichloromethane, tetrahydrofuran, etc., to form compound I-3 through Chan-Lam reaction. The Chan-Lam reaction conditions can select Cu salt as catalyst, including but not limited to copper acetate, cuprous iodide, cuprous oxide, etc., and select organic base as base, including but not limited to pyridine, triethylamine, diisopropylethylamine, etc. Compound I-3 can obtain compound I-4 through reduction conditions, and the reduction reaction is not limited to the reaction using reducing agents such as sodium borohydride, lithium borohydride, lithium aluminum hydride or DIBAL-H in ether or alcohol solvents such as tetrahydrofuran, methanol, etc. Compound (I) can be generated by the reaction of compound I-4 with compound I-6 under step c), and step c) corresponds to alkaline direct nucleophilic substitution conditions, including but not limited to sodium hydride, cesium carbonate or potassium phosphate as base in various solvents such as DMF, acetonitrile or tetrahydrofuran. Alternatively, step c) represents a metal-catalyzed coupling reaction, such as a palladium-catalyzed Buchwald reaction, a copper-catalyzed Ullmann reaction, etc. Compound (I) can be obtained by reacting compound I-4 with compound I-7 in step d) to obtain I-5, and I-5 is then reacted with compound AH, wherein compound AH represents a piperazine compound, and the secondary amine part of the molecule reacts with I-5 as a nucleophilic reagent to replace the X group in I-5, and step d) corresponds to alkaline direct nucleophilic substitution conditions, including but not limited to sodium hydride, cesium carbonate or potassium phosphate as a base in various solvents such as DMF, acetonitrile or tetrahydrofuran. Step e) represents an alkali metal-catalyzed coupling reaction, such as a palladium-catalyzed Buchwald reaction, a copper-catalyzed Ullmann reaction, etc.

[0105] The present invention also relates to compounds of general formula (I) as described above, which are prepared by the methods described above. If the preparation methods are not described in the examples, the compounds of general formula (I) and their intermediate products can be prepared by similar methods or according to the aforementioned methods. Raw materials known in the art can be obtained from commerce, or can be prepared by methods known in the art or by analogy to known methods.

[0106] It will be appreciated that the compounds of general formula (I) of this invention may be derivatized at functional groups to provide derivatives which are capable of reconversion into the parent compound in vivo.

[0107] Pharmaceutical composition

[0108] The present invention provides a method for treating a 5-GABA containing a therapeutically effective amount of α5-GABA. A Use of inverse agonist pharmaceutical compositions. Although the α5-GABA used in the treatment of the present invention AInverse agonists can be administered in the form of the raw material compound, but preferably the active ingredient, optionally in the form of a physiologically acceptable salt, is mixed with one or more additives, excipients, carriers, buffers, diluents and / or other conventional pharmaceutical adjuvants to form a pharmaceutical composition.

[0109] In a preferred embodiment, the present invention provides a pharmaceutical composition comprising an α5-GABA A inverse agonist, wherein the α5-GABA A inverse agonist is mixed with one or more pharmaceutically acceptable carriers and optionally with other therapeutic and / or prophylactic components known or used in the art. The carrier must be "acceptable", i.e., compatible with the other ingredients in the formulation and not harmful to its recipient.

[0110] The pharmaceutical compositions for use in the present invention can be those suitable for oral, rectal, bronchial, nasal, pulmonary, topical (including buccal and sublingual), transdermal, vaginal or parenteral (including intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection or infusion) administration, or those in a form suitable for administration by inhalation or spraying, including powder and liquid aerosol administration, or sustained release systems. Examples of suitable sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compounds of the present invention, wherein the matrix can be in the form of shaped articles, such as membranes or microcapsules.

[0111] Thus, the compounds for use in the present invention can be formulated into pharmaceutical compositions and unit dosage forms thereof together with conventional additives or diluents. Such forms include solids (especially in the form of tablets, filled capsules, powders and pills), and liquids (especially aqueous or non-aqueous solutions, suspensions, emulsions, elixirs), and capsules filled with the above forms, all forms for oral administration, rectal suppositories, and sterile injectable solutions for parenteral administration. Such pharmaceutical compositions and unit dosage forms thereof can include conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms can contain any suitable effective amount of the active ingredient corresponding to the desired daily dosage range.

[0112] The compounds for use in the present invention can be administered in a variety of oral and parenteral dosage forms. To those skilled in the art, the following dosage forms can contain the compounds of the present invention or their pharmaceutically acceptable salts as the active ingredient.

[0113] For preparing the pharmaceutical compositions of the compounds used in the present invention, pharmaceutically acceptable carriers can be solid or liquid. Solid forms of the preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier can be one or more substances which also act as diluents, flavoring agents, solubilizing agents, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.

[0114] In powders, the carrier is a finely divided solid which is mixed with the finely divided active ingredient.

[0115] In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compressed into the required shape and size.

[0116] Powders and tablets preferably contain from 5% or 10% to about 70% of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting waxes, cocoa butter, and the like. The term "preparations" includes those containing the active compound formulated with an encapsulating material as the carrier, the encapsulating material providing a capsule in which the active ingredient, with or without a carrier, is surrounded by the carrier so as to be bound together therewith. Similarly, preparations include cachets and lozenges. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

[0117] For preparing suppositories, first a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is melted, and then the active ingredient is uniformly dispersed therein by stirring. Then the melted and homogeneous mixture is poured into a mold of suitable size and allowed to cool and thereby solidify.

[0118] Compositions suitable for vaginal administration can be in the form of vaginal suppositories, tampons, creams, gels, pastes, foams, or sprays, and the compositions contain, in addition to the active ingredient, suitable carriers known in the art.

[0119] Liquid preparations include solutions, suspensions, and emulsions, for example, aqueous solutions or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in water-polyethylene glycol.

[0120] Thus, the compounds useful in the present invention can be formulated for parenteral administration (e.g., injection, such as rapid bolus injection or continuous infusion), and can be present in the form of unit doses, together with added preservatives, in ampoules, pre-filled syringes, small volume infusion bags or multi-dose containers. The composition can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain formulation ingredients such as suspending agents, stabilizers and / or dispersing agents. Additionally, the active ingredient can be in the form of a powder, which can be obtained by sterile solid-state isolation or by lyophilization of a solution, for reconstitution immediately before use with a suitable vehicle such as sterile, pyrogen-free water.

[0121] An aqueous solution suitable for oral administration can be prepared by dissolving the active ingredient in water and adding the desired coloring agents, flavoring agents, stabilizers and thickening agents.

[0122] An aqueous suspension suitable for oral administration can be prepared by dispersing the finely divided active ingredient in water containing a viscous substance such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.

[0123] Also included are solid preparations designed to be converted shortly before use into liquid preparations for oral administration. Such liquid preparations include solutions, suspensions and emulsions. In addition to the active ingredient, such preparations can contain coloring agents, flavoring agents, stabilizers, buffering agents, artificial and natural sweeteners, dispersing agents, thickening agents, solubilizing agents, etc.

[0124] For topical application to the epidermis, the compounds of the present invention can be formulated as ointments, creams or lotions or transdermal patches. For example, ointments and creams can be formulated with an aqueous or oily base plus a suitable thickening agent and / or gelling agent. Lotions can be formulated with an aqueous or oily base and generally also contain one or more emulsifying agents, stabilizers, dispersing agents, suspending agents, thickening agents or coloring agents.

[0125] Compositions suitable for topical oral administration include lozenges containing the active ingredient in a flavored matrix, usually sucrose and acacia or tragacanth; pastilles containing the active ingredient in an inert matrix such as gelatin and glycerin or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid vehicle.

[0126] Solutions or suspensions can be applied directly to the nasal cavity by conventional means such as with a dropper, pipette or nebulizer. The composition can be in single-dose or multi-dose form.

[0127] Respiratory administration can also be achieved by means of an aerosol, in which the active ingredient is contained in a pressurized package together with a suitable propellant, which includes chlorofluorocarbons (CFCs) such as dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide or other suitable gases. The aerosol may also suitably contain a surfactant, such as lecithin. The dose of the drug can be controlled by a metering valve.

[0128] Alternatively, the active ingredient can be in the form of a dry powder, for example a powder mixture of the compound with a suitable powder matrix such as lactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidone (PVP). The powder carrier can conveniently form a gel in the nasal cavity. The powder composition can be in unit dose form, for example in capsules or cartridges (such as gelatin capsules or cartridges), or in blister packs from which the powder can be administered via an inhaler.

