Cinnoline-2-oxide compound as well as preparation method and application thereof

Abstract

The invention relates to a cinnoline-2-oxide compound as well as a preparation method and application thereof. The cinnoline-2-oxide compound is shown as a formula (I) in the specification. According to the cinnoline-2-oxide compound, a nitrogen atom is oxidized into N-oxide to be matched with heteroaryl, good alpha2-GABAA receptor affinity activity and positive regulation activity can be achieved, meanwhile, the cinnoline-2-oxide compound has the potential of remarkably reducing and activating a PXR (pregnane X receptor), then the drug-drug interaction can be reduced, and the clinical application prospect is broad. The medicine curative effect or safety is improved. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to medicinal chemistry, in particular to a cinnoline-2-oxide compound and a preparation method and application thereof. Background Art

[0002] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system of mammals. GABA can regulate the substance of nerve conduction accordingly, so it is widely used to treat various diseases, such as pain, Alzheimer's disease, multi-infarct dementia, stroke, epilepsy, anxiety, itching or depression. In nature, there are two types of GABA receptors, one is GABA A GABA receptor A R), which is a member of the ligand-gated ion channel superfamily, and the other is GABA B GABA receptor B R), which is a member of the G protein-coupled receptor superfamily. GABA in mammals A The receptor subunits that have been discovered include α1-6, β1-4, γ1-3, δ, ε, θ and ρ1-2. Among them, the α subunit, β subunit and γ subunit form a complete functional GABA receptor. A The receptor is essential, and the alpha subunit is essential for benzodiazepines Compounds and GABA A Receptor binding is crucial.

[0003] In GABA A The drug that binds to the receptor allosteric binding site can be a positive allosteric modulator (or positive allosteric modulator) that increases receptor activity, a negative allosteric modulator (or negative allosteric modulator) that decreases receptor activity, or a neutral allosteric modulator (a compound that binds to the allosteric binding site but does not modulate receptor activity) that does not change receptor activity. Studies have shown that GABA containing the α2 subunit A Receptor (α2-GABA A Receptors) may be involved in certain pain states, and positive allosteric modulators of these receptors may be effective analgesics. A The regulatory activity of GABA receptors is currently A Modulators (such as benzodiazepines Therefore, the search for GABA is the main source of side effects (such as sedation, addiction, drowsiness, and amnesia). A receptors interact with α1-GABA A New compounds with fewer receptor-related side effects would have great therapeutic potential.

[0004] Studies have shown that orally active α2-GABAA The receptor positive allosteric modulator AZD7325 can mediate CYP3A4 enzyme induction, causing drug-drug interactions (D. Zhou, Z. Lu, Journal of Clinical Pharmacy & Therapeutics, 2014, 39 (4): 404-410). This may in turn cause adverse effects on drug metabolism, cause drug side effects, and reduce the safety of drug use. For example, if the test substance is a CYP enzyme inducer in clinical practice, it will reduce the exposure of itself (autoinduction) or other co-administered drugs (CYP enzyme substrates) in the body, thereby reducing the efficacy of the drug; at the same time, due to the promotion of the formation of metabolites, it may increase the risk of metabolite-related toxicity and adverse reactions; in addition, it can also promote the prodrug to generate more active metabolites, thereby increasing the risk. Summary of the invention

[0005] Based on this, the present invention provides a method for treating α2-GABA A The invention discloses a cinnoline-2-oxide compound which has good positive regulatory activity on the receptor and can reduce the side effects of the drug by reducing the potential of activating the pregnane X receptor (PXR), and a preparation method and use thereof.

[0006] In the first aspect of the present application, there is provided a cinnoline-2-oxide compound or a pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt thereof as shown in the following formula (I):

[0007]

[0008] Wherein, R1 is C1-C6 alkyl or C3-C6 cycloalkyl;

[0009] R2 is H, D or halogen;

[0010] Ar is n R3-substituted or unsubstituted C1-C10 heteroaryl groups;

[0011] R3 is H, D, C1-C6 alkyl, C1-C6 alkoxy, D-substituted C1-C6 alkyl, D-substituted C1-C6 alkoxy or halogen;

[0012] n is 0, 1, 2, 3 or 4.

[0013] In some embodiments, Ar is n R3-substituted or unsubstituted C2-C5 heteroaryl groups.

[0014] In some embodiments, Ar is n R3 substituted or unsubstituted pyridyl, pyrimidinyl, pyrazolyl, pyridazinyl, quinolyl, isoquinolyl, cinnolinyl, pyridoimidazolyl, indolyl, indazolyl, azaindolyl, azaindazolyl or pyridazinoimidazolyl.

[0015] In some embodiments, Ar is n R3 substituted or unsubstituted pyridyl, pyrimidinyl, pyrazolyl or pyridazinyl.

[0016] In some embodiments, Ar is one of the following groups which are substituted or unsubstituted with n R3:

[0017]

[0018] In some embodiments, R3 is H, D, C1-C3 alkyl, C1-C3 alkoxy, F, Cl or D substituted C1-C3 alkyl.

[0019] In some embodiments, the cinnoline-2-oxide compound is one of the following compounds:

[0020]

[0021]

[0022] The second aspect of the present application provides a method for preparing the cinnoline-2-oxide compound described in the first aspect, which is prepared according to method one or method two;

[0023] The method 1 comprises the following steps:

[0024] Compound A-1 is reacted with compound A-2 to prepare compound A-3;

[0025] Oxidizing compound A-3 to prepare the cinnoline-2-oxide compound;

[0026] Compound A-1

[0027] Compound A-2

[0028] Compound A-3;

[0029] The second method comprises the following steps:

[0030] Oxidizing compound A-1 to prepare compound A-4;

[0031] The compound A-4 is reacted with the compound A-2 to prepare the cinnoline-2-oxide compound;

[0032] Compound A-4;

[0033] Wherein, X is a halogen;

[0034] Optionally, in method 2, before the oxidation, the method further includes performing an amino protection reaction on compound A-1 to prepare compound A-5, then oxidizing compound A-5 to prepare compound A-6, then reacting compound A-6 with compound A-2 to prepare compound A-7, and performing a deprotection reaction on compound A-7 to prepare the cinnoline-2-oxide compound;

[0035] Compound A-5, M represents an amino protecting group;

[0036] Compound A-6.

[0037] Compound A-7.

[0038] In a third aspect of the present application, the cinnoline-2-oxide compound described in the first aspect is provided for preparing a method for treating or preventing GABA A Use in medicine for diseases related to GABA receptors; Optionally, the GABA A Receptor is α1-GABA A Receptors and / or α2-GABA A Receptor.

[0039] The fourth aspect of the present application provides the use of the cinnoline-2-oxide compounds described in the first aspect in the preparation of drugs for treating or preventing pain, Alzheimer's disease, multi-infarct dementia, stroke, epilepsy, anxiety, pruritus or depression.

[0040] During the research process, the present application discovered that the above-mentioned cinnoline-2-oxide compounds can achieve good α2-GABAA receptor affinity activity and positive regulatory activity by oxidizing a nitrogen atom into N-oxide and coordinating with a heteroaryl group. At the same time, it also has the potential to significantly reduce the activation of PXR (pregnane X receptor), thereby reducing drug-drug interactions and improving drug efficacy or safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the X-ray single crystal diffraction structure diagram of the compound prepared in the structure verification example of this application. DETAILED DESCRIPTION

[0042] The following is a further detailed description of the cinnoline-2-oxide compounds of the present invention and their preparation methods and uses in conjunction with specific examples. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0044] The optional scope of the terms "and / or", "or / and", and "and / or" used in this article includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, said any and all combinations include any combination of any two related listed items, any more related listed items, or all related listed items.

