2-oxo-coral compounds, their preparation methods and uses

Abstract

This invention relates to a cinnabar-2-oxide compound, its preparation method, and its uses. The cinnabar-2-oxide compound is shown in formula (I). By oxidizing a nitrogen atom to an N-oxide and then combining it with a heteroaryl group, the above-mentioned cinnabar-2-oxide compound achieves good α2-GABAA receptor affinity and positive regulatory activity. Simultaneously, it has the potential to significantly reduce PXR (pregnane X receptor) activation, thereby reducing drug-drug interactions and improving drug efficacy or safety.

Description

Technical Field

[0001] This invention relates to pharmaceutical chemistry, and in particular to a cinnamoline-2-oxide compound, its preparation method, and its uses. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the mammalian central nervous system. Regulating GABA can correspondingly regulate neurotransmission, and therefore it is widely used to treat various conditions, 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 of which is GABAB... A GABA receptor A R), this type of receptor is a member of the ligand-gated ion channel superfamily, another type is GABA. B GABA receptor B R), this type of receptor is a member of the G protein-coupled receptor superfamily. GABA in mammals A The receptor subunits discovered include α1-6, β1-4, γ1-3, δ, ε, θ, and ρ1-2, among which the α, β, and γ subunits form a complete functional GABA. A The acceptor is essential, while the α-subunit terephthalamide... Compounds with GABA A Receptor binding is crucial.

[0003] In GABA A Drugs bound to the receptor allosteric binding site can be positive allosteric modulators (or forward allosteric modulators) that increase receptor activity, negative allosteric modulators (or reverse allosteric modulators) that decrease receptor activity, or neutral allosteric modulators (compounds that bind to the allosteric binding site but do not modulate receptor activity) that do not alter receptor activity. Studies have confirmed that GABA containing the α2 subunit... A receptor (α2-GABA) A GABA receptors may be involved in certain pain states, and positivist modulators of these receptors may be effective analgesics. The prevailing view also holds that GABA receptors containing the α1 subunit... A The regulatory activity of receptors is currently the main focus of GABA research. A Modifiers (such as benzodiazepines) The main source of side effects (such as sedation, addiction, drowsiness, and amnesia) associated with GABA is therefore being investigated. A Receptor interaction and α1-GABA A New compounds with fewer receptor-related side effects will have great therapeutic potential.

[0004] Studies have shown that α2-GABA has oral activity.A The receptor-positive allosteric modulator AZD7325 can mediate CYP3A4 enzyme induction, leading to drug-drug interactions (D. Zhou, Z. Lu, Journal of Clinical Pharmacy & Therapeutics, 2014, 39(4):404-410). This may adversely affect drug metabolism, causing drug side effects and reducing drug safety. For example, if the test product is a CYP enzyme inducer, it will reduce the exposure of itself (self-induction) or other co-administered drugs (CYP enzyme substrates) in vivo, thereby reducing drug efficacy; at the same time, it may increase the risk of metabolite-related toxicity and adverse reactions due to promoting the generation of metabolites; in addition, it may also promote the generation of more active metabolites from prodrugs, thus increasing the risk. Summary of the Invention

[0005] Based on this, the present invention provides a method for treating α2-GABA. A The preparation methods and uses of morpholino-2-oxide compounds, which have good positive regulatory activity of receptors and can reduce drug side effects by reducing the potential to activate pregnane X receptor (PXR).

[0006] A first aspect of this application provides a cinnamoline-2-oxide compound of formula (I) or a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof.

[0007]

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

[0009] R2 is H, D, or a halogen;

[0010] Ar represents n C1-C10 heteroaryl groups that are either R3-substituted or unsubstituted;

[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 can be 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 pyridinyl, pyrimidinyl, pyrazolyl, pyridazinyl, quinolinyl, isoquinolinyl, cyclolinyl, pyridinidazoleyl, indoleyl, indazoleyl, azidoindoinyl, azidoinazoleyl, or pyridazinidazoleyl.

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

[0016] In some embodiments, Ar is one of the following groups, either R3-substituted or unsubstituted:

[0017]

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

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

[0020]

[0021]

[0022] A second aspect of this application provides a method for preparing the cyclophosphine-2-oxide compounds described in the first aspect, wherein the preparation is carried out according to method one or method two;

[0023] Method 1 includes the following steps:

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

[0025] Compound A-3 was oxidized to prepare the cenline-2-oxide compound;

[0026] Compound A-1

[0027] Compound A-2

[0028] Compound A-3;

[0029] The second method includes the following steps:

[0030] Compound A-1 was oxidized to prepare compound A-4;

[0031] Compound A-4 was reacted with compound A-2 to prepare the boryn-2-oxide compound;

[0032] Compound A-4;

[0033] Where X is a halogen;

[0034] Optionally, in method two, before the oxidation is performed, the method further includes an amino protection reaction of 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 then performing a deprotection reaction on compound A-7 to prepare the boryn-2-oxide compound.