[0129] In compositions for respiratory administration (including compositions for intranasal use), generally the compound has a small particle size, for example of the order of 5 microns or less. Such particle size can be obtained by methods known in the art, for example by micronization.

[0130] When needed, compositions suitable for sustained release of the active ingredient can be applied.

[0131] The pharmaceutical preparation is preferably in unit dose form. In such form, the preparation is subdivided into unit doses of a suitable amount of the active ingredient. The unit dose form can be an encapsulated preparation, in which a large amount of the preparation is separately contained in a sealed package, such as encapsulated tablets, capsules and powders contained in vials or ampoules. In addition, the unit dose form can be the capsule, tablet, cachet or lozenge itself, or can be any encapsulated form of an appropriate amount of the above capsules, tablets, etc.

[0132] Tablets or capsules for oral administration and liquids for intravenous administration and continuous infusions are preferred compositions. More detailed information on formulations and administration techniques can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

[0133] The amount of the active ingredient in the unit dose preparation can vary according to the specific application and the potency of the active ingredient, and can be adjusted from 0.1 mg to about 1 g. For example, in medical use, the drug can be administered one to three times a day in capsules of 0.1 to about 400 mg, and if necessary the composition can also contain other compatible therapeutic agents.

[0134] Preparation of intermediates:

[0135] Synthesis of intermediate A6:

[0136] 3-Chloro-6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazine (A6)

[0137]

[0138] Step 1: Synthesis of ethyl 1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazole-5-carboxylate (A3)

[0139] Under room temperature conditions, A1 (1 g, 3.57 mmol), A2 (675 mg, 3.92 mmol), copper acetate (778 mg, 4.28 mmol) and pyridine (338 mg, 3.46 mmol) were successively added to anhydrous dichloromethane (10 mL). At 25 °C, air was passed through with a drying tube and the reaction was carried out for 16 hours. TLC showed the formation of a new spot. The reaction solution was diluted with water (100 mL), and extracted with dichloromethane (80 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (ethyl acetate: petroleum ether = 1:20 to 1:3) to obtain the title product (156 mg, white solid).

[0140] 1 H NMR (400 MHz, DMSO-d6) δ 7.73 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.13 (t, J = 55.6 Hz, 1H), 4.18 (q, J = 7.1 Hz, 2H), 2.28 (s, 3H), 1.12 (t, J = 7.1 Hz, 3H).

[0141] Step 2: Synthesis of (1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methanol (A4)

[0142] Under ice-water bath conditions, compound A3 (150 mg, 0.563 mmol) and DIBAL-H (2.25 mL, 1 M solution in tetrahydrofuran, 2.25 mmol) were successively added to tetrahydrofuran (10 mL). The reaction was carried out at 24 °C for 24 hours. TLC showed the formation of a new spot. The reaction solution was diluted with water (100 mL), and extracted with dichloromethane (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (ethyl acetate: petroleum ether = 1:10 to 1:2) to obtain the title product (65 mg, colorless oil).

[0143] MS (ESI) m / z [M+H] + = 238.9.

[0144] Step 3: Synthesis of 3-chloro-6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazine (A6)

[0145] Under ice-water bath conditions, compound A4 (60 mg, 0.252 mmol) and sodium hydride (30 mg, 60% purity, 0.765 mmol) were successively added to DMF (3 mL), and the reaction was carried out at 0 °C for half an hour. Subsequently, compound A5 (75 mg, 0.504 mmol) was added, and the reaction was carried out at 25 °C for 3 hours. TLC showed the formation of a new spot. The reaction solution was diluted with water (10 mL) and extracted with dichloromethane (50 mL x 2). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (ethyl acetate: petroleum ether = 1:10 - 1:2) to obtain the title product (65 mg, colorless oil).

[0146] MS(ESI) m / z [M+H] + = 350.9.

[0147] Synthesis of intermediate A7:

[0148] 2-bromo-5-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyrazine (A7)

[0149]

[0150] The experimental operation was as described in Step 3 of the synthesis of intermediate A6, using A4 and 2,5-dibromopyrazine as reactants to obtain the title product.

[0151] MS(ESI) m / z [M+H] + = 395.0.

[0152] Synthesis of intermediate B4:

[0153] 3-chloro-6-((1-(4-fluorophenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazine (B4)

[0154]

[0155]

[0156] The experimental operation was as described in the synthesis of intermediate A6, with starting materials B1 and A2, to obtain the title product. MS(ESI) m / z [M+H] + = 319.1.

[0157] Synthesis of intermediate C4:

[0158] 3-chloro-6-((1-(4-(difluoromethyl)phenyl)-4-iodo-1H-pyrazol-5-yl)methoxy)pyridazine (C4)

[0159]

[0160] The experimental operation was as described for the synthesis of intermediate A6. The starting materials were A1 and C1, and the title product was obtained. MS(ESI) m / z [M+H] + = 463.0

[0161] Synthesis of intermediate D4:

[0162] 3-chloro-6-((4-chloro-1-(4-(difluoromethyl)phenyl)-1H-pyrazol-5-yl)methoxy)pyridazine (D4)

[0163]

[0164] The synthesis method was as described for the synthesis of intermediate A6. The starting materials were A1 and D1, and the title product was obtained.

[0165] MS(ESI) m / z [M+H] + = 371.0.

[0166] Example 1: Preparation method of compound 1 of the present invention

[0167] 4-(6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)piperazin-2-one (1), namely compound 1.

[0168]

[0169] Under argon protection, A6 (30 mg, 0.085 mmol) and 2-piperazinone (8.6 mg, 0.085 mmol) were dissolved in dioxane (3.0 mL). 2-Dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (4 mg, 0.0085 mmol), cesium carbonate (28 mg, 0.085 mmol) and (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II) methanesulfonate (7 mg, 0.0085 mmol) were added successively, and the mixture was stirred at 100 °C for 16 hours. The reaction was monitored by LCMS until completion. The reaction solution was diluted with dichloromethane (50 mL) and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by preparative thin-layer chromatography (petroleum ether:ethyl acetate = 0:1) to obtain the title product (14 mg, white solid).

[0170] MS(ESI) m / z [M+H]+ = 415.2.

[0171] 1 1H NMR (400 MHz, DMSO-d6) δ 8.06 (s, 1H), 7.68 (s, 4H), 7.62 (s, 1H), 7.41 (d, J = 9.6 Hz, 1H), 7.11 (t, J = 55.6 Hz, 1H), 7.03 (s, 1H), 7.04 (d, J = 9.6 Hz, 1H), 5.36 (s, 2H), 4.98 (s, 2H), 3.69 - 3.63 (m, 2H), 3.30 - 3.25 (m, 2H), 2.41 (s, 3H).

[0172] Example 2: Preparation method of the compound 2 of the present invention (S)-8-(6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)hexahydro-2H-pyrazino[1,2-a]pyrazin-1(6H)-one (2)

[0173]

[0174] Step 1: Synthesis of (S)-1-tert-butyl 3-methyl 4-(2-(((benzyloxy)carbonyl)amino)ethyl)piperazine-1,3-dicarboxylate (2-2)

[0175] At room temperature, 2-1 (1.0 g, 4.09 mmol) and N-benzyloxycarbonyl-3-bromoethylamine (1.27 g, 4.91 mmol) were dissolved in anhydrous DMF (15.0 mL), potassium carbonate (678 mg, 4.91 mmol) was added, and the reaction was carried out at 100 °C for 16 hours. TLC showed that the reaction was complete. The reaction solution was diluted with water (40 mL), extracted with dichloromethane (30 mL x 3), the organic phases were combined, washed with saturated brine (40 mL x 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by flash silica gel column chromatography (methanol:dichloromethane = 0:1 to 1:10) to obtain the title product (370 mg, yellow oil).

[0176] MS (ESI) m / z [M+H] + = 422.5.

[0177] Step 2: Synthesis of (S)-tert-butyl 9-oxohexahydro-1H-pyrazino[1,2-a]pyrazine-2(6H)-carboxylate (2-3)

[0178] At room temperature, 2-2 (370 mg, 0.87 mmol) was dissolved in methanol (6.0 mL), palladium / carbon (30 mg, 10%) was added, and the reaction was carried out under a hydrogen (15 psi) atmosphere for 16 hours. TLC showed that the reaction was complete. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the title product (210 mg, yellow oil).