[0045] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0046] In the present invention, "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "the first", "the second", "the third", "the fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0047] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0048] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0049] The percentage content involved in the present invention, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0050] The percentage concentrations involved in the present invention, unless otherwise specified, refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0051] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0052] The room temperature in the present invention generally refers to 4°C to 30°C, preferably 20±5°C.

[0053] As used herein, the term "alkyl" refers to a monovalent residue formed by the loss of a hydrogen atom from a saturated hydrocarbon containing a primary (normal) carbon atom, or a secondary carbon atom, or a tertiary carbon atom, or a quaternary carbon atom, or a combination thereof. Phrases containing this term, for example, "C1-C6 alkyl" refers to an alkyl group containing 1 to 6 carbon atoms, and each occurrence can be independently C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl. Suitable examples include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -C H(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl -1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl ( -CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3.

[0054] Herein, the term "cycloalkyl" refers to a non-aromatic hydrocarbon containing ring carbon atoms, which can be a monocyclic alkyl, a spirocyclic alkyl, or a bridged cycloalkyl. Phrases containing this term, for example, "C3-C6 cycloalkyl" refers to a cycloalkyl containing 3 to 6 carbon atoms, each occurrence of which can be independently C3 cycloalkyl, C4 cycloalkyl, C5 cycloalkyl, C6 cycloalkyl. Suitable examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. In addition, "cycloalkyl" may also contain one or more double bonds, and representative examples of cycloalkyl containing double bonds include cyclopentenyl, cyclohexenyl, cyclohexadienyl, and cyclobutadienyl.

[0055] Herein, the term "alkoxy" refers to a group having the structure -O-alkyl, i.e., an alkyl group as defined above is connected to an adjacent group of a parent core structure via an oxygen atom. Phrases containing the term, for example, "C1-C6 alkoxy" means that the alkyl portion contains 1 to 6 carbon atoms, and each occurrence can be independently C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, C6 alkoxy. Suitable examples include, but are not limited to, methoxy (-O-CH3 or -OMe), ethoxy (-O-CH2CH3 or -OEt) and tert-butoxy (-OC(CH3)3 or -OtBu).

[0056] Herein, the term "aryl" refers to an aromatic hydrocarbon group derived from an aromatic ring compound by removing a hydrogen atom. It can be a monocyclic aromatic group, a condensed aromatic group, or a polycyclic aromatic group. For polycyclic rings, at least one is an aromatic ring system.

[0057] Herein, the term "heteroaryl" refers to an aromatic group in which at least one carbon atom is replaced by a non-carbon atom, and the non-carbon atom may be an N atom, an O atom, an S atom, etc. For example, "C1-C10 heteroaryl" refers to a heteroaryl group containing 1 to 10 carbon atoms, and each occurrence may be independently a C1 heteroaryl group, a C2 heteroaryl group, a C3 heteroaryl group, a C4 heteroaryl group, a C5 heteroaryl group, a C6 heteroaryl group, a C7 heteroaryl group, a C8 heteroaryl group, a C9 heteroaryl group or a C10 heteroaryl group. Suitable examples include, but are not limited to, furanyl, benzofuranyl, thienyl, benzothienyl, pyrrolyl, pyrazolyl, triazolyl, imidazolyl, oxazolyl, oxadiazolyl, thiazolyl, tetrazolyl, indolyl, carbazolyl, pyrroloimidazolyl, pyrrolopyrrolyl, thienopyrrolyl, thienothiphenyl, furopyrrolyl, furofuranyl, thienofuranyl, benzisoxazolyl, benzisothiazolyl, benzimidazolyl, pyridoimidazolyl, indazolyl, azaindolyl, azaindazolyl, pyridazinoimidazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, quinolyl, isoquinolyl, cinnolinyl, o-naphthyl, quinoxalinyl, phenanthridinyl, primidinyl, quinazolinyl, and quinazolinonyl.

[0058] As used herein, the term "halogen" refers to F, Cl, Br or I.

[0059] As used herein, the term "pharmaceutically acceptable salt" refers to a salt suitable for use as a drug formed by any compound in the structure shown and an acid or base. Pharmaceutically acceptable salts include inorganic salts and organic salts. Among them, one type of salt is a salt formed by a compound of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid. Another type of salt is a salt formed by the compounds of the present invention and a base. The base suitable for forming the salt includes but is not limited to: alkali metal salts (e.g., sodium salts or potassium salts), alkaline earth metal salts (e.g., magnesium salts or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed by morpholine, piperazine, and lysine, respectively. The nomenclature used herein is named according to the IUPAC system nomenclature. Any open valence bonds occurring on carbon, oxygen, sulfur, or nitrogen atoms in the structure given herein indicate the presence of hydrogen atoms.

[0060] As used herein, "patient" is defined as any warm-blooded animal, such as but not limited to mice, guinea pigs, dogs, horses or humans, preferably the patient is a human.

[0061] As used herein, the term "treat" refers to any of the following: (1) alleviating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that triggers a disease; or (3) slowing the progression of one or more biological manifestations of a disease.

[0062] As used herein, the term "prevent" refers to reducing the risk of developing a disease.

[0063] As used herein, the term "drug" includes any agent, compound, composition or mixture that provides a physiological and / or pharmacological effect in vivo or in vitro, and often provides a beneficial effect. The scope of the physiological and / or pharmacological effect produced by the "drug" in vivo is not particularly limited, and may be a systemic effect or may only produce an effect locally. The activity of the "drug" is not particularly limited, and may be an active substance that can interact with other substances, or may be an inert substance that does not interact.

[0064] In some examples of the present application, a cinnoline-2-oxide compound or a pharmaceutically acceptable salt, solvate, or a pharmaceutically acceptable salt solvate thereof as shown in the following formula (I) is provided:

[0065]

[0066] Wherein, R1 is C1-C6 alkyl or C3-C6 cycloalkyl;

[0067] R2 is H, D or halogen;

[0068] Ar is n R3-substituted or unsubstituted C1-C10 heteroaryl groups;

[0069] R3 is H, D, C1-C6 alkyl, C1-C6 alkoxy, D-substituted C1-C6 alkyl, D-substituted C1-C6 alkoxy or halogen; it is understood that when there are two or more R3, each R3 may be the same or different;

[0070] n is 0, 1, 2, 3 or 4.

[0071] Without limitation, the C1-C10 heteroaryl group includes the following groups: pyridyl, pyrimidinyl, pyrazolyl, pyridazinyl, quinolyl, isoquinolyl, cinnolinyl, pyridoimidazolyl, indolyl, indazolyl, azaindolyl, azaindazolyl or pyridazinoimidazolyl. Further, the C1-C10 heteroaryl group includes one of the following groups:

[0072]

[0073] In some of the examples, Ar is n R3-substituted or unsubstituted C2-C5 heteroaryl groups.

[0074] In some of these examples, Ar is n R3 substituted or unsubstituted pyridyl, pyrimidinyl, pyrazolyl, pyridazinyl, quinolyl, isoquinolyl, cinnolinyl, pyridoimidazolyl, indolyl, indazolyl, azaindolyl, azaindazolyl or pyridazinoimidazolyl. Further, Ar is n R3 substituted or unsubstituted pyridyl, pyrimidinyl, pyrazolyl or pyridazinyl.