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

[0036] Compound A-6.

[0037] Compound A-7.

[0038] A third aspect of this application provides the cyclophosphine-2-oxide compounds described in the first aspect for use in the preparation of treatments or preventative treatments related to GABA. A Use in medicines for receptor-related diseases; alternatively, the GABA A The receptor is α1-GABA A receptors and / or α2-GABA A Receptors.

[0039] A fourth aspect of this application provides the use of the cyclophosphine-2-oxide compounds described in the first aspect in the preparation of medicaments for the treatment or prevention of pain, Alzheimer's disease, multi-infarct dementia, stroke, epilepsy, anxiety, pruritus, or depression.

[0040] During the research process, this application discovered that the above-mentioned boryn-2-oxide compounds can achieve good α2-GABAA receptor affinity and positive regulatory activity by oxidizing a nitrogen atom into an N-oxide and coordinating with a heteroaryl group. At the same time, they also have the potential to significantly reduce the activation of PXR (pregnane X receptor), thereby reducing drug-drug interactions and improving drug efficacy or safety. Attached Figure Description

[0041] Figure 1 The X-ray single-crystal diffraction structure of the compound prepared as a structural verification example of this application is shown. Detailed Implementation

[0042] The following detailed description, in conjunction with specific embodiments, illustrates the cenline-2-oxide compounds of the present invention, their preparation methods, and their uses. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this invention.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0044] The terms “and / or,” “or / and,” and “and / or” as used herein may include any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.

[0045] In this article, "one or more" refers to any one, two or more of the listed items.

[0046] In this invention, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, terms such as "first," "second," "third," and "fourth" serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0047] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0048] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0049] Unless otherwise specified, the percentage content involved in this invention refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0050] Unless otherwise specified, all percentage concentrations mentioned in this invention refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0051] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.

[0052] In this invention, room temperature generally refers to 4℃~30℃, and preferably 20±5℃.

[0053] In this document, 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, a secondary carbon atom, a tertiary carbon atom, a quaternary carbon atom, or a combination thereof. Phrases containing this term, such as "C1-C6 alkyl," refer to alkyl groups 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] In this document, the term "cycloalkyl" refers to a non-aromatic hydrocarbon containing a ring of carbon atoms, which can be monocycloalkyl, spirocycloalkyl, or bridged cycloalkyl. Phrases containing this term, such as "C3-C6 cycloalkyl," refer to cycloalkyl compounds containing 3 to 6 carbon atoms, and each occurrence can be independently C3, C4, C5, or C6 cycloalkyl. Suitable examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Additionally, "cycloalkyl" may also contain one or more double bonds; representative examples of cycloalkyl compounds containing double bonds include cyclopentenyl, cyclohexenyl, cyclohexadienyl, and cyclobutadienyl.

[0055] In this document, the term "alkoxy" refers to a group having the structure -O-alkyl, i.e., an alkyl group as defined above connected to an adjacent group in the parent nucleus via an oxygen atom. Phrases containing this term, such as "C1-C6 alkoxy," refer to alkyl moieties containing 1 to 6 carbon atoms, and each occurrence can be independently C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 alkoxy, C5 alkoxy, or 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] In this article, the term "aryl" refers to an aromatic hydrocarbon group derived from an aromatic ring compound by removing one hydrogen atom. It can be a monocyclic aryl, a fused-ring aryl, or a polycyclic aryl. For polycyclic ring species, at least one is an aromatic ring system.

[0057] In this article, the term "heteroaryl" refers to an aryl group in which at least one carbon atom is replaced by a non-carbon atom, which can 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 can be independently C1 heteroaryl, C2 heteroaryl, C3 heteroaryl, C4 heteroaryl, C5 heteroaryl, C6 heteroaryl, C7 heteroaryl, C8 heteroaryl, C9 heteroaryl, or C10 heteroaryl. Suitable examples include, but are not limited to: furanyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrroleyl, pyrazolyl, triazolyl, imidazoyl, oxazolyl, oxadiazolyl, thiazoyl, tetrazolyl, indolyl, carbazoyl, pyrroloimidazoyl, pyrrolopyrroleyl, thiophenolopyrroleyl, thiophenolothiophenyl, furanolopyrroleyl, furanolofuranyl, thiophenolofuranyl, benzoisooxazolyl, benzoisothiazoyl, benzoimidazoyl, pyridinidindolyl, indazoleyl, azaindazoleyl, pyridinidinidazoleyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, isoquinolinyl, cyclolinyl, o-diazanaphthyl, quinoxolinyl, phenanthridine, primidyl, quinazolinyl, and quinazolinoneyl.

[0058] In this article, the term "halogen" refers to F, Cl, Br, or I.

