Aromatic heterocyclic amide compounds and uses thereof

Abstract

The present invention provides aromatic heterocyclic amide compounds and uses thereof. Specifically, such compounds can effectively inhibit ASK1 kinase activity. They are compounds represented by formulas (I) to (III), or tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs of the compounds represented by formulas (I) to (III): #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry. Specifically, the present invention relates to aromatic heterocyclic amide compounds as ASK1 inhibitors. More specifically, the present invention relates to aromatic heterocyclic amide compounds as ASK1 inhibitors, preparation methods thereof, and use thereof in preparing drugs. Background Art

[0002] Apoptosis signal-regulating kinase 1 (ASK1) is a serine / threonine kinase, also known as mitogen-activated protein kinase kinase 5 (MAP3K5), and a member of the MAP3K family. ASK1 is regulated by upstream factors such as oxidative stress, calcium influx, lipopolysaccharide (LPS), reactive oxygen species (ROS), and various cytokines such as tumor necrosis factor (TNF). It activates the downstream c-Jun N-terminal kinase (JNK) and p38-MAPK signaling pathways, responding to stress by regulating cell growth, differentiation, inflammation, and apoptosis.

[0003] ASK1 activation and its signaling pathway play a crucial role in the development and progression of cardiovascular disease, neurodegenerative diseases, autoimmune diseases, inflammation, metabolic diseases, and cancer. Alzheimer's disease is characterized by the accumulation of amyloid-β peptide (Aβ) and neurofibrillary tangles. Ni Jun et al. have found that ASK1 plays a key role in Aβ-induced death of primary brain endothelial cells. Environmental MPRP, a cause of Parkinson's disease, can activate ASK1 and simultaneously phosphorylate downstream kinases, leading to cell damage. Environmentally induced Parkinson's disease is closely associated with ASK1 activation. ASK1 can promote angiotensin II-induced myocardial hypertrophy, cardiac remodeling, interstitial fibrosis, and coronary artery remodeling. It can also promote neuronal and microglial cell death after cerebral ischemia-reperfusion, thereby exacerbating stroke progression. Terada Y et al. found significant activation of ASK1 in a model of acute kidney injury induced by ischemia-reperfusion. Ni Jun et al. also found that the ASK1 signaling pathway is also involved in autoimmune responses. In ASK1 gene knockout spleen cells and bone marrow-derived dendritic cells, LPS-induced p38 signaling pathways were significantly weakened, and inflammatory factors in the cells were significantly reduced. In animal studies, ASK1 gene knockout mice were more effective than wild-type mice in resisting LPS-induced septic shock. Eapen MS et al. found that ASK1 was expressed more highly in the airway smooth muscle of patients without chronic obstructive pulmonary disease (COPD) than in those without COPD. Treatment of airway smooth muscle cells with siRNA targeting ASK1 blocked mitogen-induced cell growth. ASK1 inhibition has anti-mitotic effects and can be targeted to reduce or prevent excessive airway smooth muscle proliferation in COPD. Wilkins MR et al. found that in pulmonary hypertension, ASK1 inhibition reduced pathological remodeling of the pulmonary vasculature and right ventricle and prevented disease progression in a rodent model of pulmonary hypertension. In mouse models, a high-fat diet causes hepatic steatosis, ultimately leading to fat accumulation and fatty acid oxidation, which results in the production of ROS, which causes hepatocyte dysfunction and death. In addition, TNF is essential for hepatocyte apoptosis through the ASK1-JNK pathway. TNF-deficient mice exhibit reduced levels of hepatic steatosis and liver fibrosis. ASK1 plays a significant promoting role in high-fat diet-induced type 2 diabetes and hepatic steatosis models.

[0004] Modulating ASK1 activity may have beneficial effects in the treatment or prevention of a wide range of diseases, so developing small molecule ASK1 inhibitors is an effective method for regulating ASK1 activity. In recent years, many studies have found that ASK1 inhibitors have potential therapeutic value for chronic kidney disease (diabetic nephropathy, end-stage renal disease, renal fibrosis, etc.), cardiovascular disease (heart failure, etc.), neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), lung diseases (such as pulmonary hypertension and pulmonary fibrosis), and tumors. In particular, drugs targeting ASK1 have important clinical significance in the study of chronic liver diseases such as non-alcoholic steatohepatitis and liver fibrosis, and have achieved certain clinical efficacy.

[0005] Some ASK1 kinase small molecule inhibitors reported in the literature have potential issues such as poor molecular-level activity, suboptimal ASK1 target selectivity, and poor drug-like properties. Some compounds also have potential issues with chemical stability and in vivo metabolic stability. Therefore, further development of new and more effective ASK1 small molecule inhibitors is still needed. Summary of the Invention

[0006] The present invention aims to solve one of the above technical problems to at least some extent or at least provide a useful commercial choice.

[0007] According to one aspect of the present invention, the present invention provides a compound, which is a compound represented by formula (I), or a tautomer, stereoisomer, hydrate, solvate, salt or prodrug of the compound represented by formula (I):

[0008]

[0009] in:

[0010] X is selected from -N=, -C(R a )=;the R a Selected from halogen, C1-C3 alkyl;

[0011] R 1 independently selected from hydrogen, unsubstituted or optionally substituted with one or more R b Substituted by the following groups: C1-C6 alkyl, C3-C6 cycloalkyl; the R b Selected from halogen, C1-C3 alkyl;

[0012] R 2 Selected from halogen, C1-C6 alkyl substituted by halogen, unsubstituted or optionally substituted by one or more R c The following groups substituted: C2-C6 alkyl, 4-6 membered nitrogen-containing heterocyclic group, 7-10 membered nitrogen-containing spirocyclic group; the R c Selected from hydrogen, halogen, hydroxy, C1-C6 alkyl, -C(O)-NH-Rd , oxo; R d is selected from hydrogen, C1-C6 alkyl; preferably, the 4-6 membered nitrogen-containing heterocyclic group, 7-10 membered nitrogen-containing spirocyclic group is connected to the aryl or heteroaryl group where X is located via N;

[0013] R 3 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted by one or more halogens.

[0014] According to an exemplary embodiment of the present invention, in formula (I),

[0015] X is selected from -N=, -C(F)=;

[0016] R 1 Independently selected from isopropyl, cyclopropyl;

[0017] R 2 unsubstituted or optionally substituted with one or more R c The following groups substituted: 4-membered N-containing heterocyclic group, piperidinyl, morpholinyl, 7-10-membered nitrogen-containing spirocyclic group; the R c Selected from hydrogen, halogen, hydroxy, methyl, -C(O)-NH2, -C(O)-NH-CH3, oxo;

[0018] R 3 Selected from isopropyl, isopropyl substituted by fluorine.