[0075] In some of these examples, Ar is one of the following groups which are substituted or unsubstituted with n R3:

[0076] It is understood that n R3 can be substituted at any H group in the above groups.

[0077] In some examples, R3 is H, D, C1-C3 alkyl, C1-C3 alkoxy, F, Cl or C1-C3 alkyl substituted by D. Further, R3 is H, D, F, Cl, -CD3, methoxy or methyl.

[0078] In some of the examples, R3 is H, D, C1-C3 alkyl, C1-C3 alkoxy, F, Cl or D-substituted C1-C3 alkyl.

[0079] In some of these examples, n is 0, 1, or 2.

[0080] In some examples, R1 is C1-C4 alkyl or C3-C4 cycloalkyl. Further, R1 is C2 alkyl, C3 alkyl or cyclopropyl.

[0081] In some examples, R2 is H, D or F. Further, R2 is H or F.

[0082] Without limitation, the cinnoline-2-oxide compound is a compound as shown below:

[0083]

[0084] Some other examples of the present application provide methods for preparing the cinnoline-2-oxide compounds as described above. Without limitation, the preparation can be carried out according to method one or method two, respectively.

[0085] In some examples, the method includes the following steps:

[0086] Compound A-1 is reacted with compound A-2 to prepare compound A-3;

[0087] Oxidizing compound A-3 to prepare the cinnoline-2-oxide compound;

[0088] Compound A-1

[0089] Compound A-2

[0090] Compound A-3;

[0091] Wherein, X is a halogen.

[0092] In some examples, the second method includes the following steps:

[0093] Oxidizing compound A-1 to prepare compound A-4;

[0094] The compound A-4 is reacted with the compound A-2 to prepare the cinnoline-2-oxide compound;

[0095] Compound A-4;

[0096] Wherein, X is a halogen;

[0097] Optionally, in method 2, before the oxidation reaction, the method further includes performing an amino protection reaction on compound A-1 to prepare compound A-5, then oxidizing compound A-5 to prepare compound A-6, then reacting compound A-6 with compound A-2 to prepare compound A-7, and performing a deprotection reaction on compound A-7 to prepare the cinnoline-2-oxide compound;

[0098] Compound A-5, M represents an amino protecting group;

[0099] Compound A-6.

[0100] Compound A-7.

[0101] In some examples, the oxidation in method 1 or method 2 includes the following steps:

[0102] Compound A-3 or Compound A-1 is mixed with a peroxide and a solvent and then reacted.

[0103] In some examples, the reaction can be carried out at room temperature. Without limitation, the reaction time is 2 h to 6 h.

[0104] In some examples, the peroxide is one or more of m-chloroperbenzoic acid, peracetic acid and hydrogen peroxide.

[0105] In some examples, the solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane.

[0106] In some examples, the reaction of compound A-1 with compound A-2 in method 1 or the reaction of compound A-4 with compound A-2 in method 2 comprises the following steps:

[0107] Compound A-1 or compound A-4 is mixed with compound A-2, an inorganic base, a palladium catalyst and a solvent to carry out a reaction.

[0108] In some examples, the reaction can be carried out at 50° C. to 105° C., without limitation, and the reaction time is 0.01 h to 15 h.

[0109] In some examples, the palladium catalyst is one or both of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and methanesulfonic acid(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-3-yl)palladium(II).

[0110] In some examples, the solvent is one or more of 1,4-dioxane and water. Without limitation, the volume ratio of 1,4-dioxane to water is (0-10):1.

[0111] In some examples, the inorganic base is one or more of potassium phosphate, cesium carbonate, potassium carbonate and sodium carbonate.

[0112] In some other examples of the present application, the cinnoline-2-oxide compounds described above are also provided for preparing a method for treating or preventing GABA A The invention relates to a drug for a disease related to a GABA receptor. A Receptor is α1-GABA A Receptors and / or α2-GABA A Receptor.

[0113] Without limitation, the A Diseases associated with the receptor are pain, Alzheimer's disease, multi-infarct dementia, stroke, epilepsy, anxiety, pruritus or depression.

[0114] In some other examples of the present application, the use of the cinnoline-2-oxide compounds described above in the preparation of drugs for treating or preventing pain, Alzheimer's disease, multi-infarct dementia, stroke, epilepsy, anxiety, pruritus or depression is also provided.

[0115] The following are specific examples. The reagents and raw materials used in the examples are all commercially available. The experimental methods without specific conditions in the examples can be selected according to traditional methods and conditions, or according to the product instructions.

[0116] The synthesis routes of the compounds involved in Example 1 are summarized as follows:

[0117]

[0118] Example 1: 4-amino-7-fluoro-8-(2-fluoropyridin-3-yl)-3-(propylcarbamoyl)cinnoline 2-oxide

[0119]

[0120] Synthesis of intermediate iii:

[0121] Step 1: 2-((2-bromo-3-fluorophenyl)diazenyl)-2-cyano-N-propylacetamide

[0122] Solution A: Add 2-bromo-3-fluoroaniline (50 g, 263 mmol) to a mixed solution of water (30 mL), acetic acid (2M, 60 mL) and concentrated hydrochloric acid (60 mL), and stir at room temperature for 20 minutes. Slowly add an aqueous solution (30 mL) of sodium nitrite (21.8 g, 316 mmol) at 0°C, and stir for 30 minutes while maintaining 0°C.

[0123] Solution B: Dissolve 2-cyano-N-propylacetamide (49.7 g, 394 mmol) in ethanol (85 mL). Add an aqueous solution (85 mL) of sodium acetate (38.7 g, 472 mmol) to the solution at 0°C and stir for 20 minutes.

[0124] Mixing of solutions A and B: Add solution A to solution B at 0°C and continue stirring for 3 hours. Filter and dry the filter cake to obtain intermediate ii (51 g, yield 59%) as a reddish brown solid.

[0125] Step 2: 4-amino-8-bromo-7-fluoro-N-propylcinnoline-3-carboxamide

[0126] 2-[2-(2-bromo-3-fluorophenyl)diazenyl]-2-cyano-N-propylacetamide (50 g, 153 mmol) and aluminum chloride (61.1 g, 459 mmol) were added to toluene (200 mL) in sequence and stirred at 90°C for 3 hours. 250 mL of water was added, and then extracted with ethyl acetate (250 ml*2), concentrated, and column chromatography was performed to obtain the target product intermediate iii (45 g, yield 90%) as a brown solid.

[0127] Synthesis of target product v:

[0128] Route 1:

[0129] Step 1: 4-amino-8-bromo-7-fluoro-3-(propylcarbamoyl)cinnoline 2-oxide

[0130] 4-Amino-8-bromo-7-fluoro-N-propylcinnoline-3-carboxamide (3 g, 9.17 mmol) and 3-chloroperoxybenzoic acid (4.75 g, 27.5 mmol) were added to dichloromethane (25 mL) in sequence and stirred at room temperature for 3 hours. 150 mL of saturated aqueous sodium thiosulfate solution was added, and then extracted with dichloromethane (150 mL*2). The organic phases were combined, concentrated, and column chromatography was performed to obtain intermediate iv-1 (580 mg, yield 18%) as a yellow solid.