[0179] Step 3: Synthesis of (S)-octahydro-1H-pyrazino[1,2-a]pyrazin-1-one (2-4)

[0180] At room temperature, 2-3 (60 mg, 0.24 mmol) was dissolved in anhydrous dichloromethane (4.0 mL), and a hydrochloric acid / ethyl acetate solution (4.0 mL, 4.0 M) was slowly added dropwise. The reaction was stirred for 1 hour. TLC showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to obtain the title product (50 mg, yellow solid, hydrochloride).

[0181] Step 4: Synthesis of (S)-8-(6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)hexahydro-2H-pyrazino[1,2-a]pyrazin-1(6H)-one (2)

[0182] The experimental operation was as described in Example 1, using A6 and 2-4 as reactants to obtain the title product.

[0183] MS(ESI) m / z [M+Na] + = 492.1.

[0184] 1 H NMR (400 MHz, DMSO-d6) δ 7.66 (s, 4H), 7.59 (s, 1H), 7.39 (d, J = 9.6 Hz, 1H), 7.01 (d, J = 9.6 Hz, 1H), 6.82 (t, J = 55.6 Hz, 1H), 5.39 (s, 2H), 4.52 - 4.44 (m, 1H), 4.13 - 4.09 (m, 1H), 3.52 - 3.49 (m, 1H), 3.26 - 3.23 (m, 1H), 3.08 - 3.00 (m, 3H), 2.89 - 2.80 (m, 2H), 2.65 - 2.55 (m, 1H), 2.48 - 2.41 (m, 1H), 2.21 (s, 3H).

[0185] Example 3: Preparation method of compound 3 of the present invention

[0186] 4-(6-((1-(4-fluorophenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)piperazin-2-one (3)

[0187]

[0188] The experimental operation was as described in Example 1. Using B4 and 2-piperazinone as reactants, the title product was obtained.

[0189] MS(ESI) m / z [M+H] + = 383.2.

[0190] 1 H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H), 7.56 - 7.53 (m, 3H), 7.40 (d, J = 9.6 Hz, 1H), 7.32 (t, J = 8.8 Hz, 2H), 7.07 (d, J = 9.6 Hz, 1H), 5.31 (s, 2H), 3.99 (s, 2H) 3.68 - 3.64 (m, 2H), 3.29 - 3.25 (m, 2H), 2.12 (s, 3H).

[0191] Example 4: Preparation method of Compound 4 of the present invention (S)-8-(6-((1-(4-fluorophenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)hexahydro-2H-pyrazino[1,2-a]pyrazin-1(6H)-one (4)

[0192]

[0193] The experimental operation was as described in Example 1. Using B4 and 2-4 as reactants, the title product was obtained.

[0194] MS(ESI) m / z [M+H] + = 438.2.

[0195] 1 H NMR (400 MHz, DMSO-d6) δ 7.78 (s, 1H), 7.56 - 7.53 (m, 3H), 7.40 (d, J = 9.6 Hz, 1H), 7.31 (t, J = 8.8 Hz, 2H), 7.02 (d, J = 9.6 Hz, 1H), 5.30 (s, 2H), 4.46 - 4.42 (m, 1H), 4.03 - 3.99 (m, 1H), 3.30 - 3.27 (m, 1H), 3.09 - 3.05 (m, 1H), 2.94 - 2.85 (m, 3H), 2.66 - 2.59 (m, 2H), 2.44 - 2.40 (m, 1H), 2.29 - 2.23 (m, 1H), 2.11 (s, 3H).

[0196] Example 5: Preparation method of Compound 5 of the present invention

[0197] 4-(5-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyrazin-2-yl)piperazin-2-one (5)

[0198]

[0199] The experimental procedure was as described in Example 1. Using A7 and 2-piperazinone as reactants, the title product was obtained.

[0200] MS(ESI) m / z [M+H] + = 415.1.

[0201] 1 1H NMR (400 MHz, DMSO-d6) δ 8.10 (s, 1H), 7.91 (s, 1H), 7.83 (s, 1H), 7.70 (s, 4H), 7.62 (s, 1H), 7.10 (t, J = 55.6 Hz, 1H), 5.27 (s, 2H), 3.93 (s, 2H), 3.63 - 3.67 (m, 2H), 3.25 - 3.29 (m, 2H), 2.14 (s, 3H).

[0202] Example 6: Preparation method of Compound 6 of the present invention (S)-8-(5-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyrazin-2-yl)hexahydro-2H-pyrazino[1,2-a]pyrazin-1(6H)-one (6)

[0203]

[0204] The experimental procedure was as described in Example 1. Using A7 and Compounds 2 - 4 of the present invention as reactants, the title product was obtained. MS(ESI) m / z [M+H] + = 470.2.

[0205] 1 1H NMR (400 MHz, DMSO-d6) δ 7.91 (s, 1H), 7.82 (s, 2H), 7.71 (s, 4H), 7.62 (s, 1H), 7.10 (t, J = 55.6 Hz, 1H), 5.27 (s, 2H), 4.46 - 4.42 (m, 1H), 3.98 - 3.93 (m, 1H), 3.11 - 3.08 (m, 1H), 2.97 - 2.83 (m, 4H), 2.59 - 2.51 (m, 2H), 2.45 - 2.40 (m, 1H), 2.32 - 2.26 (m, 1H), 2.14 (s, 3H).

[0206] Example 7: Preparation method of Compound 7 of the present invention

[0207] 3 - ((1 - (4 - (difluoromethyl)phenyl) - 4 - methyl - 1H - pyrazol - 5 - yl)methoxy) - 6 - (2 - methoxypyrimidin - 5 - yl)pyridazine (7)

[0208]

[0209] At room temperature, A6 (70 mg, 0.29 mmol) and (2 - methoxypyrimidin - 5 - yl)boronic acid (45 mg, 0.29 mmol) were mixed in a mixed solvent of 1,4 - dioxane and water (5 mL / 1 mL). [1,1′ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (28 mg, 0.038 mmol) and sodium carbonate (56 mg, 0.54 mmol) were added successively, and the mixture was stirred at 95 °C for 4 hours. LCMS showed that the reaction was completed. The reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (ethyl acetate:petroleum ether = 0:1 - 2:1) to give the title product (28 mg, white solid).

[0210] MS(ESI) m / z [M + H] + = 425.1.

[0211] 1 1H NMR (400 MHz, DMSO - d6) δ 9.23 (s, 2H), 8.25 (d, J = 9.2 Hz, 1H), 7.70 (s, 4H), 7.65 (d, J = 9.2 Hz, 1H), 7.40 (s, 1H), 7.07 (t, J = 55.6 Hz, 1H), 5.58 (s, 2H), 3.40 (s, 3H), 2.18 (s, 3H).

[0212] Example 8: Preparation method of Compound 8 of the present invention

[0213] 4 - (6 - ((1 - (4 - (difluoromethyl)phenyl) - 4 - methyl - 1H - pyrazol - 5 - yl)methoxy)pyridazin - 3 - yl)pyridin - 2(1H) - one (8)

[0214]

[0215] The experimental procedure was as described in Example 7, using A6 and 2 - hydroxypyridine - 4 - boronic acid pinacol ester as reactants to give the title product.

[0216] MS(ESI) m / z [M + H] + = 410.1.

[0217] 1HNMR(400MHz,DMSO-d6)δ11.75(s,1H),8.25(d,J=9.4Hz,1H),7.70(s,4H),7.66(s,1H),7.51(d,J=7.2Hz,1H),7.36(d,J=9.4Hz,1H),7.08(t,J=55.6Hz,1H),6.99(s,1H),6.92(d,J=7.2Hz,1H),5.60(s,2H),2.18(s,3H).

[0218] Example 9: Preparation method of compound 9 of the present invention

[0219] 4-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-1-methylpyridin-2(1H)-one (9)

[0220]

[0221] The experimental operation was as described in Example 7, using A6 and (1-methyl-2-oxo-1,2-dihydropyridin-4-yl)boronic acid as reactants to obtain the title product.

[0222] MS(ESI)m / z[M+H] + =424.1.