[0019] According to an exemplary embodiment of the present invention, in formula (I),

[0020] R 2 Selected from

[0021] R 3 Selected from

[0022] According to an embodiment of the present invention, the compound of the present invention comprises a compound represented by the following formula, or a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof:

[0023]

[0024]

[0025] According to the second aspect of the present invention, the present invention provides a compound, which is a compound represented by formula (II), or a tautomer, stereoisomer, hydrate, solvate, salt or prodrug of the compound represented by formula (II):

[0026]

[0027] in:

[0028] X is selected from -N=, -C(R a )=;the R a Selected from hydrogen, halogen, C1-C3 alkyl;

[0029] R 1 independently selected from hydrogen, unsubstituted or optionally substituted with one or more R b Substituted by the following groups: C1-C6 alkyl, C3-C6 cycloalkyl; the R b is selected from halogen, C1-C3 alkyl; preferably, R 1 isopropyl, cyclopropyl;

[0030] R 2 is selected from hydrogen, halogen, C1-C6 alkyl substituted by halogen, unsubstituted or optionally substituted by one or more R c The following groups substituted: C2-C6 alkyl, 4-6 membered nitrogen-containing heterocyclic group, 7-10 membered nitrogen-containing spirocyclic group; the R c Selected from hydrogen, halogen, hydroxy, C1-C6 alkyl, -C(O)-NH-R d , oxo; R d is selected from hydrogen, C1-C6 alkyl; preferably, the 4-6 membered nitrogen-containing heterocyclic group, 7-10 membered nitrogen-containing spirocyclic group is connected to the aryl or heteroaryl group where X is located via N;

[0031] Ring A is selected from unsubstituted or optionally substituted with one or more R e Substituted groups: cycloalkyl and heterocyclic groups; R e Selected from fluorine, chlorine, bromine, C1-C3 alkyl.

[0032] According to an exemplary embodiment of the present invention, in formula (II),

[0033] X is selected from -N=, -C(F)=;

[0034] R 1 is selected from cyclopropyl;

[0035] R 2 Selected from hydrogen,

[0036] Ring A is selected from unsubstituted or optionally substituted with one or more R e Substituted 5-6 membered heterocycloalkyl.

[0037] According to an embodiment of the present invention, the compounds of the present invention further include compounds represented by the following formula, or tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs thereof:

[0038]

[0039] According to the third aspect of the present invention, the present invention provides a compound, which is a compound represented by formula (III), or a tautomer, stereoisomer, hydrate, solvate, salt or prodrug of the compound represented by formula (III):

[0040]

[0041] in:

[0042] X is selected from -N=, -C(R a )=;the R a is selected from hydrogen, halogen, C1-C3 alkyl; preferably, X is selected from -N=, -C(F)=;

[0043] Y is selected from a single bond, -C(=O)-,

[0044] Z is selected from -O- or -C(R b )2-; Each R b Independently selected from hydrogen, halogen, C1-C3 alkyl;

[0045] m is an integer selected from 0 to 6;

[0046] R 1 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted by one or more halogens;

[0047] R 2 、R 3 、R 4 Selected from hydrogen, halogen, C1-C6 alkyl, C1-C6 alkyl substituted by one or more halogens;

[0048] When Z is -C(R b )2-, R 2 You can also use R b connected to form a cycloalkyl or heterocycloalkyl group.

[0049] According to an exemplary embodiment of the present invention, the compound represented by formula (III) may further preferably be a compound represented by formula (IV):

[0050]

[0051] in:

[0052] X is selected from -N=, -C(R a )=;the R a is selected from hydrogen, halogen, C1-C3 alkyl; preferably, X is selected from -N=, -C(F)=;

[0053] m is an integer selected from 0-6; n is an integer selected from 0-6;

[0054] R 1 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted by one or more halogens; preferably, R 1 Selected from cyclopropyl, isopropyl, and cyclopropyl substituted by one fluorine group.

[0055] According to an embodiment of the present invention, the compound represented by formula (III) is selected from the following compounds:

[0056]

[0057] The compounds of the present invention may exist in tautomerism. The present invention includes all tautomeric forms of the compounds, whether in equilibrium or one form predominates, the present invention includes each tautomeric form.

[0058] According to the fourth aspect of the present invention, the present invention also provides a pharmaceutical composition comprising at least one compound of the present invention or a pharmaceutically acceptable salt, tautomer, stereoisomer, hydrate, solvate or prodrug thereof.

[0059] A "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compound to an organism.

[0060] The present invention also provides a method for treating ASK1-related diseases, comprising administering to a patient a therapeutically effective amount of the compound of the present invention or at least one of its pharmaceutically acceptable salts, tautomers, stereoisomers, hydrates, solvates or prodrugs.

[0061] The present invention also provides a method for treating ASK1-related diseases, comprising administering to a patient a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt, tautomer, stereoisomer, hydrate, solvate or prodrug thereof, or a pharmaceutical composition thereof.

[0062] The present invention also provides the use of compounds represented by formulas (I) to (IV), and compounds represented by formulas I-1 to I-10, formulas II-1 to II-3, and formula III-1, their pharmaceutically acceptable salts, tautomers, stereoisomers, hydrates, solvates or prodrugs, or pharmaceutical compositions containing compounds represented by formulas (I) to (IV), and compounds represented by formulas I-1 to I-10, formulas II-1 to II-3, and formula III-1, or their pharmaceutically acceptable salts, tautomers, stereoisomers, hydrates, solvates or prodrugs in the preparation of drugs for treating ASK1-related diseases.

[0063] In some embodiments, the ASK1-related disease is selected from cardiovascular and cerebrovascular diseases, chronic kidney diseases, lung diseases, chronic liver diseases, multiple sclerosis, metabolic diseases, stones, neurodegenerative diseases and cancer.

[0064] In some embodiments, the ASK1-related disease is selected from hyperproliferative diseases such as myocardial infarction, stroke, thrombosis, diabetic nephropathy, end-stage renal disease, renal fibrosis, cholesterol stones, cholelithiasis, pulmonary hypertension, pulmonary fibrosis, chronic obstructive pulmonary disease, acute lung injury, fatty liver hepatitis, liver fibrosis, bile acid disorders, primary sclerosing cholangitis, diabetes, Alzheimer's disease, Parkinson's disease, gastric cancer, liver cancer, polyposis, colon cancer, breast cancer, pancreatic cancer, esophageal cancer, etc.

[0065] Definitions and Explanations of Terms

[0066] Unless otherwise indicated, the definitions of groups and terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The resulting group definitions and compound structures shall fall within the scope of the description of this specification.

[0067] Unless otherwise defined, all technical and scientific terms herein have the same meanings as commonly understood by persons skilled in the art to which the claimed subject matter belongs. Unless otherwise indicated, all patents, patent applications, and publications cited herein are incorporated by reference in their entirety. If multiple definitions of a term are used herein, the definitions in this section shall prevail.

[0068] Unless otherwise indicated, conventional methods within the skill of the art, such as mass spectrometry, NMR, IR and UV / Vis spectroscopy and pharmacological methods, are used. Unless specifically defined, the terms used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceuticals and medicinal chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, as well as in the treatment of patients. For example, the manufacturer's instructions for use of the kit can be utilized, or reactions and purification can be carried out in accordance with methods well known in the art or the description of this application. The above-mentioned techniques and methods can generally be implemented according to conventional methods well known in the art, based on the descriptions in the multiple general and more specific literature cited and discussed in this specification. In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural moieties and compounds. When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents obtained when the structural formula is written from right to left. For example, CH2O is equivalent to OCH2.

[0069] When the numerical ranges described in the specification and claims of this application are understood as “integers”, they should be understood as recording the two endpoints of the range and each integer within the range. For example, “an integer from 1 to 6” should be understood as recording each integer of 0, 1, 2, 3, 4, 5, and 6. When the numerical range is understood as a “number”, it should be understood as recording the two endpoints of the range and each integer within the range and each decimal within the range. For example, “a number from 1 to 10” should be understood as recording not only each integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, but also at least the sum of each of these integers and 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9, respectively.