[0059] In this document, the term "pharmaceutically acceptable salt" refers to a salt formed by any compound in the indicated structure with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. One class of salts is the salt formed by the compounds of this invention with an acid. Acids suitable for salt formation 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 class of salts are those formed by the compounds of this invention with a base. Suitable bases for salt formation include, but are not limited to, alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (such as lower alkanol ammonium 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 from morpholine, piperazine, and lysine, respectively. Nomenclature used herein follows the IUPAC systematic nomenclature. Any open valence bonds appearing on carbon, oxygen, sulfur, or nitrogen atoms in the structures given herein indicate the presence of a hydrogen atom.

[0060] In this article, "patient" is defined as any warm-blooded animal, such as, but not limited to, mice, guinea pigs, dogs, horses, or humans, with humans being the preferred patient.

[0061] In this article, the term “treatment” means 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 the disease; or (3) slowing the development of one or more biological manifestations of a disease.

[0062] In this article, the term "prevention" refers to reducing the risk of developing a disease.

[0063] In this document, the term "drug" includes any agent, compound, composition, or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the physiological and / or pharmacological effects produced by the "drug" in vivo is not particularly limited; it may have systemic effects or only local effects. The activity of the "drug" is not particularly limited; it may be an active substance that can interact with other substances or an inert substance that does not interact with other substances.

[0064] Some examples of this application provide a cinnamoline-2-oxide compound or a pharmaceutically acceptable salt thereof, a solvate thereof, or a pharmaceutically acceptable salt solvate thereof, as shown in formula (I).

[0065]

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

[0067] R2 is H, D, or a halogen;

[0068] Ar represents n C1-C10 heteroaryl groups that are either R3-substituted or unsubstituted;

[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 more than two R3s, each R3 may be the same or different.

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

[0071] Without limitation, the C1-C10 heteroaryl groups include the following groups: pyridinyl, pyrimidinyl, pyrazolyl, pyridazinyl, quinolinyl, isoquinolinyl, cenolinyl, pyridinzimidazolyl, indoleyl, indazoleyl, azaindoleyl, azaindazoleyl, or pyridinzimidazolyl. Further, the C1-C10 heteroaryl groups include one of the following groups:

[0072]

[0073] In some of these 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 pyridinyl, pyrimidinyl, pyrazolyl, pyridazinyl, quinolinyl, isoquinolinyl, cenolinyl, pyridinzimidazolyl, indoleyl, indazoleyl, azaindoleyl, azainzolyl, or pyridazinzimidazolyl. Further, Ar is n R3-substituted or unsubstituted pyridinyl, pyrimidinyl, pyrazolyl, or pyridazinyl.

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

[0076] Understandably, n R3s can replace any H group among the above groups.

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

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

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

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

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

[0082] Without limitation, the cenline-2-oxide compounds are as follows:

[0083]

[0084] Other examples of this application provide methods for preparing cenline-2-oxide compounds as described above. Without limitation, preparation can be carried out according to either Method 1 or Method 2, respectively.

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

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

[0087] Compound A-3 was oxidized to prepare the cenline-2-oxide compound;

[0088] Compound A-1

[0089] Compound A-2

[0090] Compound A-3;

[0091] Where X is a halogen.

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

[0093] Compound A-1 was oxidized to prepare compound A-4;

[0094] Compound A-4 was reacted with compound A-2 to prepare the boryn-2-oxide compound;

[0095] Compound A-4;

[0096] Where X is a halogen;

[0097] Optionally, in method two, before the oxidation reaction, the method further includes an amino protection reaction of 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 then performing a deprotection reaction on compound A-7 to prepare the boryn-2-oxide compound.

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

[0099] Compound A-6.

[0100] Compound A-7.

[0101] In some of these examples, the oxidation described in Method 1 or Method 2 includes the following steps:

[0102] The reaction is carried out by mixing compound A-3 or compound A-1 with peroxide and solvent.

[0103] In some of these examples, the reaction can be carried out at room temperature. The reaction time is generally 2 to 6 hours.

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

[0105] In some of these 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 includes the following steps:

[0107] The reaction is carried out by mixing compound A-1 or compound A-4 with compound A-2, an inorganic base, a palladium catalyst, and a solvent.

[0108] In some of these examples, the reaction can be carried out at 50°C to 105°C, and the reaction time is unlimited, ranging from 0.01 h to 15 h.

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

[0110] In some of these 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 of these examples, the inorganic base is one or more of potassium phosphate, cesium carbonate, potassium carbonate, and sodium carbonate.

[0112] Other examples in this application also provide cenline-2-oxide compounds as described above in the preparation of treatments or preventative medications related to GABA. A Use in drugs for receptor-related diseases. Further, the GABA... A The receptor is α1-GABA A receptors and / or α2-GABA A Receptors.

[0113] Without limitation, the above is related to GABA. A Receptor-related diseases include pain, Alzheimer's disease, multi-infarct dementia, stroke, epilepsy, anxiety, itching, or depression.

[0114] Other examples of this application also provide the use of cyclophosphine-2-oxide compounds as described above in the preparation of medicaments for the treatment or prevention of pain, Alzheimer's disease, multi-infarct dementia, stroke, epilepsy, anxiety, pruritus, or depression.