[0131] Step 2: 4-amino-7-fluoro-8-(2-fluoropyridin-3-yl)-3-(propylcarbamoyl)cinnoline 2-oxide

[0132] 4-Amino-8-bromo-7-fluoro-3-(propylcarbamoyl)cinnoline 2-oxide (560mg, 1.63mmol), (2-fluoropyridin-3-yl)boronic acid (340mg, 2.44mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (240mg, 0.33mmol) and cesium carbonate (1.06g, 3.26mmol) were added to a mixed solution of 1.4-dioxane (16mL) and water (4mL) in sequence, and stirred at 100°C for 3 hours under argon protection. After concentration, 150mL of aqueous solution was added, and then extracted with dichloromethane (150mL*2), the organic phases were combined, concentrated, and column chromatography was performed to obtain the target product (105mg, yield 18%) as a yellow solid.

[0133] 1 H NMR (400MHz, DMSO-d6) δ10.02(t,J=5.5Hz,1H),9.01(bs,2H),8.58-8.50(m,1H),8.38(d,J=4.0Hz,1H),8.08(t,J=8 .2Hz,1H),7.67(t,J=9.0Hz,1H),7.54(t,J=6.0Hz,1H),3.31-3.26(m,2H),1.61-1.49(m,2H),0.91(t,J=7.1Hz,3H).

[0134] LC-MS: m / z[M+1] + =360.

[0135] Route 2:

[0136] Step 1: 4-amino-7-fluoro-8-(2-fluoropyridin-3-yl)-N-propylcinnoline-3-carboxamide

[0137] 4-Amino-8-bromo-7-fluoro-N-propylcinnoline-3-carboxamide (150 mg, 0.46 mmol), (2-fluoropyridin-3-yl)boric acid (130 mg, 0.92 mmol), cesium carbonate (450 mg, 1.38 mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium dichloride (37 mg, 0.05 mmol) were added to a mixed solvent of 1,4-dioxane (5 mL) and water (0.5 mL) in sequence, and stirred at 100° C. for 12 hours under argon protection. The reaction solution was concentrated and purified by column chromatography (petroleum ether: ethyl acetate = 2:1) to obtain intermediate iv-2 (150 mg, yield 67%) as a white solid.

[0138] Step 2: 4-amino-7-fluoro-8-(2-fluoropyridin-3-yl)-3-(propylcarbamoyl)cinnoline 2-oxide

[0139] Dissolve 4-amino-7-fluoro-8-(2-fluoropyridin-3-yl)-N-propylcinnoline-3-carboxamide (100 mg, 0.29 mmol) in dichloromethane (20 mL), add m-chloroperbenzoic acid (500 mg, 2.9 mmol) in batches and stir at room temperature for 4 hours. The reaction solution is washed with saturated sodium thiosulfate aqueous solution (20 mL) and saturated sodium bicarbonate aqueous solution (20 mL) in turn, and the organic phase is dried over anhydrous sodium sulfate, concentrated, and purified on a preparative plate (dichloromethane) to obtain the target product (27 mg, yield 26%) as a yellow solid. The NMR data is consistent with route 1.

[0140] Route 3:

[0141] Step 1: tert-butyl (8-bromo-3-((tert-butoxycarbonyl)(propyl)carbamoyl)-7-fluorocinnolin-4-yl)(tert-butoxycarbonyl)carbamate

[0142] Dissolve 4-amino-8-bromo-7-fluoro-N-propylcinnoline-3-carboxamide (50g, 150mmol), di-tert-butyl dicarbonate (100g, 450mmol), 4-dimethylaminopyridine (3.7g, 31mmol) and triethylamine (77g, 760mmol) in tetrahydrofuran (1L) and stir at room temperature overnight. Concentrate the reaction solution, dissolve in dichloromethane (500mL), wash with saturated aqueous ammonium chloride solution (500mL*3), separate the liquids, dry and concentrate the organic phase, and purify by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain a light yellow solid (80g, yield 83%). LC-MS: m / z[M+1] + =627.

[0143] Step 2: 4-(bis(tert-butoxycarbonyl)amino)-8-bromo-3-((tert-butoxycarbonyl)(propyl)carbamoyl)-7-fluorocinnoline 2-oxide

[0144] Dissolve tert-butyl (8-bromo-3-((tert-butoxycarbonyl)(propyl)carbamoyl)-7-fluorocinnolin-4-yl)(tert-butoxycarbonyl)carbamate (80g, 127mmol) in dichloromethane (1L), add m-chloroperbenzoic acid (44g, 255mmol) in batches, and stir at room temperature for 16 hours. The reaction solution is slowly added to a saturated aqueous sodium thiosulfate solution (1L), stirred to quench, separated, the organic phase is washed with a saturated aqueous sodium bicarbonate solution (1L), separated, the organic phase is dried, and purified by column chromatography (petroleum ether: ethyl acetate = 10:1 to 1:1) to obtain a light yellow solid (70g, yield 85%). LC-MS: m / z[M+1] + =643.

[0145] Step 3: 4-(bis(tert-butoxycarbonyl)amino)-3-((tert-butoxycarbonyl)(propyl)carbamoyl)-7-fluoro-8-(2-fluoropyridin-3-yl)cinnoline 2-oxide

[0146] 4-(Bis(tert-butoxycarbonyl)amino)-8-bromo-3-((tert-butoxycarbonyl)(propyl)carbamoyl)-7-fluorocinnoline 2-oxide (20 g, 31 mmol), (2-fluoropyridin-3-yl)boronic acid (8.7 g, 62 mmol), methanesulfonic acid (2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-3-yl)palladium(II) (CAS No.:1445085-82-4, 2.4 g, 3.1 mmol) and cesium carbonate (30 g, 93 mmol) were dissolved in a mixed solution of 1,4-dioxane (400 mL) and water (80 mL), and stirred at 85 degrees Celsius for 16 hours under argon protection. The aqueous phase was separated, the organic phase was concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain a yellow solid (17 g, yield 83%). LC-MS: m / z [M+1] + =660.

[0147] Step 4: 4-amino-7-fluoro-8-(2-fluoropyridin-3-yl)-3-(propylcarbamoyl)cinnoline 2-oxide

[0148] 4-(bis(tert-butoxycarbonyl)amino)-3-((tert-butoxycarbonyl)(propyl)carbamoyl)-7-fluoro-8-(2-fluoropyridin-3-yl)cinnoline 2-oxide (1g, 1.52mmol) was added to a mixed solution of dichloromethane (8mL) and trifluoroacetic acid (8mL) and stirred for 3 hours at room temperature. Concentrate, add 60ml of saturated aqueous sodium bicarbonate solution, extract with dichloromethane (60mL*2), combine the organic phases, dry over anhydrous sodium sulfate, and beat to obtain the target product as a yellow solid (447mg, yield 82%). The NMR data is consistent with Route 1.

[0149] Example 2: 4-amino-7-fluoro-8-(2-fluoropyridin-3-yl-6-deuterium)-3-(propylcarbamoyl)cinnoline 2-oxide

[0150]

[0151] Step 1: (2-fluoropyridin-3-yl-6-deuterated)boronic acid

[0152] Dissolve (2-fluoropyridin-3-yl-6-bromo)boronic acid (200 mg, 0.91 mmol) in deuterated methanol (CD3OD, 2 mL), add palladium carbon (30 mg, palladium carbon was stirred in deuterated methanol for 24 hours), stir at room temperature for 2 hours under deuterium atmosphere, filter through celite pad, concentrate the organic phase and use it directly in the next step.