[0070] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0071] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a non-toxic acid or base, including salts of inorganic acids and bases, and organic acids and bases. Salts derived from inorganic bases include, but are not limited to, metal salts formed with Al, Ca, Li, Mg, K, Na, and Zn; salts derived from organic bases include, but are not limited to, salts of primary, secondary, or tertiary amines, including naturally occurring substituted or unsubstituted amines, cyclic amines, and basic ion exchange resins, such as ammonium, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, caffeine, procaine, choline, betaine, benzylpenicillin, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, or organic salts formed with polyamine resins; Salts derived from inorganic and organic acids include, but are not limited to, organic salts formed from sulfuric, phosphoric, nitric, hydrobromic, hydrochloric, formic, acetic, propionic, benzenesulfonic, benzoic, phenylacetic, salicylic, alginic, anthranilic, camphoric, citric, ethylenesulfonic, formic, fumaric, furoic, gluconic, glucuronic, glutamic, glycolic, isethionic, lactic, maleic, malic, mandelic, mucic, pamoic, pantothenic, stearic, succinic, sulfanilic, tartaric, p-toluenesulfonic, malonic, 2-hydroxypropionic, oxalic, glycolic, glucuronic, galacturonic, citric, lysine, arginine, aspartic, cinnamic, p-toluenesulfonic, methanesulfonic, ethanesulfonic, or trifluoromethanesulfonic acids.

[0072] In addition to pharmaceutically acceptable salts, the present invention also contemplates other salts that may serve as intermediates in the purification of compounds or in the preparation of other pharmaceutically acceptable salts or that may be useful in the identification, characterization, or purification of the compounds of the present invention.

[0073] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, diastereomers, and conformers. The stereochemical definitions and conventions used herein are generally those of SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.

[0074] According to the selection of raw materials and methods, the compounds of the present invention can exist in the form of one of possible isomers or their mixture, for example as pure optical isomers, or as isomer mixtures, such as as racemic and diastereomeric mixtures, depending on the number of asymmetric carbon atoms. When describing a compound with optical activity, prefixes D and L or R and S are used to represent the absolute configuration of the molecule with respect to the chiral center (or multiple chiral centers) in the molecule. The prefixes D and L or (+) and (-) are symbols for specifying the rotation of plane polarized light caused by the compound, where (-) or L represent that the compound is left-handed. Compounds prefixed with (+) or D are dextrorotatory. With respect to a given chemical structure, except that these stereoisomers are mirror images of each other, these stereoisomers are identical. Specific stereoisomers may also be referred to as enantiomers, and the mixture of the isomers is commonly referred to as a mixture of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process. Many geometric isomers of alkenes, C=N double bonds, etc. can also exist in the compounds described herein, and all such stable isomers are contemplated by the present invention. When the compounds described herein contain olefinic double bonds, unless otherwise specified, such double bonds include both E and Z geometric isomers. If the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents may be in either the cis- or trans- configuration.

[0075] When bonds to chiral carbon atoms in formulae of the present invention are depicted as straight lines, it is understood that both the (R) and (S) configurations of the chiral carbon atoms and the enantiomerically pure compounds and mixtures thereof are encompassed within the scope of the formulae. The diagrammatic representations of racemates and enantiomerically pure compounds herein are adapted from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, wedge-shaped bonds and dashed bonds are used to represent the absolute configuration of a stereocenter.

[0076] Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral preparations, or resolved using conventional techniques. Compounds of the invention containing asymmetrically substituted carbon atoms can be separated in optically active form or racemic form. Resolution of a racemic mixture of a compound can be carried out by any of a number of methods known in the art. An exemplary method includes fractional recrystallization using a chiral resolving acid that is an optically active, salified organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or various optically active camphorsulfonic acids such as the D and L forms of β-camphorsulfonic acid. Other resolving agents suitable for fractional crystallization methods include α-methyl-benzylamine (e.g., S and R forms or diastereoisomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, etc. The resolution of the racemic mixture can also be carried out by eluting on a column filled with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). High performance liquid chromatography (HPLC) can also be used to carry out supercritical fluid chromatography (SFC). The selection of specific methods and elution conditions, chromatographic column selection can be selected by those skilled in the art according to the structure of the compound and test results. Further, optically pure starting materials or reagents of known configuration can also be used to obtain any enantiomer or diastereomer of the compound described in the present invention through stereoorganic synthesis.

[0077] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom between two positions in a molecule. Compounds of the present invention may exhibit tautomerism. Tautomeric compounds can exist as two or more interconvertible species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture with physical and chemical properties consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. The present invention encompasses all tautomeric forms of the compounds.

[0078] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compound to an organism.

[0079] With respect to a drug or pharmacologically active agent, the terms "effective dose," "effective amount," or "therapeutically effective amount" refer to a non-toxic amount of the drug or agent sufficient to achieve the intended effect. For oral dosage forms of the present invention, an "effective amount" of an active substance in a composition refers to the amount required to achieve the intended effect when used in combination with another active substance in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, as well as the specific active substance. The appropriate effective amount in each individual case can be determined by those skilled in the art through routine experimentation.

[0080] The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

[0081] The term "solvate" refers to a compound of the present invention or a salt thereof including a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. When the solvent is water, it is a hydrate.

[0082] The term "prodrug" refers to a compound of the present invention that can be converted to a biologically active compound under physiological conditions or by solvolysis. Prodrugs of the present invention are prepared by modifying functional groups within the compound. These modifications can be removed by conventional procedures or in vivo to yield the parent compound. Prodrugs include compounds in which a hydroxyl group or an amino group within a compound of the present invention is attached to any group. When a prodrug of a compound of the present invention is administered to a mammalian subject, the prodrug is cleaved to form a free hydroxyl group or a free amino group, respectively.

[0083] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0084] The term "excipient" refers to a pharmaceutically acceptable inert ingredient. Examples of the term "excipient" include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, and diluents. Excipients enhance the handling properties of pharmaceutical formulations, i.e., by increasing flowability and / or cohesiveness, making the formulation more suitable for direct compression. Examples of typical "pharmaceutically acceptable carriers" suitable for the above-mentioned preparations are: sugars, such as lactose, sucrose, mannitol and sorbitol; starches, such as corn starch, tapioca starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinyl pyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal salts of stearic acid, such as magnesium stearate and calcium stearate; stearic acid; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; nonionic, cationic and anionic surfactants; ethylene glycol polymers; fatty alcohols; and cereal hydrolyzed solids and other non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants and the like excipients commonly used in pharmaceutical preparations.

[0085] The term "C1-C6 alkyl" is understood to mean preferably a straight-chain or branched saturated monovalent hydrocarbon radical having 1 to 6 carbon atoms; the term "C2-C6 alkyl" is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 2, 3, 4, 5 or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or the like, or isomers thereof. In particular, the radical has 1, 2, 3, 4, 5, 6 carbon atoms ("C1-C6 alkyl"), for example methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, more particularly the radical has 1, 2 or 3 carbon atoms ("C1-C3 alkyl"), for example methyl, ethyl, n-propyl or isopropyl.

[0086] As used herein, the term "halo" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0087] "Haloalkyl" refers to a saturated aliphatic hydrocarbon group, including branched and straight chains, having the specified number of carbon atoms, substituted with one or more halogens (e.g., -CvFw, where v = 1 to 3, w = 1 to (2v+1)). Examples of haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl.

[0088] The term "C3-C6 cycloalkyl" is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0089] The term "4-6 membered nitrogen-containing heterocyclic group" is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 4-6 ring atoms, at least one of which is N, and may further contain 0-5 heteroatoms independently selected from N, O and S. In particular, the heterocyclic group may include, but is not limited to, a 4-membered ring such as azetidinyl or oxetanyl; a 5-membered ring such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl. According to the present invention, the heterocyclic group is non-aromatic.