[0115] The following are specific examples. All reagents and raw materials used in the examples are commercially available. For experimental methods that do not specify specific conditions in the examples, traditional methods and conditions can be followed, or the product instructions can be followed.

[0116] The synthetic routes of the compounds involved in Example 1 are summarized below:

[0117]

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

[0119]

[0120] Synthesis of intermediate iii:

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

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

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

[0124] Mixing 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%), a reddish-brown solid.

[0125] Step 2: 4-Amino-8-bromo-7-fluoro-N-propylcenline-3-carboxamide

[0126] 2-[2-(2-bromo-3-fluorophenyl)diazeninyl]-2-cyano-N-propylacetamide (50 g, 153 mmol) and aluminum chloride (61.1 g, 459 mmol) were added sequentially to toluene (200 mL), and the mixture was stirred at 90 °C for 3 hours. 250 mL of water was added, followed by extraction with ethyl acetate (250 mL x 2). The extract was concentrated, and column chromatography was used to obtain intermediate iii (45 g, 90% yield) as a brown solid.

[0127] Synthesis of target product v:

[0128] Route 1:

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

[0130] 4-Amino-8-bromo-7-fluoro-N-propylbenzyl-3-carboxamide (3 g, 9.17 mmol) and 3-chloroperoxybenzoic acid (4.75 g, 27.5 mmol) were added sequentially to dichloromethane (25 mL) and stirred at room temperature for 3 hours. 150 mL of saturated aqueous sodium thiosulfate solution was added, followed by extraction with dichloromethane (150 mL x 2). The organic phases were combined, concentrated, and subjected to column chromatography to give intermediate IV-1 (580 mg, 18% yield) as a yellow solid.

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

[0132] 4-Amino-8-bromo-7-fluoro-3-(propylcarbamoyl)cenline 2-oxide (560 mg, 1.63 mmol), (2-fluoropyridin-3-yl)boronic acid (340 mg, 2.44 mmol), [1,1'-bis(diphenylphosphine)ferrocene]dichloropalladium(II) (240 mg, 0.33 mmol), and cesium carbonate (1.06 g, 3.26 mmol) were added sequentially to a mixed solution of 1,4-dioxane (16 mL) and water (4 mL). The mixture was stirred at 100 °C for 3 hours under argon protection. The solution was concentrated, and 150 mL of aqueous solution was added. The mixture was then extracted with dichloromethane (150 mL x 2). The organic phases were combined, concentrated, and subjected to column chromatography to obtain the target product (105 mg, 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-propylcenline-3-carboxamide

[0137] 4-Amino-8-bromo-7-fluoro-N-propylcenolin-3-carboxamide (150 mg, 0.46 mmol), (2-fluoropyridin-3-yl)boronic acid (130 mg, 0.92 mmol), cesium carbonate (450 mg, 1.38 mmol), and 1,1-bis(diphenylphosphine)ferrocene palladium dichloride (37 mg, 0.05 mmol) were sequentially added to a mixed solvent of 1,4-dioxane (5 mL) and water (0.5 mL). The mixture was 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 give 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)zoline 2-oxide

[0139] 4-Amino-7-fluoro-8-(2-fluoropyridin-3-yl)-N-propylcenline-3-carboxamide (100 mg, 0.29 mmol) was dissolved in dichloromethane (20 mL), and m-chloroperoxybenzoic acid (500 mg, 2.9 mmol) was added in portions. The mixture was stirred at room temperature for 4 hours. The reaction solution was washed successively with saturated sodium thiosulfate aqueous solution (20 mL) and saturated sodium bicarbonate aqueous solution (20 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by preparative plate (dichloromethane) to the target product (27 mg, 26% yield), which was a yellow solid. The NMR data were consistent with those of route 1.

[0140] Route 3:

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

[0142] 4-Amino-8-bromo-7-fluoro-N-propylbenzyl-3-carboxamide (50 g, 150 mmol), di-tert-butyl dicarbonate (100 g, 450 mmol), 4-dimethylaminopyridine (3.7 g, 31 mmol), and triethylamine (77 g, 760 mmol) were dissolved in tetrahydrofuran (1 L) and stirred overnight at room temperature. The reaction mixture was concentrated, dissolved in dichloromethane (500 mL), washed with saturated ammonium chloride aqueous solution (500 mL * 3), separated, dried, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to give a pale yellow solid (80 g, 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-fluorodecamorpholine 2-oxide

[0144] (8-Bromo-3-((tert-butoxycarbonyl)(propyl)carbamoyl)-7-fluorodendrolin-4-yl)(tert-butoxycarbonyl)carbamate tert-butyl ester (80 g, 127 mmol) was dissolved in dichloromethane (1 L), and m-chloroperoxybenzoic acid (44 g, 255 mmol) was added in portions. The mixture was stirred at room temperature for 16 hours. The reaction solution was slowly added to a saturated sodium thiosulfate aqueous solution (1 L), and the mixture was stirred until quenched. The mixture was separated, and the organic phase was washed with a saturated sodium bicarbonate aqueous solution (1 L). The organic phase was separated, dried, and purified by column chromatography (petroleum ether:ethyl acetate = 10:1 to 1:1) to give a pale yellow solid (70 g, 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)zoline 2-oxide