[0153] Step 2: 4-amino-7-fluoro-8-(2-fluoropyridin-3-yl-6-deuterium)-3-(propylcarbamoyl)cinnoline 2-oxide

[0154] The experimental operation was the same as that of Example 1, Route 3 (Step 3, Step 4) to obtain the target product as a light yellow solid (400 mg, yield 73%).

[0155] 1 HNMR(400MHz,DMSO-d6)10.02(br.s.,1H),9.00(br.s.,2H),8.57-8.50(m,1H),8.14-8.06(m,1H),7.6 8(t,J=9.0Hz,1H),7.55(d,J=6.8Hz,1H),3.31-3.22(m,2H),1.63-1.50(m,2H),0.92(t,J=7.1Hz,3H).

[0156] LC-MS: m / z[M+1] + =361.

[0157] Example 3: 4-amino-7-fluoro-8-(2-(methyl-d3)pyridin-3-yl)-3-(propylcarbamoyl)cinnoline 2-oxide

[0158]

[0159] Step 1: (Methyl-d3)zinc(II) iodide

[0160] Iodomethane-d (2 g, 13.8 mmol), zinc powder (1.35 g, 20.7 mmol) and iodine (0.18 g, 0.69 mmol) were added to N,N-dimethylacetamide (8 mL) in sequence, and stirred at room temperature for 3 hours under argon protection. The reaction solution was directly used for the next step.

[0161] Step 2: 3-Bromo-2-(methyl-d3)pyridine

[0162] 3-Bromo-2-iodopyridine (1.8 g, 6.34 mmol), tetrakis(triphenylphosphine)palladium (370 mg, 0.32 mmol) and (methyl-d3)zinc(II) iodide (1.33 g, 6.34 mmol) were added to N,N-dimethylacetamide (5 mL) in sequence, and stirred at 40°C for 5 hours under argon protection. The target product (800 mg, yield 72%) was obtained as a yellow liquid by distillation under reduced pressure at 80°C. LCMS: m / z[M+H]+=175.

[0163] Step 3: 3-bromo-2-(methyl-d3)pyridine (800 mg, 4.56 mmol), potassium acetate (960 mg, 9.6 mmol), boronic acid naphthalene (2430 mg, 9.6 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium dichloromethane complex (CAS No.: 95464-05-4, 200 mg, 0.25 mmol) were added to ethylene glycol dimethyl ether (20 mL) in sequence, and stirred at 80 ° C for 5 hours under argon protection. Concentrate, add water (50 mL), and extract with ethyl acetate (50 mL*2). The organic layer was concentrated and the target compound was obtained as a black oil by column chromatography (500 mg, yield 49%).

[0164] Step 4: The experimental operation is the same as that of Route 3 (Step 3 and Step 4) in Example 1 to obtain the target product.

[0165] 1 HNMR(400MHz,DMSO-d6)10.08(s,1H),9.01(s,1H),8.60-8.54(m,1H),8.52-8.42(m,1H),7.71 -7.60(m,2H),7.40-7.31(m,1H),3.31-3.23(m,2H),1.63-1.48(m,2H),0.92(t,J=7.1Hz,3H).

[0166] LC-MS: m / z[M+1] + =359.

[0167] Example 4: 4-amino-7-fluoro-8-(2-chloro-pyridin-3-yl)-3-(propylcarbamoyl)cinnoline 2-oxide

[0168]

[0169] The experimental operation is the same as that of Route 3 (Step 3 and Step 4) in Example 1 to obtain the target product.

[0170] 1HNMR(400MHz,DMSO-d6)9.98(s,1H),8.98(s,1H),8.60-8.50(m,1H),7.95(d,J=7.3Hz,1H),7.6 7(t,J=8.8Hz,1H),7.62-7.55(m,1H),3.32-3.23(m,2H),1.63-1.51(m,2H),0.96-0.88(m,3H).

[0171] LC-MS: m / z[M+1] + =376.

[0172] Example 5: 4-amino-7-fluoro-8-(2,4-dimethoxypyrimidin-5-yl)-3-(propylcarbamoyl)cinnoline 2-oxide

[0173]

[0174] The experimental operation is the same as that of Route 3 (Step 3 and Step 4) in Example 1 to obtain the target product.

[0175] 1 H NMR(400MHz,DMSO-d6)10.07(s,1H)8.99(s,2H)8.37-8.50(m,1H)8.27(s,1H)7.53-7.6 5(m,1H)3.99(s,3H)3.86(s,3H)3.22-3.31(m,2H)1.49-1.60(m,2H)0.85-0.98(m,3H).

[0176] LC-MS: m / z[M+1] + =403.

[0177] Example 6: 4-amino-7-fluoro-8-(1-methyl-1H-pyrazol-4-yl)-3-(propylcarbamoyl)cinnoline 2-oxide

[0178]

[0179] The experimental operation is the same as that of Route 3 (Step 3 and Step 4) in Example 1 to obtain the target product.

[0180] 1 H NMR(400MHz,DMSO-d6)10.32(s,1H)9.04(s,2H)8.31(s,1H)8.18-8.25(m,1H)8.03(s,1 H)7.53-7.60(m,1H)3.95(s,3H)3.28-3.34(m,2H)1.53-1.62(m,2H)0.94-0.88(m,3H).

[0181] LC-MS: m / z[M+1] + =345.

[0182] Example 7: 4-amino-7-fluoro-8-(5-fluoro-6-methoxypyridin-3-yl)-3-(ethylcarbamoyl)cinnoline 2-oxide

[0183]

[0184] Step 1: 4-amino-8-bromo-7-fluorocinnoline-3-carboxylic acid

[0185] Dissolve 4-amino-8-bromo-7-fluoro-N-propylcinnoline-3-carboxamide (5 g, 15 mmol) in aqueous sulfuric acid solution (80%, 50 mL) and stir at 100°C for 16 hours. Pour into 500 mL of ice water, a large amount of solid precipitates, filter and dry to obtain the title product as a brown solid (4 g, crude product).

[0186] Step 2: 4-amino-8-bromo-N-ethyl-7-fluorocinnoline-3-carboxamide

[0187] 4-Amino-8-bromo-7-fluorocinnoline-3-carboxylic acid (2g, 7mmol), HATU (5.7g, 15mmol), ethylamine hydrochloride (1.2g, 15mmol) and triethylamine (3g, 30mmol) were added to N'N-dimethylformamide (20mL) in sequence and stirred at room temperature for 16 hours. Water (50mL) was added and extracted with ethyl acetate (50mL*2), the organic phase was concentrated and the title compound was obtained by column chromatography as a yellow solid (1.5g, yield 68%).

[0188] Step 3: The experimental operation is the same as that of Route 3 in Example 1 to obtain the target product.

[0189] 1 HNMR(400MHz, DMSO-d6)10.02(s,1H),8.99(s,2H),8.46(dd,J=5.4,9.3Hz,1H),8.30(d,J=2.9Hz,1H), 7.80(dd,J=2.9,8.3Hz,1H),7.63-7.53(m,1H),3.78(s,3H),3.38-3.33(m,2H),1.14(t,J=7.3Hz,3H).

[0190] LC-MS: m / z[M+1] + =376.