[0090] The term "spirocyclyl" is understood to mean a polycyclic group in which the rings share a carbon atom (called a spiro atom). These rings may contain one or more double bonds, but none of the rings have a completely conjugated π electron system. Spirocyclyls are classified as monospirocyclyls, dispirocyclyls, or polyspirocyclyls depending on the number of spiro atoms shared between the rings. Non-limiting examples of spirocyclyls include:

[0091]

[0092] The above definition of the term "spirocyclic group" is also applicable to other terms containing it, such as the term "7-10 membered nitrogen-containing spirocyclic group" and the like.

[0093] The term "7-10 membered nitrogen-containing spirocyclic group" should be understood as a spirocyclic group containing 7-10 ring atoms (at least one of which is N), including "7-10 membered saturated nitrogen-containing spirocyclic group" and "7-10 membered unsaturated nitrogen-containing spirocyclic group". Optionally, the ring atoms (such as carbon atoms or sulfur atoms) in the cyclic structure may be oxo-substituted.

[0094] The term "aryl" is understood to mean any stable 6-10 membered monocyclic or bicyclic aromatic group, for example, phenyl, naphthyl, tetrahydronaphthyl, indanyl or biphenyl.

[0095] The term "heteroaryl" refers to an aromatic ring group formed by replacing at least one carbon atom in the ring with a heteroatom selected from nitrogen, oxygen or sulfur, which can be a 5-12 membered heteroaryl, preferably a 5-7 membered monocyclic structure or a 7-12 membered bicyclic structure, preferably a 5-6 membered heteroaryl. For example: pyridyl, pyrimidinyl, pyridazin-3(2H)-one, furyl, thienyl, thiazolyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,4-triazolyl, 1,2,3-triazolyl, tetrazolyl, indazolyl, isoindazolyl, indolyl, isoindolyl, benzofuranyl, benzothienyl, benzo[d][1,3]dioxolanyl, benzothiazolyl, benzoxazolyl, quinolyl, isoquinolyl, quinazolinyl, etc.

[0096] Beneficial effects

[0097] The inventors surprisingly discovered that the compounds prepared by the present invention exhibit significant ASK1 inhibitory activity, significantly improving upon existing compounds. Compared to other ASK1 inhibitors, the representative compounds of the present invention exhibited superior inhibitory effects on α-SMA and COL1A1 gene expression in HSC cells. They also possessed superior hepatic metabolic stability, slower metabolism in humans, and higher exposure levels. Compared to control compounds, the compounds of the present invention exhibited weaker CYP inhibitory activity and improved drugability.

[0098] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. DETAILED DESCRIPTION

[0099] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0100] Embodiments of the present invention provide methods for preparing compounds represented by formulas (I) to (IV), and compounds represented by formulas I-1 to I-10, formulas II-1 to II-3, and formula III-1, and pharmaceutically acceptable salts, tautomers, stereoisomers, hydrates, solvates, or prodrugs thereof, and use of compounds represented by formulas (I) to (IV), and compounds represented by formulas I-1 to I-10, formulas II-1 to II-3, and formula III-1, or pharmaceutically acceptable salts, tautomers, stereoisomers, hydrates, solvates, or prodrugs thereof, in the preparation of drugs for treating diseases associated with ASK1.

[0101] The reaction solvents used in each reaction step described in the present invention are not particularly limited. Any solvent that can dissolve the starting materials to a certain extent and does not inhibit the reaction is included in the present invention. In addition, many similar modifications, equivalent substitutions, or solvents, solvent combinations, and different ratios of solvent combinations equivalent to those described in the present invention are considered to be within the scope of the present invention.

[0102] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). The units of NMR shifts are 10 -6 The solvents for NMR measurements are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and tetramethylsilane (TMS) is the internal standard.

[0103] Liquid chromatography-mass spectrometry (LC-MS) was performed on a Waters Acquity H-class UPLC-QDA mass spectrometer using an ACQUITY UPLC BEHC18, 2.1 x 50 mm, 1.7 μm column. Gradient elution conditions were: 95% to 5% solvent A1 and 5% to 95% solvent B1, followed by 95% B1 and 5% A1 for 0.5 min, at a flow rate of 1.0 mL / min. Percentages represent the volume percentage of a particular solvent relative to the total solvent volume. Solvent A1: 0.1% formic acid in water; Solvent B1: 0.1% formic acid in acetonitrile. Percentages represent the volume percentage of the solute relative to the total solvent volume.

[0104] The abbreviations of the present invention are defined as follows:

[0105] Symbol or unit:

[0106] IC 50 : Half-maximal inhibitory concentration, which refers to the concentration at which half of the maximum inhibitory effect is achieved

[0107] M: mol / L, for example, 1M sodium hydroxide aqueous solution means a sodium hydroxide aqueous solution with a molar concentration of 1 mol / L

[0108] Reagents:

[0109] AcOH: acetic acid

[0110] DIPEA: also written as DIEA, diisopropylethylamine, also known as N,N-diisopropylethylamine

[0111] DMF: N,N-dimethylformamide

[0112] iPrNH2: Isopropylamine

[0113] K2CO3: Potassium carbonate

[0114] KI: Potassium iodide

[0115] MeOH: methanol

[0116] Pd / C, Pd-C: Palladium on carbon

[0117] Pd(dppf)Cl2:[1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride

[0118] THF: Tetrahydrofuran

[0119] Unless otherwise indicated, the compounds exemplified herein are named and numbered using ChemBioDraw Ultra 13.0. Comparative Example 1: Control compound and its preparation

[0120]

[0121] The control compound was synthesized with reference to patent application WO2013112741A1.

[0122] The control compounds in the following test examples all refer to the compounds described in Comparative Example 1.

[0123] Example 1: Synthesis of target compound I-1

[0124] 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)benzamide (Compound I-1)

[0125]

[0126] The synthetic route of target compound I-1 is as follows:

[0127]

[0128] Step 1: Synthesis of methyl 2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)-5-nitrobenzoate (I-1B)

[0129] 3-Methyl-3-hydroxyazetidine hydrochloride (0.63 g, 5 mmol) was added portionwise to a solution of methyl 2,4-difluoro-5-nitrobenzoate (1.1 g, 5 mmol) and DIEA (0.97 g, 7.5 mmol) in THF (15 mL) at 0°C. The mixture was stirred at 0°C for 1 h. After completion of the reaction, the mixture was diluted with distilled water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined and washed with saturated brine (70 mL × 2). The organic phases were separated and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (petroleum ether:ethyl acetate (v / v) = 3:1) to afford methyl 2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)-5-nitrobenzoate (1.5 g, 4.4 mmol, 88% yield) as a yellow solid.

[0130] Step 2: Synthesis of methyl 5-amino-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)benzoate (I-1C)

[0131] Pd-C (150 mg) was carefully added to a solution of methyl 2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)-5-nitrobenzoate (1.5 g, 4.4 mmol) in MeOH (20 ml). The mixture was allowed to react overnight at room temperature under a hydrogen atmosphere. After the reaction was complete, the mixture was filtered and the filtrate was concentrated. The residue was purified on a silica gel column (petroleum ether:ethyl acetate (v / v) = 1:1) to afford methyl 5-amino-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)benzoate (1.2 g, 4.7 mmol) as a yellow solid.