[0146] 4-(bis(tert-butoxycarbonyl)amino)-8-bromo-3-((tert-butoxycarbonyl)(propyl)carbamoyl)-7-fluorodecaline 2-oxide (20 g, 31 mmol), (2-fluoropyridin-3-yl)boronic acid (8.7 g, 62 mmol), methanesulfonic acid (2-dicyclohexylphosphine-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°C for 16 hours under argon protection. The aqueous phase was separated, the organic phase was concentrated, and purified by column chromatography (petroleum ether:acetic acid = 10:1) to give 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)zoline 2-oxide

[0148] 4-(bis(tert-butoxycarbonyl)amino)-3-((tert-butoxycarbonyl)(propyl)carbamoyl)-7-fluoro-8-(2-fluoropyridin-3-yl)cenline 2-oxide (1 g, 1.52 mmol) was added to a mixed solution of dichloromethane (8 mL) and trifluoroacetic acid (8 mL) and stirred at room temperature for 3 hours. The solution was concentrated, extracted with 60 mL of saturated sodium bicarbonate solution, and then extracted with dichloromethane (60 mL * 2). The organic phases were combined, dried over anhydrous sodium sulfate, and slurried to obtain the target product as a yellow solid (447 mg, yield 82%). The NMR data were consistent with those of route 1.

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

[0150]

[0151] Step 1: (2-Fluoropyridin-3-yl-6-deuterium)boronic acid

[0152] (2-Fluoropyridin-3-yl-6-bromo)boronic acid (200 mg, 0.91 mmol) was dissolved in deuterated methanol (CD3OD, 2 mL), and palladium on carbon (30 mg, pre-stirred in deuterated methanol for 24 hours) was added. The mixture was stirred at room temperature for 2 hours under a deuterium atmosphere. The solution was filtered through diatomaceous earth, and the organic phase was concentrated and used directly for the next step.

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

[0154] The experimental procedure was the same as in Example 1, Route 3 (steps 3 and 4), and the target product, a light yellow solid (400 mg, yield 73%), was obtained.

[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)zoline 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 sequentially to N,N-dimethylacetamide (8 mL), and the mixture was stirred for 3 hours at room temperature under argon protection. The reaction solution was used directly 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 sequentially added to N,N-dimethylacetamide (5 mL) under argon protection and stirred at 40 °C for 5 hours. The mixture was then distilled under reduced pressure at 80 °C to obtain the target product (800 mg, 72% yield) as a yellow liquid. 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), berberine diboronate (2430 mg, 9.6 mmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (CAS No.: 95464-05-4, 200 mg, 0.25 mmol) were sequentially added to ethylene glycol dimethyl ether (20 mL). Under argon protection, the mixture was stirred at 80 °C for 5 hours. The mixture was concentrated, and extracted with water (50 mL) and ethyl acetate (50 mL * 2). The organic layer was concentrated, and column chromatography yielded the target compound as a black oil (500 mg, yield 49%).

[0164] Step 4: The experimental procedure is the same as in Example 1, Route 3 (Step 3 and Step 4), 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)zoline 2-oxide

[0168]

[0169] The experimental procedure was the same as in Example 1, Route 3 (steps 3 and 4), and the target product was obtained.

[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)zoline 2-oxide

[0173]

[0174] The experimental procedure was the same as in Example 1, Route 3 (steps 3 and 4), and the target product was obtained.

[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)zoline 2-oxide

[0178]

[0179] The experimental procedure was the same as in Example 1, Route 3 (steps 3 and 4), and the target product was obtained.

[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)zoline 2-oxide

[0183]

[0184] Step 1: 4-Amino-8-bromo-7-fluorodeoxyline-3-carboxylic acid

[0185] 4-Amino-8-bromo-7-fluoro-N-propylbenzyl-3-carboxamide (5 g, 15 mmol) was dissolved in an aqueous sulfuric acid solution (80%, 50 mL) and stirred at 100 °C for 16 hours. The solution was poured into 500 mL of ice water, and a large amount of solid precipitated out. The solid was filtered and dried to obtain the title product, a brown solid (4 g, crude product).

[0186] Step 2: 4-Amino-8-bromo-N-ethyl-7-fluorocenzoline-3-carboxamide

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

[0188] Step 3: The experimental procedure is the same as in Example 1, Route 3, 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)zoline 2-oxide

[0192]

[0193] The experimental procedure was the same as in Example 1, Route 3 (steps 3 and 4), and the target product was obtained.

[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)zoline 2-oxide

[0197]

[0198] The experimental procedure was the same as in Example 7, and the target product was obtained.