[0191] Example 8: 4-amino-7-fluoro-8-(2-fluoro-6-methylpyridin-3-yl)-3-(ethylcarbamoyl)cinnoline 2-oxide

[0192]

[0193] The experimental operation is the same as that of Route 3 (Step 3 and Step 4) in Example 1 to obtain the target product.

[0194] 1 HNMR(400MHz, DMSO-d6)9.99(s,1H),8.99(s,2H),8.49(dd,J=5.6,9.0Hz,1H),7.92(t,J=8.6Hz,1H ),7.64(t,J=9.0Hz,1H),7.40-7.32(m,1H),3.39-3.34(m,2H),2.53(s,3H),1.14(t,J=7.1Hz,3H).

[0195] LC-MS: m / z[M+1] + =360.

[0196] Example 9: 4-amino-3-(cyclopropylcarbamoyl)-7-fluoro-8-(2-methoxypyridin-3-yl)cinnoline 2-oxide

[0197]

[0198] The experimental operation was the same as in Example 7 to obtain the target product.

[0199] 1 HNMR(400MHz, DMSO-d6)9.93(d,J=3.4Hz,1H),8.82(s,2H),8.41(dd,J=5.9,8.8Hz,1H),8.28(d,J=3.9Hz,1H),7.66(d,J=7 .3Hz,1H),7.58(t,J=9.0Hz,4H),7.18-7.07(m,1H),3.77(s,3H),2.89-2.84(m,1H),0.78-0.72(m,2H),0.59-0.53(m,2H).

[0200] LC-MS: m / z[M+1] + =370.

[0201] Example 10: 4-amino-3-(cyclopropylcarbamoyl)-7-fluoro-8-(3,6-dimethoxypyridazin-4-yl)cinnoline 2-oxide

[0202]

[0203] The experimental operation was the same as in Example 7 to obtain the target product.

[0204] 1 HNMR(400MHz, DMSO-d6)9.80(s,1H),8.80(s,2H),8.49(dd,J=5.9,9.3Hz,1H),7.63(t,J=9.0Hz,1H), 7.32-7.26(m,1H),4.02(s,3H),3.87(s,3H),2.90-2.85(m,1H),0.79-0.70(m,2H),0.59-0.55(m,2H).

[0205] LC-MS: m / z[M+1] + =401

[0206] Embodiment 11: 4-Amino-8-(2-fluoropyridin-3-yl)-3-(propylcarbamoyl)cinnoline 2-oxide

[0207]

[0208] The experimental operation was the same as that of Example 1, route 3, to obtain the target product.

[0209] 1 H NMR(400MHz,DMSO-d6)10.18(s,1H)8.98(s,2H)8.38-8.44(m,1H)8.29-8.36(m,1H)7.96-8.07(m,1H)7.83- 7.88(m,1H)7.60-7.71(m,1H)7.45-7.55(m,1H)3.32-3.26(dm,2H)1.58-1.52(m,2H)0.91(t,J=7.34Hz,3H).

[0210] LC-MS: m / z[M+1] + =342.

[0211] Example 12: 4-amino-8-(2-(methyl-d3)pyridin-3-yl)-3-(propylcarbamoyl)cinnoline 2-oxide

[0212]

[0213] The experimental operation was the same as that of Example 1, route 3, to obtain the target product.

[0214] 1H NMR(400MHz,DMSO-d 6)10.24(s,1H)9.01(s,2H)8.53-8.48(m,1H)8.30-8.39(m,1H)7.72-7.65(m,2H)7.54-7 .60(m,1H)7.27-7.35(m,1H)3.31-3.25(m,2H)1.49-1.62(m,2H)0.90(t,J=7.34Hz,3H).

[0215] LC-MS: m / z[M+1] + =341.

[0216] Example 13: 4-amino-3-(cyclopropylcarbamoyl)-8-(3,6-dimethoxypyridazin-4-yl)cinnoline 2-oxide

[0217]

[0218] The experimental operation was the same as in Example 7 to obtain the target product.

[0219] 1 H NMR(400MHz, DMSO-d6)10.01(s,1H)8.85(m,2H)8.35-8.41(m,1H)7.73-7.81(m,1H)7.57-7.68( m,1H)7.20(s,1H)4.01(s,3H)3.83(s,3H)2.83-2.93(m,1H)0.70-0.80(m,2H)0.50-0.64(m,2H).

[0220] LC-MS: m / z[M+1] + =383

[0221] Structural verification example: 4-amino-8-(3,5-difluorophenyl)-7-fluoro-3-(propylcarbamoyl)cinnoline 2-oxide

[0222]

[0223] The synthesis method is similar to that of Route 2 of Example 1, except that (2-fluoropyridin-3-yl)boric acid is replaced by (3,5-difluorophenyl)boric acid.

[0224] Structural identification: The crystal was determined by single crystal X-ray diffraction to determine the structure. Figure 1 As shown in Tables 1 and 2.

[0225] Table 1

[0226]

[0227] Table 2

[0228]

[0229]

[0230] Test example:

[0231] The α, β and γ subunits form a complete functional GABA A In this test example, the liposome transfection method (the method can be referred to Felgner, PL, et al. Proceedings of the National Academy of Sciences, 1987, 84: 7413-7417) was used to construct and screen for stable expression of α2-GABA A Receptor (α2-GABA A R) HEK293 cells. α2-GABA A The R-HEK293 cell model simultaneously expresses the α2 subunit (protein sequence see GenBank accession number: NM_000807.4), the β3 subunit (protein sequence see GenBank accession number: NM_000814.5) and the γ2 subunit (protein sequence see GenBank accession number: NM_000816.3).

[0232] The above cell lines were subcultured. A R-HEK293 cells were used to investigate the effects of compounds on GABA A Benzodiazepine receptors (BZD) Binding site affinity test. α2-GABA A During the subculturing process of R-HEK293 cells, some suspended cells were plated on glass slides pretreated with Poly-D-Lysine for electrophysiological testing (the method can be found in paragraph 0586 of patent CN 107344936 A).

[0233] (1) Compounds for α2-GABA A Receptor affinity activity

[0234] Through competition 3 H-flunitrazepam (isotope 3 H-labeled flunitrazepam) and stably expressed human α2-GABA A The binding site of the membrane protein BZD in R-HEK293 cells was used to determine the effect of the compounds on α2-GABA A Receptor affinity.

[0235] Membrane preparation: cells were suspended in 50mM Tris-HCl buffer (pH=7.4), homogenized on ice for 20 seconds 10 times by homogenizer, and centrifuged at 4°C, 1000g for 10min. The supernatant was taken and the above steps were repeated. The supernatant was centrifuged at 4°C (33800g; Thermo, rotor: A27-8x50) for 60min, and the precipitate was resuspended in Tris buffer (50mMTris-HCl, 10mMMgCl2, 0.5mM EDTA, 10% glycerol). The protein content was determined (BCA (bicinchoninic acid) protein quantification test method based on copper ion reduction method, BCA kit purchased from Pierce), 1mL aliquots were prepared, and stored at -80°C.