[0132] Step 3: Synthesis of methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)benzoate (I-1D)

[0133] A reaction solution of DMF (10 mL) containing methyl 5-amino-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)benzoate (1.0 g, 3.9 mmol), 2-bromo-1-cyclopropylethanone (1.27 g, 7.8 mmol), KI (129 mg, 0.8 mmol), and K2CO3 (1.1 g, 7.8 mmol) was stirred at 70 °C for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column purification (petroleum ether: ethyl acetate (V / V) = 1:1) to give a yellow solid 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)benzoic acid methyl ester (1.0 g, 3.0 mmol, yield 76%).

[0134] Step 4: Synthesis of methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)benzoate (I-1E)

[0135] Methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)benzoate (1.0 g, 3.0 mmol) was dissolved in formamide (5 ml, 1.550 mmol), heated to 180°C in a microwave oven, and stirred for 60 min. After the reaction was complete, the mixture was diluted with distilled water (20 mL) and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (petroleum ether:ethyl acetate (v / v) = 1:1) to obtain methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)benzoate (200 mg, 0.58 mmol, 19.3% yield) as a yellow solid.

[0136] Step 5: Synthesis of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)benzamide (I-1)

[0137] Potassium tert-butoxide (260 mg, 2.32 mmol) was added to a DMF (3 ml) solution containing methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)benzoate (200 mg, 0.58 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (141 mg, 0.70 mmol), and the mixture was stirred at room temperature for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (dichloromethane: methanol (V / V) = 10:1) to obtain 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)benzamide.

[0138] 1 H NMR(400MHz,DMSO-d6)δ10.30(d,1H),8.87(s,1H),8.17(d,1H),8.01(t,1H),7.85(d,1H),7.60(s,1H),7.53(d,1H),7.06(s,1H),6.55( d,1H),5.61-5.54(m,1H),5.51(s,1H),3.45(dd,4H),1.87-1.83(m,1H),1.46(d,6H),1.33(s,3H),0.83-0.79(m,2H),0.70-0.66(m,2H).

[0139] LC-MS, M / Z(ESI):517.4[M+H] + .

[0140] Example 2: Preparation of target compound I-2

[0141]

[0142] The synthetic route of target compound I-2 is as follows:

[0143]

[0144] Step 1: Synthesis of methyl 2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)-5-nitrobenzoate (I-2B)

[0145] N-Methylpiperidine-4-carboxamide hydrochloride (5.18 g, 29.0 mmol) was added dropwise to a solution of methyl 2,4-difluoro-5-nitrobenzoate (6 g, 27.6 mmol) and DIEA (7.86 g, 60.8 mmol) in THF (80 mL) at 0°C and stirred for 2 h at 0°C. After the reaction was complete, the mixture was diluted with water (80 mL) and extracted with ethyl acetate (80 mL × 3). The organic phases were combined and washed with saturated brine (70 mL × 2). The organic phases were separated and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column to obtain methyl 2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)-5-nitrobenzoate (5 g, 53.3% yield) as a yellow solid.

[0146] LC-MS, M / Z(ESI):340.3[M+H] + .

[0147] Step 2: Synthesis of methyl 5-amino-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (I-2C)

[0148] Pd / C (0.5 g, 10%) was carefully added to a solution of methyl 2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)-5-nitrobenzoate (5.0 g, 14.74 mmol) in methanol (30 mL). The mixture was allowed to react at room temperature for 16 hours under a hydrogen atmosphere. After completion of the reaction, the mixture was filtered and concentrated directly. The residue was purified on a silica gel column to afford methyl 5-amino-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (0.8 g, 17.6% yield) as a yellow solid.

[0149] LC-MS, M / Z(ESI):310.3[M+H] + .

[0150] Step 3: Synthesis of methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (I-2D)

[0151] 2-Bromo-1-cyclopropylethanone (1.16 g, 6.47 mmol) was added dropwise to a DMF (10 mL) solution containing methyl 5-amino-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (0.8 g, 2.59 mmol), potassium iodide (0.472 g, 2.84 mmol), and potassium carbonate (0.536 g, 6.47 mmol). The reaction was continued at 70°C for 16 h. After the reaction, water (50 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography to give a yellow solid methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (0.77 g, yield 76%).

[0152] LC-MS, M / Z(ESI):392.4[M+H] + .

[0153] Step 4: Synthesis of methyl 5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (I-2E)

[0154] Under nitrogen, methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (0.77 g, 1.967 mmol) was dissolved in acetic acid (6 mL) and potassium thiocyanate (0.382 g, 3.93 mmol) was added. The reaction was stirred at 110°C overnight. After completion of the reaction, the mixture was diluted with water (30 mL) and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column to afford methyl 5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (0.7 g, 82% yield) as a yellow solid.

[0155] LC-MS, M / Z(ESI):433.4[M+H] + .

[0156] Step 5: Synthesis of methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (I-2F)

[0157] Methyl 5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (0.7 g, 1.967 mmol) was dissolved in acetic acid (6 mL). Water (1 mL) was added, followed by the slow dropwise addition of hydrogen peroxide (0.367 g, 3.24 mmol, 30%). The reaction was stirred at 50°C for 1 h. After the reaction is completed, water (30 mL) is added to dilute the mixture, and the mixture is extracted with ethyl acetate (20 mL × 3). The organic phases are combined, washed with saturated brine (20 mL), separated, and the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by silica gel column to give a yellow solid 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoic acid methyl ester (0.47 g, yield 72.5%).

[0158] LC-MS, M / Z(ESI):401.4[M+H] + .

[0159] Step 6: Synthesis of 1-(2-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoro-4-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N-methylpiperidine-4-carboxamide (I-2)

[0160] Potassium tert-butoxide (135 mg, 1.199 mmol) was added to a DMF (2 mL) solution containing methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (120 mg, 0.300 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (73.8 mg, 0.360 mmol), and the mixture was stirred at room temperature for 2 h. After the reaction is completed, water (10 mL) is added to dilute the mixture, and the mixture is extracted with dichloromethane (20 mL×3). The organic phases are combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by silica gel plate to obtain 1-(2-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoro-4-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N-methylpiperidine-4-carboxamide.

[0161] 1H NMR(400MHz,DMSO-d6)δ10.65(s,1H),8.87(s,1H),8.19(d,1H),8.02(t,1H) ,7.90-7.82(m,2H),7.73(d,1H),7.61(d,1H),7.23(s,1H),7.10(d,1H),5.67 -5.59(m,1H),2.92(d,2H),2.63-2.52(m,5H),2.22-2.10(m,1H),1.90-1.83( m,1H),1.68-1.54(m,4H),1.44(d,6H),0.84-0.77(m,2H),0.72-0.66(m,2H).

[0162] LC-MS, M / Z(ESI):572.5[M+H] +

[0163] Example 3: Preparation of target compound I-3

[0164]

[0165] The synthetic route of the target compound is as follows:

[0166]

[0167] Step 1: Synthesis of 2-chloro-4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropyridine (I-3B)

[0168] 8-Hydroxyquinoline (0.338 g, 2.331 mmol), cesium carbonate (11.39 g, 35.0 mmol), and cuprous iodide (0.167 g, 1.165 mmol) were added sequentially to a solution of 2-chloro-5-fluoro-4-iodopyridine (3 g, 11.65 mmol) and 4-cyclopropyl-1H-imidazole (1.386 g, 12.82 mmol) in n-butyronitrile (50 ml). The mixture was stirred at 65°C for 16 h under nitrogen. After the reaction, distilled water (50 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (70 mL × 3). The organic phases were combined, washed with saturated brine (50 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate (V / V) = 3:1) to give 2-chloro-4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropyridine (0.9 g, 3.79 mmol, yield 32.5%) as a yellow solid.