[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-dimethoxypyridazine-4-yl)zoline 2-oxide

[0202]

[0203] The experimental procedure was the same as in Example 7, and the target product was obtained.

[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] Example 11: 4-Amino-8-(2-Fluoropyridin-3-yl)-3-(propylcarbamoyl)zoline 2-oxide

[0207]

[0208] The experimental procedure was the same as route 3 in Example 1, and the target product was obtained.

[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)zoline 2-oxide

[0212]

[0213] The experimental procedure was the same as route 3 in Example 1, and the target product was obtained.

[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-dimethoxypyridazine-4-yl)zoline 2-oxide

[0217]

[0218] The experimental procedure was the same as in Example 7, and the target product was obtained.

[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)zoline 2-oxide

[0222]

[0223] Its synthesis method is similar to Route 2 in Example 1, except that (2-fluoropyridin-3-yl)boronic acid is replaced with (3,5-difluorophenyl)boronic acid.

[0224] Structural identification: The structure of the crystal was determined using single-crystal X-ray diffraction. 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 Receptors are essential. In this test case, α2-GABA was constructed and screened stably expressing receptors using liposome transfection (the method can be found in Felgner, PL, et al. Proceedings of the National Academy of Sciences, 1987, 84:7413-7417). A receptor (α2-GABA) A HEK293 cells (R). α2-GABA A The R-HEK293 cell model simultaneously expresses the α2 subunit (protein sequence in GenBank accession number: NM_000807.4), β3 subunit (protein sequence in GenBank accession number: NM_000814.5), and γ2 subunit (protein sequence in GenBank accession number: NM_000816.3).

[0232] The above cell lines were passaged. α2-GABA was amplified. A R-HEK293 cells were used to target GABA compounds. A Benzodiazepine receptors (BZD) binding site affinity test. α2-GABA A During passage, some R-HEK293 cells were plated on slides pretreated with Poly-D-Lysine for electrophysiological testing (the method can be found in paragraph 0586 of patent CN 107344936 A).

[0233] (1) The compound on 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 compound's effect on α2-GABA. A Receptor affinity.

[0235] Membrane preparation: Cells were suspended in 50 mM Tris-HCl buffer (pH 7.4) and homogenized on ice for 20 seconds 10 times. The mixture was then centrifuged at 1000 g for 10 min at 4 °C. The supernatant was collected, and the above steps were repeated. The supernatant was centrifuged at 4 °C (33800 g; Thermo Fisher Scientific, rotor: A27-8x50) for 60 min. The precipitate was resuspended in Tris buffer (50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM EDTA, 10% glycerol). Protein content was determined (using the BCA (dioctanoic acid) protein quantification method based on copper ion reduction; the BCA kit was purchased from Pierce Biotech). 1 mL 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 membrane. 3 The concentration of H-flunitrazepam was 1 nM, and the concentration of the analyte was 1 x 10⁻⁶. -5 -10 -6 Within the M range. Flumazenil was used as a control. 1 μL of 2 mM flumazenil (final concentration 10 μM) was added to the low control (LC) wells, and 1 μL of dimethyl sulfoxide (DMSO) was added to the high control (HC) wells. The final concentration of the target membrane protein was 5 μg / well. All test compound samples were stored in 10 mM DMSO solution. The working concentration of the samples was achieved by diluting all samples to 0.2 mM with DMSO, followed by a 4-fold serial dilution, for a total of 8 concentration gradients. The 96-well plate was sealed with sealing film and incubated on a shaker at room temperature for 1 hour. Simultaneously, the GF / C filter plate was soaked in immersion buffer (0.3% PEI (polyethyleneimine, purchased from Sigma-Aldrich, model: P314), stored at 4°C) for at least 0.5 hours. After incubation, cells were collected onto a GF / C filter plate using a cell collector and washed four times with wash 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. Residual radioactivity of the filter membrane was detected using liquid scintillation counting. 50 μL of scintillation buffer was added to each well, and the plate was sealed. Microbeta was used for further analysis. 2 (Microplate counter, purchased from PerkinElmer, model: CNLL0153) Reading. Calculate the sample pair. 3 H-flunitrazepam and GABA AThe inhibitory activity against receptor membrane protein binding was determined by calculating the IC50 of each test sample using dose-response curve fitting (GraphPadPrism 5 software). 50 and through IC 50 Calculate the K of the sample i To evaluate the relationship between the compound and α2-GABA. A Binding ability of the receptor BZD site.