[0223] 1 H NMR(400MHz,DMSO-d6)δ8.26(d,J=9.2Hz,1H),7.84(d,J=7.2Hz,1H),7.70(s,4H),7.66(s,1H),7.37(d,J=9.2Hz,1H),6.95 - 7.23(m,3H),5.60(s,2H),3.47(s,3H),2.18(s,3H). Example 10: Preparation method of compound 10 of the present invention

[0224] 1-(4-(Difluoromethyl)phenyl)-5-(((6-(3-oxopiperazin-1-yl)pyridazin-3-yl)oxy)methyl)-1H-pyrazole-4-carbonitrile (10)

[0225]

[0226] Step 1: 5-(((6-Chloropyridazin-3-yl)oxy)methyl)-1-(4-(difluoromethyl)phenyl)-1H-pyrazole-4-carbonitrile (10-1)

[0227] At room temperature, cuprous cyanide (77 mg, 0.86 mmol) and C4 (200 mg, 0.43 mmol) were mixed into DMF (5 mL), and stirred at 140 °C for 4 hours. LCMS showed the formation of the product. The reaction solution was cooled to room temperature, filtered, and the filtrate was diluted with ethyl acetate (100 mL) and washed with saturated brine (100 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (ethyl acetate: petroleum ether = 0:1 to 1:3) to obtain the title product (80 mg, yellow solid).

[0228] MS(ESI) m / z [M+H] + = 362.1.

[0229] Step 2: 1-(4-(Difluoromethyl)phenyl)-5-(((6-(3-oxopiperazin-1-yl)pyridazin-3-yl)oxy)methyl)-1H-pyrazole-4-carbonitrile (10)

[0230] The experimental operation was as described in Example 1, using 10-1 and 2-piperazinone as reactants to obtain the title product.

[0231] MS(ESI) m / z [M+H] + = 426.1.

[0232] 1 1H NMR (400 MHz, DMSO-d6) δ 8.35 (s, 1H), 8.06 (s, 1H), 7.77 (s, 4H), 7.42 (d, J = 9.6 Hz, 1H), 7.13 (t, J = 55.6 Hz, 1H), 7.07 (d, J = 9.6 Hz, 1H), 5.59 (s, 2H), 3.96 (s, 2H) 3.67 - 3.65 (m, 2H), 3.28 - 3.24 (m, 2H).

[0233] Example 11: Preparation method of compound 11 of the present invention

[0234] 1-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-N-methyl-1H-imidazole-4-carboxamide (11)

[0235]

[0236] Step 1: Methyl 1-(6-chloropyridazin-3-yl)-1H-imidazole-4-carboxylate (11-2)

[0237] Under an ice bath, A5 (1 g, 6.7 mmol) and 11-1 (844 mg, 6.7 mmol) were dissolved in DMF (15 mL), sodium hydride (348 mg, 8.7 mmol) was added, and the mixture was stirred at room temperature for 16 hours. LCMS showed the formation of the product. The reaction solution was diluted with water (50 mL), and extracted with DCM (50 mL x 3). The organic phase was concentrated under reduced pressure to obtain the title product (1 g, crude product).

[0238] MS(ESI) m / z [M+H] + = 239.0.

[0239] Step 2: 1-(6-chloropyridazin-3-yl)-1H-imidazole-4-carboxylic acid (11-3)

[0240] 11-2 (1 g, 4.2 mmol) was dissolved in 1,4-dioxane (5 mL), 1M sodium hydroxide solution (6.3 mL, 6.3 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The precipitated solid was filtered, and the filter cake was dried under reduced pressure to obtain the title product (500 mg, white solid).

[0241] MS(ESI) m / z [M+H] + = 224.0.

[0242] Step 3: 1-(6-chloropyridazin-3-yl)-N-methyl-1H-imidazole-4-carboxamide (11-4)

[0243] 11-3 (150 mg, 0.67 mmol), methylamine hydrochloride (67 mg, 1.00 mmol) and HATU (381 mg, 1.00 mmol) were dissolved in DMF (5 mL), DIEA (174 mg, 1.34 mmol) was added, and the mixture was stirred at room temperature for 16 hours. LCMS showed the completion of the reaction. The reaction solution was diluted with ethyl acetate (50 mL), and washed with saturated brine (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain the title product (100 mg, white solid).

[0244] MS(ESI) m / z [M+H] + = 238.0.

[0245] Step 4: 1-(6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-N-methyl-1H-imidazole-4-carboxamide (11)

[0246] Under an ice bath, A4 (100 mg, 0.42 mmol) was dissolved in a mixed solution of anhydrous DCM and anhydrous DMF (5 mL / 5 mL), sodium hydride (25 mg, 0.63 mmol) was added, and the mixture was stirred at 0 °C for 10 minutes. Then 11-4 (119 mg, 0.50 mmol) was added, and the mixture was stirred at room temperature for 16 hours. LCMS showed the formation of the product. The reaction solution was poured into ice water (20 mL), and extracted with ethyl acetate (30 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase C18 flash preparative chromatography (acetonitrile: water = 0 - 45%) to obtain the title product (25 mg, white solid).

[0247] MS(ESI) m / z [M+H] + = 440.1.

[0248] 1 H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 8.41 (s, 1H), 8.27 (d, J = 9.6 Hz, 1H), 8.18 (s, 1H), 7.72 - 7.66 (m, 5H), 7.56 (d, J = 9.6 Hz, 1H), 7.09 (t, J = 55.6 Hz, 1H), 5.58 (s, 2H), 2.77 (d, J = 4.8 Hz, 3H), 2.19 (s, 3H).

[0249] Example 12: Preparation method of compound 12 of the present invention

[0250] 1-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-1H-pyrazole-4-carbonitrile (12)

[0251]

[0252] Step 1: 1-(6-Bromopyridazin-3-yl)-1H-pyrazole-4-carbonitrile (12-3)

[0253] 12-1 (1 g, 4.2 mmol) and 12-2 (391 mg, 4.2 mmol) were dissolved in DMF (15 mL), cesium carbonate (2.7 g, 8.4 mmol) was added, and the mixture was stirred at room temperature for 16 hours. LCMS showed the formation of the product. The mixture was diluted with water (50 mL) and extracted with DCM (50 mL x 2). The organic phase was concentrated under reduced pressure to obtain the title product (1 g, crude product).

[0254] MS(ESI) m / z [M+H] + = 250.0.

[0255] Step 2: 1-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-1H-pyrazole-4-carbonitrile (12)

[0256] At room temperature, A4 (80 mg, 0.33 mmol) and 12-3 (125 mg, 0.50 mmol) were dissolved in acetonitrile (5 mL), cesium carbonate (215 mg, 0.66 mmol) was added, and the mixture was stirred at 60 °C for 16 h. LCMS showed the formation of the product. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase C18 flash preparative chromatography (acetonitrile: water = 0 - 65%) to give the title product (21 mg, yellow solid).

[0257] MS (ESI) m / z [M+H] + = 408.1.

[0258] 1 1H NMR (400 MHz, DMSO-d6) δ 9.42 (s, 1H), 8.50 (s, 1H), 8.21 (d, J = 9.6 Hz, 1H), 7.71 - 7.66 (m, 5H), 7.54 (d, J = 9.6 Hz, 1H), 7.09 (t, J = 55.6 Hz, 1H), 5.60 (s, 2H), 2.19 (s, 3H).

[0259] Example 13: Preparation method of compound 13 of the present invention (6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridin-3-yl)(morpholino)methanone (13)

[0260]

[0261] Step 1: 6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)nicotinic acid (13-2)

[0262] A4 (100 mg, 0.42 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL), cooled to 0 °C in an ice bath, sodium hydride (34 mg, 0.84 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Then 13-1 (108 mg, 0.63 mmol) was added, and the mixture was stirred at room temperature for 2 h. LCMS showed the formation of the product. The reaction solution was poured into ice water (20 mL), and extracted with ethyl acetate (30 mL x 3). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give the title product (70 mg, yellow solid). MS (ESI) m / z [M+H] + = 360.1.

[0263] Step 2: (6 - ((1 - (4 - (difluoromethyl)phenyl)-4 - methyl - 1H - pyrazol - 5 - yl)methoxy)pyridin - 3 - yl)(morpholino)methanone (13)

[0264] Dissolve 13 - 2 (70 mg, 0.19 mmol), morpholine (33 mg, 0.38 mmol) and HATU (144 mg, 0.38 mmol) in DMF (5 mL), add DIEA (49 mg, 0.38 mmol), and stir at room temperature for 2 hours. LCMS shows that the reaction is complete. The reaction solution is diluted with ethyl acetate (50 mL) and washed with saturated brine (50 mL x 3). The organic phase is dried over anhydrous sodium sulfate and filtered, and the filtrate is concentrated under reduced pressure. The residue is purified by preparative thin - layer chromatography (petroleum ether:ethyl acetate = 0:1) to obtain the title product (19 mg, white solid).