[0236] Radioligand competition binding assay: This assay was performed in a 200 μL system (96-well plate) containing 100 μL of cell membranes. 3 The concentration of H-flunitrazepam was 1 nM and the concentration of the test compound was 1 x 10 -5 -10 -6 M range. Flumazenil was used as a control. 1 μL 2mM flumazenil (final concentration 10 μM) was added to the low signal control well (Low control, LC), and 1 μL dimethyl sulfoxide was added to the high signal control well (High control, HC). The final concentration of the target membrane protein was 5 μg / well. The storage solution of all test compound samples was 10 mM dimethyl sulfoxide solution. The sample working concentration was to dilute all samples to 0.2 mM with dimethyl sulfoxide, and then perform 4-fold continuous gradient dilution, for a total of 8 concentration gradients. Seal the 96-well plate with a sealing film and incubate it on a shaker at room temperature for 1 hour. At the same time, soak the GF / C filter plate with a plate soaking buffer (0.3% PEI (polyethyleneimine, purchased from: Sigma-Aldrich, model: P314), stored at 4°C) for at least 0.5 hours. After the incubation, the cells were collected on the GF / C filter plate using a cell harvester and washed 4 times with a plate washing buffer (50 mM Tris-HCl, pH 7.4, stored at 4°C). After drying in a 50°C oven for 1 hour, the bottom of the dried GF / C filter plate was sealed with a membrane and the residual radioactivity of the membrane was detected by liquid scintillation counting. 50 μL of scintillation fluid was added to each well and sealed. Microbeta 2 (Microplate counter, purchased from PerkinElmer, model: CNLL0153) reading. Calculate the sample to be tested 3 H-flunitrazepam and GABA AThe inhibitory activity of receptor membrane protein binding was calculated by dose-effect curve fitting (GraphPadPrism 5 software) for each sample to be tested. 50 and through IC 50 Calculate the K of the sample i , thereby evaluating the interaction of compounds with α2-GABA A Binding capacity of receptor BZD site.

[0237] Assay compounds and expression of human α2-GABA A Representative test results obtained by the method of binding affinity of the BZD site of R-HEK293 cell membrane protein are shown in Table 1.

[0238] (2) Effects of compounds on different GABA subtypes A Functional activity of receptors

[0239] Electrophysiological detection of the effect of the drug on α1-GABA A Receptors and α2-GABA A The specific method is as follows:

[0240] Compound concentration setting: The final concentration of the compounds used in compound screening was 100 nM. A receptor cell lines, GABA concentration is 0.05-0.07 μM (about EC 2～4 );For the expression of α2-GABA A receptor cell lines, GABA concentration is 0.10-0.11 μM (about EC 7～8 ). The whole-cell patch clamp technique was used for electrophysiological experiments. The method can be referred to the method reported in the literature (Nickolls, SA, et al. British Journal of Pharmacology, 2018, 175: 708-725). The composition of the extracellular solution (ECS) for electrophysiology is as follows: 150mM NaCl, 5mM KCl, 2.5mM CaCl2, 1mM MgCl2, 10mM HEPES and 10mM glucose (pH 7.4); the formula of the electrode internal solution (ICS) for electrophysiology is as follows: 140mM CsCl, 11mM EGTA, 10mM HEPES, 2mM CaCl2, 1mM MgCl2, 4mM MgATP, 2mM TEA (pH 7.3). GABA (γ-aminobutyric acid) powder was prepared into a stock solution with pure water and then diluted in ECS; the compound was first prepared into a 4mM stock solution with dimethyl sulfoxide and then gradually diluted into the corresponding concentration of GABA-ECS. All solutions were freshly prepared before electrophysiological testing.

[0241] Electrophysiological signals were collected using an EPC 10 amplifier and PatchMaster software (HEKA) or an Axon 700B amplifier and Clampex software (AXON). Recording electrodes were made of borosilicate glass with an electrode resistance of 4 to 6 MΩ. Extracellular drug delivery was performed using an ALA-VC-8PG TM System. A single independently grown cell was selected, and the glass electrode was well sealed with the cell before breaking the membrane to form a whole-cell mode. The cell membrane potential was clamped at -60mV and recorded in Gap-free mode. During the experiment, extracellular fluid was first applied to the cell for about 20 seconds. After the baseline (I prebaseline ) stabilized, the extracellular fluid was switched to GABA-ECS. At this point, the current induced by GABA (I gaba After about 10 to 20 seconds, when the current is stable, the extracellular fluid is switched to a mixed solution of the compound and GABA-ECS until the current (I treatment Finally, the solution was switched to the extracellular solution and the test was terminated after recording for 20 to 40 seconds. gaba -I prebaseline ) Only cells with an absolute value greater than 40 pA and a relatively stable current within 10 to 20 seconds were used for compound testing.

[0242] α1-GABA A R and α2-GABA A The experimental results of the positive allosteric regulation of R were analyzed using PatchMaster v2x90.1 or PatchMaster v2x90.3 software (EPC 10 amplifier) ​​and Clampex 10.6 software (Axon 700B amplifier). The current value of each stage was calculated as the average value of the current after stabilization under the corresponding conditions. We defined the control current as I gaba -I prebaseline , the current after compound treatment is defined as I treatment -I prebaseline The allosteric modulatory activity of the compound is expressed as a percentage and calculated according to the following formula: Functional activity = [(I treatment -I gaba ) / (I gaba -I prebaseline)] × 100%. If the value is negative, it means that the regulatory effect of the test compound on the GABA receptor is reverse allosteric regulation. If the value is positive, it means that the regulatory effect of the test compound on the GABA receptor is positive allosteric regulation. For the convenience of comparison, the positive compound CVL-865 (ie, Example 4 of WO2014091368A1) is used as the reference compound for normalization, that is, positive allosteric regulation activity = [functional activity of the compound / functional activity of the reference compound] × 100%.

[0243] Effects of each compound on α2-GABA A Receptor affinity activity and α1-GABA A , α2-GABA A The results of the positive allosteric regulation activity of the receptor are shown in Table 3 below.

[0244] It should be noted that under different detection systems, the absolute values ​​of the functional activities of the compounds will have certain differences and are not comparable. Therefore, in order to facilitate comparison, the present invention simultaneously detects the control and the compounds involved under the same detection system, and uses the positive compound CVL-865 as a reference for normalization, that is, positive allosteric regulatory activity = [functional activity of the compound / functional activity of the reference compound] × 100%. The specific results are shown in Table 3 below.

[0245]

[0246] Table 3

[0247]

[0248] Note: “-” means not tested.

[0249] (3) Potential of compounds to activate pregnane X receptor (PXR)

[0250] 1. Purpose

[0251] The aim of this study was to evaluate the potential of test compounds to activate PXR in vitro and thereby induce drug metabolizing enzymes.

[0252] 2. Equipment, Materials, and Reagents

[0253] 2.1 Cells: DPX2 cells were purchased from Puacyp.

[0254] 2.2 Materials and instruments are shown in Table 4 below:

[0255] Table 4

[0256]

[0257] 3. Experimental Procedure

[0258] 3.1 Cell seeding

[0259] 1) Prepare DPX2 cell complete culture medium: DPX2 culture medium containing 10% fetal bovine serum.

[0260] 2) DPX2 cells were cultured in T-75 cell culture flasks at 37° C., 5% CO 2 , and 95% relative humidity. When the cell confluence reached 80-90%, the cells were passaged and plated.

[0261] 3) Rinse the cells in the T-75 flask with 10 mL of PBS. Remove the liquid and add 3-5 mL of trypsin to digest the cells. Incubate at 37°C until the cells are detached and suspended, then add excess complete medium to inactivate trypsin.