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

[0238] (2) The effect of compounds on different subtypes of GABA A receptor functional activity

[0239] The effect of the test drug on α1-GABA was detected by electrophysiological methods. A Receptors and α2-GABA A Positive regulation of receptor activity. Specific methods are as follows:

[0240] Compound concentration setting: The final concentration of all compounds used in the compound screening was 100 nM. For expression of α1-GABA... A The receptor's cell line had a GABA concentration of 0.05–0.07 μM (approximately EC50). 2～4 For expressing α2-GABA A The receptor's cell line had a GABA concentration of 0.10–0.11 μM (approximately EC50). 7～8 Electrophysiological experiments were performed using the whole-cell patch-clamp technique, which can be referred to the method reported in the literature (Nickolls, SA, et al. British Journal of Pharmacology, 2018, 175:708-725). The extracellular fluid (ECS) for electrophysiology consisted of the following: 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4); the intraelectrode solution (ICS) for electrophysiology consisted of the following: 140 mM CsCl, 11 mM EGTA, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, 4 mM MgATP, and 2 mM TEA (pH 7.3). GABA (γ-aminobutyric acid) powder was prepared into a stock solution using pure water and then diluted in ECS. The compound was first prepared into a 4 mM stock solution using dimethyl sulfoxide, and then gradually diluted into GABA-ECS of the corresponding concentrations. All solutions were freshly prepared before electrophysiological testing.

[0241] Electrophysiological signal acquisition was performed 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 a resistance of 4–6 MΩ. Extracellular drug delivery was performed using ALA-VC-8PG. TM System. Single, independently growing cells were selected, and after a good seal between the glass electrode and the cell, the membrane was ruptured to form a whole-cell model. The cell membrane potential was clamped at -60 mV and recorded in gap-free mode. During the experiment, extracellular fluid was applied for approximately 20 seconds. The baseline (I0) was then established. prebaseline Once stable, the extracellular fluid was switched to GABA-ECS. At this point, a current (Ig) induced by GABA could be detected. gaba After approximately 10–20 seconds, once the current has stabilized, the extracellular fluid is switched to a mixed solution of the compound and GABA-ECS until a current jointly induced by the compound and GABA can be detected (IC). treatment Finally, switch the solution to extracellular fluid and record for 20–40 seconds to terminate the experiment. Only when the baseline is stable and the control current magnitude (I) is the result obtained. gaba -I prebaseline Only cells with an absolute value greater than 40 pA and a relatively stable current for 10 to 20 seconds are used for compound testing.

[0242] α1-GABA A R and α2-GABA A The experimental results for the positive allosteric regulation activity 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 values ​​at each stage were calculated as the average of the currents after stabilization under the corresponding conditions. We defined the control current as I0. gaba -I prebaseline The current after compound treatment is defined as I. treatment -I prebaseline The allosteric regulatory activity of a compound is expressed as a percentage and calculated using the following formula: Functional activity = [(I treatment -I gaba ) / (I gaba -I prebaselineThe value is calculated as follows: [[functional activity of the compound / functional activity of the reference compound] × 100%]. If the value is negative, it indicates that the regulatory effect of the test compound on the GABA receptor is inverse allosteric regulation. If the value is positive, it indicates that the regulatory effect of the test compound on the GABA receptor is positive allosteric regulation. For ease of comparison, the positive compound CVL-865 (i.e., WO2014091368A1, Example 4) is used as the reference compound, and normalization is performed, i.e., positive allosteric regulation activity = [functional activity of the compound / functional activity of the reference compound] × 100%.

[0243] Each compound on α2-GABA A Receptor affinity activity and α1-GABA A α2-GABA A The results of the receptor's positive allosteric regulatory activity are shown in Table 3 below.

[0244] It should be noted that the absolute values ​​of the functional activities of compounds may differ under different detection systems, making them incomparable. Therefore, for ease of comparison, this invention simultaneously detects the control and the involved compounds under the same detection system, and uses the positive compound CVL-865 as a reference for normalization, i.e., positive allosteric regulation 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: "-" indicates that no detection was performed.

[0249] (3) The potential of compounds to activate the pregnane X receptor (PXR).

[0250] 1. Purpose

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

[0252] 2. Equipment, materials and reagents

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

[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 complete cell culture medium: DPX2 medium containing 10% fetal bovine serum.

[0260] 2) Under conditions of 37℃, 5% CO2, and 95% relative humidity, DPX2 cells were cultured in T-75 cell culture flasks. When the cell confluence reached 80-90%, the cells were passaged and seeded into plates.

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

[0262] 4) Transfer the cell suspension to a centrifuge tube and centrifuge at 150g for 5 minutes. After removing the supernatant, resuspend the cells in culture medium to a final concentration of 4.0 × 10⁻⁶ cells / mL. 5 cells / mL. Add 100 μL of cell suspension to a 96-well cell plate. Incubate the cell plate at 37°C, 5% CO2, 95% CO2 for 24 hours.

[0263] 3.2 Compound incubation

[0264] 1) Prepare stock solutions of the test compounds and inducers (control drugs) using DMSO, and prepare working solutions of the corresponding concentrations using 37°C administration medium. The final concentrations of the positive control rifampin were 1 μM, 10 μM, and 20 μM, the final concentration of the negative control propranolol was 10 μM, the blank control was 0.1% DMSO, and the final working concentrations of the test compounds were 1 μM and 10 μM. The final concentration of DMSO was 0.1%.