[0265] MS(ESI) m / z [M + H] + = 429.1.

[0266] 1 1H NMR (400 MHz, DMSO - d6) δ 8.22 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.69 (s, 4H), 7.63 (s, 1H), 7.08 (t, J = 55.6 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 5.38 (s, 2H), 3.62 - 3.58 (s, 4H), 3.34 - 3.28 (m, 4H), 2.15 (s, 3H).

[0267] Example 14: Preparation method of compound 14 of the present invention

[0268] 4 - (6 - ((4 - chloro - 1 - (4 - (difluoromethyl)phenyl)-1H - pyrazol - 5 - yl)methoxy)pyridazin - 3 - yl)piperazin - 2 - one (14)

[0269]

[0270] The experimental operation is as described in Example 1, using D4 and 2 - piperazinone as reactants to obtain the title product.

[0271] MS(ESI) m / z [M + H] + = 435.1.

[0272] 11H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H), 7.99 (s, 1H), 7.72 (s, 4H), 7.41 (d, J = 9.6 Hz, 1H), 7.09 (d, J = 9.6 Hz, 1H), 7.05 (t, J = 55.6 Hz, 1H), 5.39 (s, 2H), 3.98 (s, 2H), 3.73 - 3.61 (m, 2H), 3.30 - 3.23 (m, 2H).

[0273] Example 15: Preparation method of the compound 15 of the present invention (S)-8-(6-((4-chloro-1-(4-(difluoromethyl)phenyl)-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)hexahydro-2H-pyrazino[1,2-a]pyrazin-1(6H)-one (15)

[0274]

[0275] The experimental operation was as described in Example 1, using D4 and 2-4 as reactants to obtain the title product.

[0276] MS (ESI) m / z [M+H] + = 490.2.

[0277] 1 1H NMR (400 MHz, CD3OD) δ 7.79 (s, 1H), 7.69 (s, 4H), 7.41 (d, J = 9.6 Hz, 1H), 7.02 (d, J = 9.6 Hz, 1H), 6.84 (t, J = 56.0 Hz, 1H), 5.43 (s, 2H), 4.54 - 4.43 (m, 1H), 4.20 - 3.99 (m, 1H), 3.59 - 3.44 (m, 1H), 3.28 - 3.20 (m, 1H), 3.10 - 2.96 (m, 3H), 2.92 - 2.77 (m, 2H), 2.68 - 2.54 (m, 1H), 2.52 - 2.35 (m, 1H).

[0278] Example 16: Preparation method of the compound 16 of the present invention

[0279] 1-(6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-N-ethyl-1H-imidazole-4-carboxamide (16)

[0280]

[0281] Step 1: Synthesis of methyl 1-(6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-1H-imidazole-4-carboxylate (16-1)

[0282] Dissolve A4 (800 mg, 3.36 mmol) in THF (10 mL), cool down to 0 °C, then add sodium hydride (161 mg, 6.72 mmol). After stirring for 10 minutes, add 11-2 (1.2 g, 5.04 mmol), and react the reaction mixture at room temperature for 16 h. Pour the reaction mixture into ice water (20 mL) to quench, and extract with ethyl acetate (30 mL × 3). Dry the organic phase over anhydrous sodium sulfate and filter. Concentrate the filtrate under reduced pressure to obtain the title product (500 mg, yellow solid).

[0283] MS (ESI) m / z [M+H] + = 441.1.

[0284] Step 2: 1-(6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-1H-imidazole-4-carboxylic acid (16-2)

[0285] Dissolve 16-1 (500 mg, 1.14 mmol) in a mixed solution of THF (5 mL) and water (5 mL), add NaOH (91.2 mg, 2.28 mmol), and stir the reaction mixture at room temperature for 1 h. Detect the completion of the reaction by LCMS. Pour the reaction mixture into ice water, adjust the pH to 3 with 2 M hydrochloric acid, and then extract with a mixed solution of dichloromethane / methanol (10:1) (30 mL × 3). Dry the organic phase over anhydrous sodium sulfate and filter. Concentrate the filtrate under reduced pressure to obtain the title product (400 mg, yellow solid).

[0286] MS (ESI) m / z [M+H] + = 427.1.

[0287] Step 3: 1-(6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-N-ethyl-1H-imidazole-4-carboxamide (16)

[0288] 16-2 (35 mg, 0.082 mmol), ethylamine hydrochloride (13.4 mg, 0.16 mmol) and HATU (47 mg, 0.12 mmol) were added to DCM (5 mL) for dissolution, and TEA (24.9 mg, 0.25 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 3 h. The reaction was monitored by LCMS until completion. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative thin-layer chromatography (petroleum ether: ethyl acetate = 0:1) to obtain the title product (17 mg, white solid).

[0289] MS (ESI) m / z [M+H] + = 454.2.

[0290] 1 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.39 (s, 1H), 8.27 (d, J = 9.4 Hz, 1H), 8.20 (t, J = 6.0 Hz, 1H), 7.68 (s, 4H), 7.66 (s, 1H), 7.56 (d, J = 9.4 Hz, 1H), 7.09 (t, J = 55.6 Hz, 1H), 5.57 (s, 2H), 3.32 - 3.16 (m, 2H), 2.18 (s, 3H), 1.10 (t, J = 7.2 Hz, 3H).

[0291] Example 17: Preparation method of compound 17 of the present invention

[0292] 1-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-N-isopropyl-1H-imidazole-4-carboxamide (17)

[0293]

[0294] The experimental procedure was as described in Example 16, using 16-2 and isopropylamine as reactants to obtain the title product.

[0295] MS (ESI) m / z [M+H] + = 468.2.

[0296] 11H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.40 (s, 1H), 8.27 (d, J = 9.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.70 (s, 4H), 7.66 (s, 1H), 7.56 (d, J = 9.4 Hz, 1H), 7.09 (t, J = 55.6 Hz, 1H), 5.57 (s, 2H), 4.15 - 4.01 (m, 1H), 2.18 (s, 3H), 1.17 (d, J = 6.6 Hz, 6H).

[0297] Example 18: Preparation method of compound 18 of the present invention

[0298] 1-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-N-cyclopropyl-1H-imidazole-4-carboxamide (18)

[0299]

[0300] The experimental operation was as described in Example 16, using 16-2 and cyclopropylamine as reactants to obtain the title product.

[0301] MS (ESI) m / z [M+H] + = 466.2.

[0302] 1 1H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H), 8.41 (s, 1H), 8.27 (d, J = 9.4 Hz, 1H), 8.17 (d, J = 4.8 Hz, 1H), 7.70 (s, 4H), 7.66 (s, 1H), 7.56 (d, J = 9.4 Hz, 1H), 7.08 (t, J = 55.6 Hz, 1H), 5.57 (s, 2H), 2.94–2.75 (m, 1H), 2.18 (s, 3H), 0.79 - 0.49 (m, 4H).

[0303] Example 19: Preparation method of compound 19 of the present invention

[0304] 1-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-N-(oxetan-3-yl)-1H-imidazole-4-carboxamide (19)

[0305]

[0306] The experimental operation was as described in Example 16. Using 16-2 and 3-aminooxetane as reactants, the title product was obtained. MS(ESI) m / z [M+H] + = 482.2.

[0307] 1 H NMR (400 MHz, DMSO-d6) δ 8.93 (d, J = 6.8 Hz, 1H), 8.64 (s, 1H), 8.45 (s, 1H), 8.27 (d, J = 9.4 Hz, 1H), 7.70 (s, 4H), 7.66 (s, 1H), 7.56 (d, J = 9.4 Hz, 1H), 7.08 (t, J = 55.6 Hz, 1H), 5.57 (s, 2H), 5.15–4.89 (m, 1H), 4.81–4.56 (m, 4H), 2.69 (s, 3H).

[0308] Example 20 : Preparation method of compound 20 of the present invention

[0309] 4-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-1-ethylpiperazin-2-one (19)

[0310]

[0311] Step 1: tert-Butyl 4-ethyl-3-oxopiperazine-1-carboxylate (20-2)

[0312] At room temperature, 20-1 (1 g, 4.99 mmol) and NaH (399 mg, 9.98 mmol) were added to ACN (30 mL), and iodoethane (1.17 g, 7.49 mmol) was slowly added dropwise. The mixture was stirred at room temperature overnight. The reaction solution was quenched with saturated ammonium chloride (30 mL) and extracted with ethyl acetate (100 mL). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 2 / 1) to obtain the title product (450 mg, yellow liquid).