[0169] 1H NMR (400MHz, CDCl3) δ8.40 (s, 1H), 7.90 (s, 1H), 7.36 (d, J = 4.0Hz, 1H), 7.09 (d,J=4.0Hz,1H),1.88-1.92(m,1H),0.90-0.94(m,2H),0.82-0.89(m,2H).

[0170] Step 2: Synthesis of 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropyridinecarbonitrile (I-3C)

[0171] Zn(CN)2 (0.415 g, 3.53 mmol) and Pd(Ph3P)4 (0.583 g, 0.505 mmol) were added sequentially to a DMF (10 ml) solution containing 2-chloro-4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropyridine (1.2 g, 5.05 mmol). The mixture was stirred in a microwave at 120°C for 40 min under nitrogen protection. After the reaction, distilled water (50 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate (V / V) = 3:1) to give a yellow solid 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropyridinecarbonitrile (0.7 g, 3.07 mmol, yield 60.7%).

[0172] LC-MS, M / Z(ESI):229.2[M+H] + .

[0173] Step 3: Synthesis of 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropicolinic acid (I-3D)

[0174] A solution of 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropicolinonitrile (750 mg, 3.29 mmol) in concentrated hydrochloric acid (4438 μL, 52.6 mmol) was heated to 70°C and stirred for 3 hours. After the reaction was complete, the reaction solution was concentrated to afford a yellow solid, 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropicolinic acid (700 mg, 2.83 mmol). The crude product was used directly in the next reaction.

[0175] Step 4: Synthesis of methyl 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropicolinate (I-3E)

[0176] Thionyl chloride (0.413 ml, 5.66 mmol) was added dropwise to a solution of 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropicolinic acid (700 mg, 2.83 mmol) in MeOH (10 mL) at 0°C. The mixture was stirred at 50°C for 5 h. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The residue was purified on a silica gel column (petroleum ether:ethyl acetate (v / v) = 1:1) to afford methyl 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropicolinate (410 mg, 1.569 mmol, 55.4% yield) as a pale yellow solid.

[0177] LC-MS, M / Z(ESI):262.3[M+H] + .

[0178] Step 5: Synthesis of methyl 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(3-hydroxy-3-methylazetidin-1-yl)picolinate (I-3F)

[0179] 3-Methylazetidin-3-ol (284 mg, 2.297 mmol, hydrochloride) and K2CO3 (529 mg, 3.83 mmol) were added sequentially to a DMF (3 ml) solution containing 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropicolinate (200 mg, 0.766 mmol) and stirred at 90°C for 16 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate (V / V) = 3:1) to give a yellow solid 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(3-hydroxy-3-methylazetidin-1-yl)picolinic acid methyl ester (135 mg, 0.411 mmol, yield 53.7%).

[0180] 1 H NMR (400MHz, DMSO-d6) δ8.03(s,1H),7.69(s,2H),7.15(d,J=4.0Hz,1H),5.56(s,1H),3.81(s,3H),3.57(dd,J=8.0Hz,12.0Hz4H),1.81-1.88(m,1H),1 1.34(s,3H),0.78-0.83(m,2H),0.66-0.69(m,2H).

[0181] Step 6: Synthesis of 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(3-hydroxy-3-methylazetidin-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)picolinamide (I-3)

[0182] Potassium tert-butoxide (54.7 mg, 0.487 mmol) was added to a DMF (2 mL) solution containing methyl 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(3-hydroxy-3-methylazetidin-1-yl)picolinate (40 mg, 0.122 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (29.7 mg, 0.146 mmol), and the mixture was stirred at room temperature for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (dichloromethane: methanol (V / V) = 10:1) to obtain 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(3-hydroxy-3-methylazetidin-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)picolinamide.

[0183] 1 H NMR (400MHz, DMSO-d6) δ10.33(s,1H),8.90(s,1H),8.31(d,J=8.0Hz,1H),8.10( s,1H),8.05(t,J=8.0Hz,1H),7.85(d,J=4.0Hz,1H),7.82(s,1H),7.74(s,1H),7. 72(s,1H),5.57(s,1H),5.43-5.50(m,1H),3.60(dd,J=8.0Hz,16.0Hz4H),1.86( m,1H),1.51(d,J=4.0Hz,6H),1.36(s,3H),0.81-0.84(m,2H),0.69-0.72(m,2H).

[0184] LC-MS, M / Z(ESI):500.3[M+H] + .

[0185] Example 4: Preparation of target compound I-4

[0186]

[0187] The synthetic route of the target compound is as follows:

[0188]

[0189] Step 1: Synthesis of methyl 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(4-(methylcarbamoyl)piperidin-1-yl)picolinate (I-4B)

[0190] N-Methylpiperidine-4-carboxamide hydrochloride (137 mg, 0.766 mmol) and potassium carbonate (265 mg, 1.914 mmol) were added sequentially to a solution of 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropicolinate (100 mg, 0.383 mmol) in DMF (5 mL) and stirred at 90°C for 16 h. After completion of the reaction, the mixture was diluted with distilled water (20 mL) and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column to afford methyl 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(4-(methylcarbamoyl)piperidin-1-yl)picolinate (70 mg, 47.7% yield) as a yellow solid.

[0191] LC-MS, M / Z(ESI):384.3[M+H] + .

[0192] Step 2: 4-(4-cyclopropyl-1H-imidazol-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridine-2-

[0193] Synthesis of 5-(4-(methylcarbamoyl)piperidin-1-yl)pyridineamide (I-4)

[0194]

[0195] Under nitrogen protection, potassium tert-butoxide (82 mg, 0.730 mmol) was added to a DMF (2 mL) solution containing methyl 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(4-(methylcarbamoyl)piperidin-1-yl)picolinate (70 mg, 0.183 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (40.8 mg, 0.201 mmol) and stirred at room temperature for 2 h. After the reaction is completed, distilled water (10 mL) is added to dilute the mixture, and the mixture is extracted with dichloromethane (20 mL × 3). The organic phases are combined, washed with saturated brine (10 mL), separated, and the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by silica gel plate to obtain 4-(4-cyclopropyl-1H-imidazol-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(4-(methylcarbamoyl)piperidin-1-yl)picolinamide.

[0196] 1 H NMR(400MHz,DMSO-d6)δ10.65(s,1H),8.87(s,1H),8.19(d,1H),8.02(t,1H) ,7.90-7.82(m,2H),7.73(d,1H),7.61(d,1H),7.23(s,1H),7.10(d,1H),5.67 -5.59(m,1H),2.92(d,2H),2.63-2.52(m,5H),2.20-2.10(m,1H),1.90-1.83( m,1H),1.67-1.55(d,4H),1.44(d,6H),0.84-0.77(m,2H),0.72-0.65(m,2H).

[0197] LC-MS, M / Z(ESI):572.5[M+H] + .

[0198] Example 5: Preparation of target compound I-5

[0199]

[0200] The synthetic route of target compound I-5 is as follows:

[0201]

[0202] Step 1: Synthesis of methyl 2-fluoro-5-nitro-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzoate (I-5B)

[0203] Synthesis steps: Refer to the synthesis of intermediate 1-11B in Example 11.

[0204] Step 2: Synthesis of methyl 5-amino-2-fluoro-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzoate (I-5C)

[0205] Pd-C (570 mg, 5.36 mmol) was carefully added to a solution of methyl 2-fluoro-5-nitro-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzoate (5.7 g, 19.24 mmol) in MeOH (20 mL). The mixture was allowed to react overnight at room temperature under a hydrogen atmosphere. After the reaction was complete, the reaction mixture was filtered and the filtrate was concentrated. The residue was purified on a silica gel column (petroleum ether:ethyl acetate (v / v) = 1:1) to afford methyl 5-amino-2-fluoro-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzoate (540 mg, 2.028 mmol, 10.54% yield) as a yellow solid.