[0262] 4) Transfer the cell suspension to a centrifuge tube and centrifuge at 150 g for 5 minutes. After removing the supernatant, resuspend the cells in culture medium to a final concentration of 4.0 × 10 5 cells / mL. Take 100 μL of the cell suspension and add it to a 96-well cell plate. Place the cell plate in a 37°C, 5% CO2, 95% incubator for 24 hours.

[0263] 3.2 Compound incubation

[0264] 1) Prepare the stock solutions of the test compound and the inducer (control drug) with DMSO, and prepare the working solutions of the corresponding concentrations with the administration medium at 37°C. The final concentrations of the positive control drug rifampicin are 1μM, 10μM and 20μM, respectively, and the final concentration of the negative control drug propranolol is 10μM. The blank control is 0.1% DMSO, and the final working concentrations of the test compound are 1μM and 10μM. The final concentration of DMSO is 0.1%.

[0265] 2) Take out the cell culture plate from the incubator, remove the liquid, add 100 μL of working solution to each well, replace the working solution prepared with new dosing culture medium every 24 hours, and put the cell culture plate back into the incubator for incubation for 48 hours.

[0266] 3) Observe the cell morphology and monolayer integrity to ensure that the cell quality meets the experimental standards.

[0267] 3.3 Quantitative determination of PXR activity

[0268] 1) 48 hours after administration, samples were prepared for determination of PXR activity.

[0269] 2) Thaw CellTiter-Fluor at room temperature TM Cell Viability Assay Kit and ONE-Glo TMLuciferase Assay Kit. Take out 10μL GF-AFC Substrate and add it to 10mL Assay Buffer to form a 2× reaction solution. Then add 10mL PBS to dilute it to a 1× reaction solution. TM Transfer all Luciferase Transfer Assay Substrate powder to ONE-Glo Luciferase Assay Buffer.

[0270] 3) Take the cell culture plate out of the incubator and remove the working solution of the dosing medium in each well. Wash the cell culture plate once with preheated PBS and use a multi-channel pipette to add 1×CellTiter-Fluor TM The cell viability assay reagent reaction solution was added to each well of a 96-well cell culture plate at 50 μL and incubated in a 37°C incubator for 30 minutes.

[0271] 4) Take out the cell culture plate from the incubator, cool it to room temperature, and use a microplate reader to measure fluorescence at an excitation wavelength of 400 nM and an emission wavelength of 505 nM.

[0272] 5) Use a multi-channel pipette to transfer ONE-Glo TM Luciferase detection reagent was added to each well of a 96-well cell culture plate at 50 μL, and the plate was gently shaken to mix the reagents in the wells. After 5 minutes, the fluorescence value of each well was read using an ELISA reader.

[0273] 3.4 Data Analysis

[0274] All calculations were performed using Microsoft Excel.

[0275] 1) Cell viability (%) can be calculated by the following formula:

[0276] Cell viability (%) = (RFU 受试化合物 / RFU 空白对照 )×100

[0277] 2) Normalized luciferase activity was determined by RLU / RFU, where RFU represents the activity of the luciferase enzyme using CellTiter-Fluor TM Relative fluorescence values ​​of compounds at different concentrations measured by cell viability assay reagent (triplicate), RLU means using ONE-Glo TM The relative fluorescence values ​​of the compounds at different concentrations measured by luciferase assay reagent (triplicate). The RLU and RFU data of the blank control are the average values ​​of the three replicates of the blank control samples.

[0278] The mRNA fold induction was determined by the following formula:

[0279] Induction fold = (RLU 受试化合物 / RFU 受试化合物 ) / (RLU 空白对照 / RFU 空白对照 )

[0280] 3) The control percentage (%) of the test compound and the positive compound (rifampicin) can be calculated by the following formula: Control percentage (%) of the test compound and the positive compound = (induction multiple 受试化合物 / Induction multiple 阳性对照组 )×100. The specific results are shown in Table 5 below.

[0281] Table 5

[0282]

[0283] It can be seen that compared with the positive compounds and the compounds of Comparative Examples 2-4, the compounds of the present application have a smaller potential for activating PXR in vitro and a smaller risk of potential drug-drug interaction.

[0284] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0285] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in detail, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description can be used to interpret the contents of the claims.

Claims

1. A cinnoline-2-oxide compound represented by the following formula (I) or a pharmaceutically acceptable salt, solvate, or a solvate of a pharmaceutically acceptable salt thereof, in, R1 is C1-C6 alkyl or C3-C6 cycloalkyl; R2 is H, D or halogen; Ar is n R3-substituted or unsubstituted C1-C10 heteroaryl groups; R3 is H, D, C1-C6 alkyl, C1-C6 alkoxy, D-substituted C1-C6 alkyl, D-substituted C1-C6 alkoxy or halogen; n is 0, 1, 2, 3 or 4.

2. The cinnoline-2-oxide compound according to claim 1, characterized in that: Ar is a C2-C5 heteroaryl group which is substituted or unsubstituted with n R3.

3. The cinnoline-2-oxide compound according to claim 1, characterized in that: Ar is pyridyl, pyrimidinyl, pyrazolyl, pyridazinyl, quinolyl, isoquinolyl, cinnolinyl, pyridoimidazolyl, indolyl, indazolyl, azaindolyl, azaindazolyl or pyridazinoimidazolyl substituted or unsubstituted by n R3.

4. The cinnoline-2-oxide compound according to claim 3, characterized in that: Ar is a pyridyl group, a pyrimidinyl group, a pyrazolyl group or a pyridazinyl group which may be substituted or unsubstituted by n R3.

5. The cinnoline-2-oxide compound according to claim 1, characterized in that: Ar is one of the following groups which are substituted or unsubstituted with n R3:

6. The cinnoline-2-oxide compound according to claim 1, characterized in that: R3 is H, D, C1~C3 alkyl, C1~C3 alkoxy, F, Cl or D-substituted C1~C3 alkyl.

7. The cinnoline-2-oxide compound according to any one of claims 1 to 6, characterized in that: The cinnoline-2-oxide compound is one of the following compounds:

8. The method for preparing cinnoline-2-oxide compounds according to any one of claims 1 to 7, characterized in that: Prepare according to method 1 or method 2; The method 1 comprises the following steps: Compound A-1 is reacted with compound A-2 to prepare compound A-3; Oxidizing compound A-3 to prepare the cinnoline-2-oxide compound; The second method comprises the following steps: Oxidizing compound A-1 to prepare compound A-4; The compound A-4 is reacted with the compound A-2 to prepare the cinnoline-2-oxide compound; Wherein, X is a halogen; Optionally, in method 2, before the oxidation, the method further includes performing an amino protection reaction on compound A-1 to prepare compound A-5, then oxidizing compound A-5 to prepare compound A-6, then reacting compound A-6 with compound A-2 to prepare compound A-7, and performing a deprotection reaction on compound A-7 to prepare the cinnoline-2-oxide compound; M represents an amino protecting group; 9. The cinnoline-2-oxide compound according to any one of claims 1 to 7 for use in the preparation of a method for treating or preventing GABA A Use in medicine for diseases related to GABA receptors; Optionally, the GABA A Receptor is α1-GABA A Receptors and / or α2-GABA A Receptor.

10. Use of the cinnoline-2-oxide compound according to any one of claims 1 to 7 in the preparation of a medicament for treating or preventing pain, Alzheimer's disease, multi-infarct dementia, stroke, epilepsy, anxiety, pruritus or depression.

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