[0313] MS(ESI) m / z [M+H] + = 229.1.

[0314] Step 2: 1-Ethylpiperazin-2-one (20-3)

[0315] 20-2 (450 mg, 1.97 mmol) was dissolved in DCM (15 mL), and HCl / Dioxane (2 mL) was added dropwise. The mixture was stirred at room temperature for 2 hours. LCMS showed the formation of the product. The reaction solution was concentrated under reduced pressure to obtain the crude title product (250 mg, yellow solid). The crude product was directly used in the next step.

[0316] MS(ESI) m / z [M+H] + = 129.1.

[0317] Step 3: 4-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-1-ethylpiperazin-2-one (20)

[0318] The experimental operation was as described in Example 1. Using A6 and 20-3 as reactants, the title product was obtained.

[0319] MS(ESI) m / z [M+H] + = 443.2.

[0320] 1 1H NMR (400 MHz, CDCl3) δ 7.81 - 7.37 (m, 5H), 7.09 - 6.84 (m, 2H), 6.65 (t, J = 56.0 Hz, 1H), 5.40 (s, 2H), 4.07 (s, 2H), 3.93 (s, 2H), 3.50 (s, 4H), 2.19 (s, 3H), 1.19 (s, 3H).

[0321] Example 21: Preparation method of compound 21 of the present invention

[0322] 4-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-1-methylpiperazin-2-one (21)

[0323]

[0324] The experimental operation was as described in Example 1. Using 20-1 and methyl iodide as starting materials, the title product was obtained.

[0325] MS(ESI) m / z [M+H] + = 429.3.

[0326] 1 1H NMR (400 MHz, DMSO-d6) δ 7.69 (s, 4H), 7.67 - 7.57 (m, 2H), 7.27 (d, J = 9.6 Hz, 1H), 7.09 (t, J = 55.6 Hz, 1H), 5.36 (s, 2H), 4.10 (s, 2H), 3.91 - 3.72 (m, 2H), 3.51 - 3.38 (m, 2H), 2.89 (s, 3H), 2.15 (s, 3H).

[0327] Example 22: Preparation method of compound 22 of the present invention

[0328] 4-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-1-cyclopropylpiperazin-2-one (22)

[0329]

[0330] Step 1: tert-Butyl 4-cyclopropyl-3-oxopiperazine-1-carboxylate (22-2)

[0331] At room temperature, 22-1 (1 g, 4.27 mmol), cyclopropylboronic acid (0.73 g, 8.54 mmol), copper(II) acetate (850 mg, 4.27 mmol), pyridine (1.01 g, 12.8 mmol) and cesium carbonate (3.48 g, 10.7 mmol) were added to toluene (15 mL), and the mixture was stirred at 110 °C overnight. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by column chromatography (PE / EA = 3 / 1) to obtain the title product (300 mg, pale yellow liquid).

[0332] MS(ESI) m / z [M+H] + = 275.1.

[0333] Step 2: 1-Cyclopropylpiperazin-2-one (22-3)

[0334] At room temperature, 22-2 (300 mg, 1.09 mmol) and wet Pd / C (60 mg, 0.56 mmol, purity: 10%) were added to methanol (10 mL), and the mixture was stirred at 27 °C overnight in a hydrogen atmosphere. The reaction mixture was filtered, and the filtrate was concentrated to obtain the title product (140 mg, colorless liquid).

[0335] MS(ESI) m / z [M+H] + = 141.1.

[0336] Step 3: 4-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-1-cyclopropylpiperazin-2-one (22)

[0337] The experimental procedure was as described in Example 1, using A6 and 22-3 as reactants to obtain the title product.

[0338] MS(ESI) m / z [M+H] + = 455.2.

[0339] 1HNMR(400MHz,CDCl3)δ7.64(d,J=8.4Hz,2H),7.58(s,1H),7.55(d,J=8.4Hz,2H),6.97(d,J=9.6Hz,1H),6.92(d,J=9.6Hz,1H),6.65(t,J=56.4Hz,1H),5.40(s,2H),4.07(s,2H),3.96 - 3.80(m,2H),3.54 - 3.36(m,2H),2.94 - 2.73(m,1H),2.18(s,3H),0.94 - 0.82(m,2H),0.77 - 0.65(m,2H).

[0340] Example 23: Preparation method of compound 23 of the present invention

[0341] 6-(6-((1-(4-(Difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)pyrimidin-4(3H)-one (23)

[0342]

[0343] Step 1: Synthesis of ethyl 3-(6-chloropyridazin-3-yl)-3-oxopropionate (23-2)

[0344] Under nitrogen protection, compound 23-1 (2 g, 12.6 mmol) and CDI (2.46 g, 15.1 mmol) were dissolved in THF (30 mL), stirred for 2 h, potassium monoethyl malonate (1.65 g, 12.6 mmol) and magnesium chloride (1.20 g, 12.6 mmol) were added, and the reaction was continued to stir at 25 °C for 16 h. The reaction was monitored by LCMS until completion. The reaction solution was quenched with water (100 mL), diluted with EA (100 mL), separated, and the aqueous phase was extracted with EA (100 mL x 2). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain the title product (1.1 g, yellow solid).

[0345] MS(ESI)m / z[M+H] + = 229.1.

[0346] Step 2: Synthesis of ethyl 3-(6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)-3-oxopropionate (23-3)

[0347] The experimental operation was as described in Step 3 of the synthesis of Intermediate A6. Using A4 and 23-2 as reactants, the title product was obtained. Step 3: Synthesis of 6-(6-((1-(4-(difluoromethyl)phenyl)-4-methyl-1H-pyrazol-5-yl)methoxy)pyridazin-3-yl)pyrimidin-4(3H)-one (23)

[0348] Under nitrogen protection, compound 23-2 (14.2 mg, 0.32 mmol) was dissolved in methanol (4 mL), sodium methoxide (34.8 mg, 0.64 mmol) was added, and the reaction was stirred at 50 °C for 16 h. LCMS showed the formation of the product. The reaction solution was quenched by adding water (20 mL), diluted with EA (50 mL), separated, and the aqueous phase was extracted with EA (30 mL x 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative thin-layer chromatography (dichloromethane:methanol = 10:1), and the crude product was further purified by reversed-phase C18 flash preparative chromatography (acetonitrile:water = 0-45%) to obtain the title product (3 mg, white solid).

[0349] MS(ESI) m / z [M+H] + = 411.1.

[0350] 1 1H NMR (400 MHz, CD3OD) δ 8.43–8.09 (m, 2H), 7.68–7.59 (m, 4H), 7.56 (s, 1H), 7.23 (d, J = 8.8 Hz, 1H), 7.18 (s, 1H), 6.88 (t, J = 56.0 Hz, 1H), 5.57 (s, 2H), 2.16 (s, 3H).

[0351] Example 24: Comparison of the effects of the compounds of the present invention with other compounds

[0352] Biological assay method:

[0353] Recent research results have shown that GABA A receptors mediate at least two inhibitory modes, phasic inhibition and tonic inhibition. Intrasynaptic GABA A receptors, due to the synchronous release of GABA-containing vesicles in the synapse caused by action potentials, result in a sharp increase in the GABA concentration in the synaptic cleft to the millimolar level, thereby causing synchronous activation and rapid desensitization of postsynaptic GABA A receptors, forming phasic inhibition. While extrasynaptic GABA A receptors are usually in a low concentration of GABA of several tens of nanomolar to several millimolar that persistsA In an environment, GABA with high affinity for GABA A receptors are continuously and asynchronously activated, forming tonic inhibition. Phasic inhibition and tonic inhibition jointly regulate neural excitability and signal transmission. (Farrant, M. et al., Nat Rev Neurosci, 2005, 6, 215 - 229). Yeung JY et al. disclosed that low concentrations of GABA are more likely to activate α5 - GABA A receptors (Yeung JY et al. Mol Pharmacol, 2003, 63, 2 - 8). K.Y. Lee reported that sustained high - affinity GABA A currents activated by low concentrations of GABA were detected on isolated DRG cells cultured for 24 hours. (Lee, K.Y. et al, Neuroscience 2012, 208, 133 - 142). In 2013, I. Lecker et al. disclosed that the α5 - GABA A receptor inverse agonist L - 655,708 dose - dependently inhibited the currents induced by low concentrations of GABA (5, 50 and 500 nM). When the GABA concentration increased to 1 μM, the highest concentration of L - 655,708 could only inhibit 15% of the current. When the GABA concentration continued to increase, L - 655,708 had no inhibitory effect on the currents induced by GABA. (Lecker, I. et al, British Journal of Anaesthesia, 2013, 110(S1), i73 - i81).