[0206] Step 3: Synthesis of methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzoate (I-5D)

[0207] A reaction solution of DMF (10 ml) containing methyl 5-amino-2-fluoro-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzoate (490 mg, 1.840 mmol), 2-bromo-1-cyclopropylethanone (600 mg, 3.68 mmol), KI (367 mg, 2.208 mmol), and K2CO3 (381 mg, 2.76 mmol) was stirred at 70°C for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column purification (petroleum ether: ethyl acetate (V / V) = 1:1) to give a yellow solid 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzoic acid methyl ester (570 mg, 1.636 mmol, yield 89%).

[0208] LC-MS, M / Z(ESI):349.3[M+H] + .

[0209] Step 4: Synthesis of methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzoate (I-5E)

[0210] Methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzoate (540 mg, 1.550 mmol) was dissolved in formamide (5 ml, 1.550 mmol), heated to 180°C in a microwave oven, and stirred for 60 min. After the reaction was completed, distilled water (20 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column purification (petroleum ether: ethyl acetate (V / V) = 1:1) to give a yellow solid 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzoic acid methyl ester (280 mg, 0.783 mmol, yield 50.5%).

[0211] LC-MS, M / Z(ESI):358.4[M+H] +

[0212] Step 5: Synthesis of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzamide (I-5)

[0213] Potassium tert-butoxide (54.7 mg, 0.487 mmol) was added to a DMF (3 mL) solution containing 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzamide (150 mg, 0.420 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (85 mg, 0.420 mmol), and the mixture was stirred at room temperature for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (dichloromethane: methanol (V / V) = 10:1) to obtain 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(2-oxa-6-azaspiro[3.3]hept-6-yl)benzamide.

[0214] 1H NMR (400MHz, DMSO-d6) δ10.33(d,J=4.0Hz,1H),8.86(s,1H),8.16(d,J=8.0Hz,1H),8.00(d,J=4.0Hz,1H),6.54(d,J=16.0Hz,1H ),5.53-5.60(s,1H),4.60(s,4H),3.78(s,4H),1.82-1.89(m,1H),1.44(d,J=8.0Hz,6H),0.81-0.83(m,2H),0.68-0.72(m,2H).

[0215] LC-MS, M / Z(ESI):529.4[M+H] + .

[0216] Example 6: Preparation of target compound I-6

[0217]

[0218] The synthetic route of the target compound is as follows:

[0219]

[0220] Step 1: Synthesis of methyl 2-fluoro-5-nitro-4-(1-oxo-2,8-diazaspiro[4.5]decane-8-yl)benzoate (I-6B)

[0221] 2,8-Diazaspiro[4.5]decan-1-one hydrochloride (1.85 g, 9.7 mmol) was added dropwise to a THF (30 mL) solution containing methyl 2,4-difluoro-5-nitrobenzoate (2.1 g, 9.7 mmol) and DIEA (1.88 ml, 14.6 mmol) at 0°C. The mixture was stirred at 0°C for 1 h. After the reaction, distilled water (50 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (70 mL × 2), separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (petroleum ether: ethyl acetate (V / V) = 3:1) to give a yellow solid 2-fluoro-5-nitro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)benzoic acid methyl ester (3.0 g, 8.5 mmol, yield 88%).

[0222] Step 2: Synthesis of methyl 5-amino-2-fluoro-4-(1-oxo-2,8-diazaspiro[4.5]decane-8-yl)benzoate (I-6C)

[0223] Pd-C (300 mg) was carefully added to a solution of methyl 2-fluoro-5-nitro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)benzoate (3.0 g, 8.5 mmol) in MeOH (30 ml). The mixture was allowed to react overnight at room temperature under a hydrogen atmosphere. After the reaction was complete, the mixture was filtered and the filtrate was concentrated. The residue was purified on a silica gel column (petroleum ether:ethyl acetate (v / v) = 1:1) to afford methyl 5-amino-2-fluoro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)benzoate (2.0 g, 6.2 mmol, 73.2% yield) as a yellow solid.

[0224] Step 3: Synthesis of methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(1-oxo-2,8-diazaspiro[4.5]decane-8-yl)benzoate (I-6D)

[0225] A reaction solution of DMF (10 mL) containing methyl 5-amino-2-fluoro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)benzoate (2.0 g, 6.2 mmol), 2-bromo-1-cyclopropylethanone (2.0 g, 12.4 mmol), KI (514 mg, 3.1 mmol), and K2CO3 (1.28 g, 9.3 mmol) was stirred at 70 °C for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column purification (petroleum ether: ethyl acetate (V / V) = 1:1) to give a yellow solid 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)benzoic acid methyl ester (1.6 g, 4.0 mmol, yield 70%).

[0226] Step 4: Synthesis of methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(1-oxo-2,8-diazaspiro[4.5]decane-8-yl)benzoate (I-6E)

[0227] Methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)benzoate (1.6 g, 4.0 mmol) was dissolved in formamide (5 mL), heated to 180°C in a microwave oven, and stirred for 60 min. After the reaction is completed, distilled water (20 mL) is added to dilute the mixture, and the mixture is extracted with ethyl acetate (30 mL × 3). The organic phases are combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by silica gel column chromatography (petroleum ether: ethyl acetate (V / V) = 1:1) to give a yellow solid 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)benzoic acid methyl ester (41 mg, 0.099 mmol, yield 2.5%).

[0228] Step 5: Synthesis of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(1-oxo-2,8-diazaspiro[4.5]decane-8-yl)benzamide (I-6)

[0229] Potassium tert-butoxide (44 mg, 0.40 mmol) was added to a DMF (3 mL) solution containing methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)benzoate (41 mg, 0.099 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (20 mg, 0.099 mmol), and the mixture was stirred at room temperature for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (dichloromethane: methanol (V / V) = 10:1) to obtain 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(1-oxo-2,8-diazaspiro[4.5]decane-8-yl)benzamide.

[0230] 1H NMR(400MHz,DMSO-d6)δ10.62(s,1H),8.83(s,1H),8.15(d,1H),7.99(t, 1H),7.85(d,2H),7.57(d,2H),7.21(s,1H),7.08(s,1H),5.63-5.57(m,1 H),3.12(t,2H),2.86(d,2H),2.25(t,2H),1.91-1.88(m,3H),1.68-1.64 (m,2H),1.40(d,6H),1.29(d,2H),0.79-0.75(m,2H),0.66-0.64(m,2H).

[0231] LC-MS, M / Z(ESI):584.5[M+H] + .

[0232] Example 7: Preparation of target compound I-7

[0233]

[0234] The synthetic route of target compound I-7 is as follows:

[0235]

[0236] Step 1: Synthesis of methyl 2-fluoro-5-nitro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (I-7B)

[0237] 2,8-Diazaspiro[4.5]decan-3-one (3.73 g, 24.2 mmol) was added to a solution of methyl 2,4-difluoro-5-nitrobenzoate (5 g, 23.0 mmol) and diisopropylethylamine (3.27 g, 25.3 mmol) in tetrahydrofuran (80 mL) at 0°C. The mixture was stirred at 0°C for 2 h. After completion of the reaction, the mixture was diluted with water (80 mL) and extracted with ethyl acetate (80 mL × 3). The organic phases were combined, washed with saturated brine (70 mL × 2), separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column to obtain methyl 2-fluoro-5-nitro-4-(3-oxo-2,8-diazaspiro[4.5]decan-8-yl)benzoate (6 g, 74.2% yield) as a yellow solid.