[0265] 2) Remove the cell culture plate from the incubator, remove the liquid, add 100 μL of working solution to each well, replace the working solution with fresh drug administration medium every 24 hours, and put the cell culture plate back into the incubator for 48 hours.

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

[0267] 3.3 Quantitative determination of PXR activity

[0268] 1) Samples were prepared 48 hours after drug administration for the determination of PXR activity.

[0269] 2) Thaw CellTiter-Fluor at room temperature TM Cell viability assay kit and ONE-Glo TMLuciferase assay kit. Add 10 μL of GF-AFC Substrate to 10 mL of Assay Buffer to form a 2× reaction solution, then dilute with 10 mL of PBS to a 1× reaction solution. Add ONE-Glo TM All Luciferase Transfer Assay Substrate powder was transferred to ONE-Glo Luciferase Assay Buffer.

[0270] 3) Remove the cell culture plate from the incubator and remove the working solution from each well of the drug-treated medium. After washing the cell culture plate once with preheated PBS, use a multichannel pipette to transfer 1×CellTiter-Fluor... TM The cell viability assay reagent reaction solution was added to each well of a 96-well cell culture plate at a rate of 50 μL, and incubated at 37°C for 30 minutes.

[0271] 4) Remove the cell culture plate from the incubator and cool it to room temperature. Use a microplate reader to perform fluorescence measurement 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 Add 50 μL of luciferase assay reagent to each well of a 96-well cell culture plate. Gently shake the plate to mix the reagent in the wells. After 5 minutes, read the fluorescence value of each well using a microplate reader.

[0273] 3.4 Data Analysis

[0274] All calculations were performed using Microsoft Excel.

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

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

[0277] 2) Standardized luciferase activity was determined by RLU / RFU, where RLU indicates the ratio of CellTiter-Fluoride enzyme activity. TM The relative fluorescence values ​​(in triplicate) of the compound at different concentrations measured by the cell viability assay reagent. RLU indicates the use of ONE-Glo TM The relative fluorescence values ​​of the compound at different concentrations measured by the luciferase assay reagent (three parallels). The RLU and RFU data for the blank control are the average of three parallel samples of the blank control.

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

[0279] Induction factor = (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 percentage (%) of the test compound and the positive control compound (rifampin) can be calculated using the following formula: Percentage (%) of the test compound and the positive control compound = (Induction fold) 受试化合物 / Induction fold 阳性对照组 ()×100, the specific results are shown in Table 5 below.

[0281] Table 5

[0282]

[0283] It is evident that, compared with the positive compound and the comparative compounds 2-4, the compound of this application has a lower potential for activating PXR in vitro and a lower potential drug-drug interaction risk.

[0284] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely illustrative of several implementations of the present invention, facilitating a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A cinnoline-2-oxide compound of the following formula (I) or a pharmaceutically acceptable salt thereof, (I) wherein R1 is C1-C6 alkyl or C3-C6 cycloalkyl; R2 is H, D or halogen; Ar is n R3 substituted or unsubstituted pyridyl, pyrimidyl, pyrazolyl or pyridazinyl; 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 compound according to claim 1, wherein Ar is n R3 substituted or unsubstituted one of the following groups: 。 3. The compound according to claim 1, wherein R3 is H, D, C1-C3 alkyl, C1-C3 alkoxy, F, Cl or D substituted C1-C3 alkyl.

4. The compound according to any one of claims 1 to 3, wherein The cinnoline-2-oxide compound is one of the following compounds: 。 5. A process for the production of the compound of the cinnoline-2-oxide series according to any one of claims 1 to 4, characterized in that, Prepared according to Method One or Method Two; The Method One comprises the following steps: reacting compound A-1 with compound A-2 to prepare compound A-3; oxidizing compound A-3 to prepare the cinnoline-2-oxide compound; Compound A-1 Compound A-2 Compound A-3; The Method Two comprises the following steps: oxidizing compound A-1 to prepare compound A-4; reacting compound A-4 with compound A-2 to prepare the cinnoline-2-oxide compound; Compound A-4; wherein X is halogen; Compound A-5, M represents an amino protecting group; Compound A-6; Compound A-7.

6. The method for preparing cinnamyl-2-oxide compounds according to claim 5, characterized in that, In the Method Two, before the oxidation, further comprising amino protection reaction on compound A-1 to prepare compound A-5, then oxidizing compound A-5 to prepare compound A-6, and then reacting compound A-6 with compound A-2 to prepare compound A-7, and deprotection reaction on compound A-7 to prepare the cinnoline-2-oxide compound.

7. Use of the cinnoline-2-oxide compound according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment or prevention of a disease associated with GABA A receptors.

7. Use of the cinnoline-2-oxide compound according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment or prevention of a disease associated with GABA A receptors.

8. Use according to claim 7, characterized in that, The GABA A receptor is an αl-GABA A receptor and / or an α2-GABA A receptor. 9.Use of the cinnoline-2-oxide compound of any one of claims 1-4 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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