[0354] Effect Examples

[0355] I. Affinity Activity of the Compounds of the Present Invention for Different Subtypes of GABA A Receptors

[0356] By competing 3 the binding of H - flunitrazepam to HEK293 cells stably expressing human α1β3γ2, α2β3γ2, α3β3γ2 and α5β3γ2 receptors to determine the affinity of the compounds for each subtype of GABA A receptors.

[0357] The cells were suspended in 50 mM Tris-HCl buffer (pH = 7.4), homogenized 10 times for 20 seconds on ice using a homogenizer, and centrifuged at 4 °C, 1000 g for 10 min. The supernatant was taken and the above steps were repeated. The supernatant was centrifuged at 4 °C (33800 g; Thermo, rotor: A27-8x50) for 60 min, and the precipitate was resuspended in Tris buffer (50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM EDTA, 10% glycerol). Protein was measured (BCA method, Pierce), 1 ml aliquots were prepared and stored at -80 °C.

[0358] The radioligand competition binding assay was carried out in a 200 μL system (96-well plate), which contained 100 μL of cell membrane. 3 The concentration of H-flunitrazepam was 1 nM, and the concentration of the test compound was in the range of 1 x 10 -5 -10 -6 M. Flumazenil was used as a control. 1 μL of 2 mM flumazenil (final concentration 10 μM) was added to the low signal control well (Low control, LC), and 1 μL of DMSO was added to the high signal control well (High control, HC). The final concentration of the target membrane protein was 5 μg / well. The stock solutions of all test compound samples were 10 mM. The working concentration of the samples was obtained by diluting all samples with DMSO to 0.2 mM and then performing 4-fold serial dilutions for a total of 8 concentration gradients. The 96-well plate was sealed with a sealing film and then incubated on a shaker at room temperature for 1 hour. Meanwhile, the GF / C filter plate was soaked in the soaking buffer (0.3% PEI, stored at 4 °C) for at least 0.5 hours. After the binding incubation was completed, the cells were collected onto the GF / C filter plate using a cell harvester and washed 4 times with the washing buffer (50 mM Tris-HCl, pH 7.4, stored at 4 °C). After drying in an oven at 50 °C for 1 hour, the bottom of the dried GF / C filter plate was sealed with a film, and the residual radioactivity on the filter membrane was detected by liquid scintillation counting. 50 μL of scintillation fluid was added to each well and sealed, and Microbeta2 was used for reading. Calculate the inhibitory activity of the test samples on the binding of 3 H-flunitrazepam to the GABA A receptor membrane protein, calculate the IC 50 of each test sample by dose-effect curve fitting (GraphPad Prism5 software), and calculate the Ki of the sample through IC 50 to evaluate the binding ability of the sample to each subtype of the GABA A receptor.

[0359] By the above determination of the expression of human GABAA Representative test results obtained by the method of binding affinity of HEK293 cells of the receptor are shown in the following table.

[0360] Table 1 Affinity activity of compounds for α5-GABA A receptor

[0361]

[0362] As can be seen from Table 1, the compounds of the present invention will 3 H-flunitrazepam is displaced from the human α5-GABA A receptor with a Ki value of 100 nM or less, indicating that the compounds of the present invention have good affinity for the α5-GABA A receptor. In a preferred example, the compound displaces 3 H-flunitrazepam from the human α5-GABA A receptor with a Ki < 10 nM, indicating that the compound of the present invention has a strong affinity for the α5-GABAA subunit.

[0363] Table 2 Affinity activity of compounds for different subtypes of GABA A receptor

[0364]

[0365]

[0366] Note: "-" indicates that this experiment was not detected;

[0367] As can be seen from Table 2, in a preferred example, the compounds of the present invention have selectivity for the affinity of different subtypes of GABA A receptor, and can better selectively bind to the α5 subunit relative to the α1, α2, and α3-GABA A subunit receptors, and have a higher affinity selectivity for the α5-GABAA subunit. Compared with the examples in Patent WO202016443, the compounds of the present invention have a higher affinity selectivity; especially for the α1-GABA subunit receptor that can cause sedative side effects, the compounds of the present invention have a lower affinity and can better avoid the side effects caused by the activation or inhibition of the α1-GABA subunit receptor.

[0368] II. Inverse agonistic activity of the compounds of the present invention against the α5-GABA A receptor

[0369] The present invention detects the test drug against α5-GABA by electrophysiological methods AInverse agonistic efficiency of the receptor. The specific method is as follows:

[0370] Co-express different subunits of the GABA A receptor in the HEK293 cell line to construct a fully functional GABA A receptor. The α subunit, β subunit, and γ subunit are essential for forming a complete functional GABA A receptor. In this example, the present invention established the following cell model: Co-express the α5 subunit (protein sequence see GenBank accession number: NM_000810.3), β3 subunit (protein sequence see GenBank accession number: NM_000814.5), and γ2 subunit (protein sequence see GenBank accession number: NM_000816.3) in the HEK293 cell line, and screen out monoclonal stable transfected cell lines. This cell line expresses a fully functional α5-GABA A receptor.

[0371] Culture the monoclonal stable transfected HEK293 cell line expressing the α5-GABA A receptor on a 10 cm culture dish, and passage the cells when they grow to 80%-90%. When passaging, first aspirate the medium, then add 3 mL of DPBS phosphate buffer (Gibco TM ) to the culture dish, gently shake the culture dish, and then aspirate the DPBS. Add 1 mL of TrypLE Express, Gibco TM , and digest at 37 °C for 1-2 minutes. Then add 3 mL of complete medium (DMEM + 10% FBS (Gibco TM )) to disperse the cells on the bottom of the culture dish, transfer them to a 15 mL centrifuge tube (Corning), and centrifuge at 200 g for 3 minutes. Discard the supernatant, add 4 mL of complete medium, gently pipette to resuspend the cells for standby. For cell passage, dilute the cell suspension at a ratio of 1:5 or 1:10. For preparing cells for electrophysiological detection, after diluting the cell suspension at a ratio of 1:12, add it to a 24-well plate (Corning TM ) containing glass slides pre-treated with Poly-D-Lysine, and perform the experiment after the cells adhere. The culture time of cells for electrophysiology does not exceed 48 hours.

[0372] Drug concentration setting: The final concentration of the drugs used in drug screening was 100 nM for all drugs, and the GABA concentration was 0.05 μM. The whole-cell patch-clamp technique was used for electrophysiological experiments. This method can refer to the method reported in the literature (I. Lecker, Y. Yin, D. S. Wang and B. A. Orser, British Journal of Anaesthesia, 2013, 110(S1), i73-i81). The composition of the extracellular solution for electrophysiology was as follows: 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 10 mM glucose (pH 7.4); the formula of the intracellular solution for the electrophysiological electrode was as follows: 140 mM CsCl, 11 mM EGTA, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, 4 mM MgATP, 2 mM TEA (pH 7.3). Signal acquisition used an EPC 10 amplifier and PatchMaster software (HEKA) or an Axon 700B amplifier and Clampex software (AXON). The recording electrode was pulled from borosilicate glass, and the electrode resistance was 4 - 6 MΩ. Extracellular drug administration used an ALA-VC-8PG TM system. During recording, a single independently growing cell was selected. During the recording process, the cell membrane potential was clamped at -60 mV. During the experiment, the extracellular solution was applied extracellularly for about 20 seconds first. After the baseline was stable, the extracellular solution was switched to GABA. At this time, the current induced by GABA could be detected. After about 20 - 40 seconds, when the current was stable, the extracellular solution was switched to the corresponding drug solution to detect the effect of the drug. Finally, the solution was switched to the extracellular solution, and the experiment was terminated when the baseline returned to the level before drug administration. Only the data with a baseline less than -120 pA and capable of recovery after drug addition were analyzed subsequently. GABA was diluted in the extracellular solution at a final concentration of 0.05 μM. Then, the drug was diluted to the required concentration in the extracellular solution containing GABA.

[0373] The analysis of the experimental results used PatchMaster software. During analysis, the leakage current (I leak ), the GABA current before drug addition (I pre ), and the GABA current after drug addition (I post ) were measured respectively. The drug effect was calculated by the following formula: Inverse agonistic efficiency (%) = 100 - 100*(I post - I leak ) / (I pre - I leak ).

[0374] Screening results of compounds:

[0375] Table 3: Inverse agonistic activity of the compound against α5-GABA A receptor

[0376]

[0377]

[0378] Note: "-" indicates that this experiment was not detected;

[0379] As can be seen from Table 3, in the preferred examples, the compound of the present invention has strong inverse agonistic activity against α5-GABA A receptor; compared with the examples in Patent WO202016443, the compound of the present invention has comparable inverse agonistic activity against α5-GABA A receptor.

[0380] III. Solubility study of the compounds of the present invention

[0381] The kinetic solubility of the compound was evaluated by measuring the solubility of the DMSO stock solution of the compound in FaSSIF solution.

[0382] Preparation of FaSSIF-V2 solution: Weigh FaSSIF-V2 (53.7 mg) and add it to 15 mL of buffer solution (1.39 g of sodium hydroxide, 2.22 g of maleic acid and 4.01 g of sodium chloride, dissolved in 0.9 L of purified water, adjust the pH to 6.5 with NaOH or HCl), and make up the solution to 30 mL after dissolution, mix well and set aside.

[0383] Weigh 0.3 mg of the test sample, add 1.5 mL of FaSSIF-V2 solution, shake it manually until evenly mixed, and then oscillate it at 37 °C and 1000 rpm for several hours (set the oscillation time according to the detection requirements). Take it out, shake it manually until evenly mixed, filter the sample, discard 0.5 mL of the initial filtrate, and retain the subsequent filtrate. Pipette 400 μL of the subsequent filtrate and dilute it with 400 μL of acetonitrile, mix well, and use it as the test solution. Prepare two parallel samples. Determine the concentration by HPLC-MS method. According to the chromatogram obtained from the standard curve solution, use the concentration-peak area to make a regression equation, substitute the peak area of the sample to obtain the sample concentration, multiply by 2 to get the actual sample concentration, and calculate the average value of the two samples, which is the solubility of the sample.

[0384] IV. Table 4: Kinetic solubility of the compound

[0385]

[0386] As can be seen from Table 4, in the preferred examples, compared with Example 2 in Patent WO202016443, the compounds of the present invention have higher solubility, better pharmaceutical properties, and are more conducive to drug development.

[0387] In summary, the above are only the preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art can use the disclosed technical content to make changes or equivalent changes to equivalent embodiments, but as long as they do not depart from the technical solution content of the present invention, any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A pyrazole ring compound having the following general formula I, or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are each independently selected from hydrogen, halogen, cyano, C 1-6 alkyl, C 3-6 cycloalkyl, and the C 1-6 alkyl and C 3-6 cycloalkyl are optionally substituted with 1-3 R'; R’ is independently selected from hydrogen, halogen or C 1-3 alkyl; X1 and X2 are each independently selected from N or CH; A is selected from -C(O)NR3R4; a 5- to 6-membered heteroaryl; a 3- to 6-membered heterocycloalkyl; the 5- to 6-membered heteroaryl and 3- to 6-membered heterocycloalkyl may each optionally be substituted by 1 to 3 R; R is independently selected from hydrogen, oxo, halogen, cyano, hydroxy, amino, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylamino-C(O)-, C 3-6 cycloalkylamino-C(O)-, 3- to 6-membered heteroalkylamino-C(O)- or C 3-6 cycloalkyl, wherein the C 1-6 alkyl, C 1-6 alkoxy, C 1-6 alkylamino-C(O)-, C 3-6 cycloalkyl is optionally substituted with 1-3 R'; R3 and / or R4 are each independently selected from hydrogen or C 1-6 alkyl groups, each of which may optionally be substituted by 1-3 Rs; R3 and R4 together with the connected N atom may form a 4- to 7-membered heterocycle, the 4- to 7-membered heterocycle may include 1, 2 or 3 heteroatoms selected from N, O or S as ring atoms, and the 4- to 7-membered heterocycle may each optionally be substituted by 1 to 3 R.

2. The pyrazole ring compound according to claim 1, wherein The R1 is selected from F or -CHF2.

3. The pyrazole ring compound according to claim 1, characterized in that, The R2 is selected from -Me, Cl or CN.

4. The pyrazole ring compound according to claim 1, characterized in that, The R3 and / or R4 are each independently selected from H, Me, Et or i-Pr; R3 and R4 together with the connected N atom may form a morpholine ring or a piperazine ring; and they may each optionally be substituted by 1 to 3 R.

5. The pyrazole ring compound according to claim 1, characterized in that, The 5- to 6-membered heteroaryl is selected from imidazolyl, pyrazolyl, pyridyl and pyrimidinyl; the imidazolyl, pyrazolyl, pyridyl and pyrimidinyl may each optionally be substituted by 1 to 3 R.

6. The pyrazole ring compound according to claim 1, wherein The 3- to 6-membered heterocycloalkyl is selected from piperazinyl; the piperazinyl may each optionally be substituted by 1 to 3 R.

7. The pyrazole ring compound according to claim 1, wherein Each R is independently selected from hydrogen, oxo, halogen, cyano, hydroxy, amino C 1-4 alkyl, C 1-4 alkoxy, C 1-4 alkylamino-C(O)-, C 3-6 cycloalkylamino-C(O)- or 3-6-membered nitrogen-containing heterocycloalkylamino-C(O)-, and the 3-6-membered nitrogen-containing heterocycloalkylamino-C(O)- may optionally be substituted with 1-3 R'.

8. The pyrazole ring compound according to any one of claims 1, 4, 5 and 6, characterized in that A is selected from 9. A pyrazole ring compound having the following general formula I, or a pharmaceutically acceptable salt thereof, wherein, R1 and R2 are each independently selected from hydrogen, halogen, cyano, C 1-6 alkyl, C 3-6 cycloalkyl, and the C 1-6 alkyl and C 3-6 cycloalkyl are optionally substituted by 1-3 R'; R’ is independently selected from hydrogen, halogen or C 1-3 alkyl; X1 and X2 are each independently selected from N or CH; A is selected from 10. The pyrazole ring compound according to claim 9, wherein, The R1 is selected from F or -CHF2.

11. The pyrazole ring compound according to claim 9 or 10, characterized in that, The R2 is selected from -Me, Cl or CN.

12. A pyrazole ring compound selected from any one of the following compounds:

13. A composition, characterized in that, The composition comprises the compound according to any one of claims 1 to 7, 9 - 11, 12.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound according to any one of claims 1 to 7, 9 - 11, 12 and one or more of a pharmaceutically acceptable carrier, diluent or excipient.

15. Use of the compound according to any one of claims 1 to 7, 9 - 11, 12, the composition according to claim 13, or the pharmaceutical composition according to claim 14 in the preparation of an α5-GABA A receptor modulator.

16. The use of the compound according to any one of claims 1 to 7, 9-11, 12 or the composition according to claim 13 in the preparation of a medicament for treating or preventing a disease associated with the α5-GABA A receptor.

17. Use of the compound according to any one of claims 1 to 7, 9 - 11, 12 or the composition according to claim 13 in the preparation of a drug for treating or preventing a disease; the disease is selected from pain, Alzheimer's disease, multi-infarct dementia and stroke.

18. The application according to claim 17, wherein The pain is neuropathic pain, inflammatory pain and cancer pain.

19. The application according to claim 17, characterized in that, The pain is selected from: headache, facial pain, neck pain, shoulder pain, back pain, chest pain, abdominal pain, back pain, lumbar pain, lower limb pain, muscle and bone pain, vascular pain, gout, arthritis pain, visceral pain, pain caused by infectious diseases, polyostotic pain, pain related to sickle cell anemia, autoimmune diseases, multiple sclerosis or inflammation, chronic pain caused by injury or surgery, nociceptive pain, painful diabetes, trigeminal neuralgia, lumbar or cervical radiculopathy pain, glossopharyngeal neuralgia, autonomic reflex pain, reflex sympathetic dystrophy, nerve root avulsion, cancer, chemical injury, toxin, nutritional deficiency, viral or bacterial infection, pain related to degenerative osteoarthropathy.

20. The application according to claim 19, characterized in that, The pain caused by infectious diseases is AIDS or postherpetic neuralgia.

Images