[0238] LC-MS, M / Z(ESI):352.3[M+H] + .

[0239] Step 2: Synthesis of methyl 5-amino-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (I-7C)

[0240] Pd / C (0.6 g, 10%) was carefully added to a solution of methyl 2-fluoro-5-nitro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (6.0 g, 17.18 mmol) in methanol (30 mL). The mixture was allowed to react at room temperature for 16 hours under a hydrogen atmosphere. After the reaction was complete, the reaction mixture was filtered and concentrated directly. The residue was purified on a silica gel column to afford methyl 5-amino-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (4.1 g, 74.7% yield) as a yellow solid.

[0241] LC-MS, M / Z(ESI):322.3[M+H] + .

[0242] Step 3: Synthesis of methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (I-7D)

[0243] 2-Bromo-1-cyclopropylethanone (2.54 g, 15.56 mmol) was added dropwise to a DMF (10 mL) solution containing methyl 5-amino-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (2 g, 6.22 mmol), potassium iodide (1.14 g, 6.85 mmol), and potassium carbonate (2.15 g, 15.56 mmol). The reaction was continued at 70°C for 16 h. After the reaction, water (50 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column to give a yellow solid 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoic acid methyl ester (0.41 g, yield 16.3%).

[0244] LC-MS, M / Z(ESI):404.4[M+H] + .

[0245] Step 4: Synthesis of methyl 5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (I-7E)

[0246] Under nitrogen protection, methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (0.77 g, 1.01 mmol) was dissolved in acetic acid (6 mL), potassium thiocyanate (0.2 g, 2.03 mmol) was added, and the reaction was stirred at 110 ° C overnight. After the reaction, water (30 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column to give a yellow solid 5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoic acid methyl ester (0.33 g, yield 73.1%).

[0247] LC-MS, M / Z(ESI):445.5[M+H] + .

[0248] Step 5: Synthesis of methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (I-7F)

[0249] Methyl 5-(4-cyclopropyl-2-mercapto-1H-imidazol-1-yl)-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (0.33 g, 0.732 mmol) was dissolved in acetic acid (6 mL). Water (1 mL) was added, followed by the slow dropwise addition of hydrogen peroxide (0.5 mL, 30%). The reaction was stirred at 50°C for 1 h. After the reaction is completed, water (30 mL) is added to dilute the mixture, and the mixture is extracted with ethyl acetate (20 mL × 3). The organic phases are combined, washed with saturated brine (20 mL), separated, and the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by silica gel column to give a yellow solid 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoic acid methyl ester (0.18 g, yield 58.8%).

[0250] LC-MS, M / Z(ESI):413.4[M+H] + .

[0251] Step 6: Synthesis of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzamide (I-7)

[0252] Potassium tert-butoxide (54.4 mg, 0.485 mmol) was added to a DMF (2 mL) solution containing methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzoate (50 mg, 0.121 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (27.1 mg, 0.133 mmol), and the mixture was stirred at room temperature for 2 h. After the reaction is completed, water (10 mL) is added to dilute the mixture, and the mixture is extracted with dichloromethane (20 mL × 3). The organic phases are combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by silica gel plate to obtain 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(3-oxo-2,8-diazaspiro[4.5]dec-8-yl)benzamide.

[0253] 1 H NMR(400MHz,DMSO-d6)δ10.65(s,1H),8.86(s,1H),8.18(d,1H),8.01(t, 1H),7.86(d,1H),7.62(d,1H),7.52(s,1H),7.28(s,1H),7.10(d,1H),5. 68-5.56(m,1H),3.03(s,2H),2.75-2.65(m,4H),2.05(s,2H),1.90-1.80 (m,1H),1.54(s,4H),1.43(d,6H),0.86-0.78(m,2H),0.72-0.64(m,2H).

[0254] LC-MS, M / Z(ESI):584.4[M+H] + .

[0255] Example 8: Preparation of target compound I-8

[0256]

[0257] The synthetic route of target compound I-8 is as follows:

[0258]

[0259] Step 1: Synthesis of 6-aminopyrazine-2-carbohydrazide (I-8B)

[0260] Methyl 6-aminopyrazine-2-carboxylate (2 g, 13.06 mmol) was dissolved in methanol, followed by the addition of hydrazine hydrate (2.45 g, 39.2 mmol). The reaction was heated to 80°C and stirred at this temperature for 4 hours. After slowly cooling to room temperature, the precipitated solid was filtered and the filter cake collected to afford the title compound, 6-aminopyrazine-2-carbohydrazide, as a yellow solid (1.6 g, 80% yield).

[0261] LC-MS, M / Z (ESI): 154.2 [M+H] + .

[0262] Step 2: Synthesis of (E)-N'-(6-(2-((E)-(dimethylamino)methylene)hydrazine-1-carbonyl]pyrazin-2-yl)-N,N-dimethylformamide (I-8C)

[0263] At room temperature, 6-aminopyrazine-2-carbohydrazide from the previous step was dissolved in N,N-dimethylformamide dimethyl acetal (40 mL) and heated under reflux with stirring for 18 hours. After the reaction system was cooled to room temperature, it was concentrated under reduced pressure to give crude (E)-N'-(6-(2-((E)-(dimethylamino)methylene)hydrazine-1-carbonyl]pyrazin-2-yl)-N,N-dimethylformamide (2.3 g), which was used in the next step without purification.

[0264] LC-MS, M / Z(ESI):264.3[M+H] + .

[0265] Step 3: Synthesis of 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyrazin-2-amine (I-8D)

[0266] The crude product of (E)-N'-(6-(2-((E)-(dimethylamino)methylene)hydrazine-1-carbonyl]pyrazin-2-yl)-N,N-dimethylformamide (2.3 g, 8.85 mmol) from the previous step was dissolved in acetonitrile (30 mL). Acetic acid (4 mL) and isopropylamine (2.62 g, 44.2 mol) were added sequentially, and the mixture was stirred under reflux overnight. After cooling, the reaction system was concentrated under reduced pressure and the organic solvent was concentrated. Tetrahydrofuran (20 mL) and 1M aqueous sodium hydroxide solution were added to the reaction flask to adjust the pH to 8.0, and the mixture was stirred at room temperature overnight. The organic solvent was removed under reduced pressure, the aqueous phase was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. The organic solvent was concentrated under reduced pressure and separated by column chromatography to obtain the title compound, 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyrazin-2-amine (0.3 g, 16.6% yield).

[0267] LC-MS, M / Z (ESI): 205.2 [M+H] + .

[0268] Step 4: Synthesis of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyrazin-2-yl)-4-methylbenzamide (I-8)

[0269] Potassium tert-butoxide (188 mg, 1.68 mmol) was added to a DMF (3 ml) solution containing methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methylbenzoate (115 mg, 0.42 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyrazin-2-amine (85 mg, 0.420 mmol), and the mixture was stirred at room temperature for 3 h. After the reaction is completed, distilled water (10 mL) is added to dilute the mixture, and the mixture is extracted with ethyl acetate (20 mL × 3). The organic phases are combined, washed with saturated brine (10 mL), separated, and the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by silica gel column (dichloromethane: methanol (V / V) = 10:1) to obtain 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyrazin-2-yl)-4-methylbenzamide.

[0270] 1 H NMR(400MHz,DMSO-d6)δ9.36(s,1H),9.04(s,1H),8.93(s,1H),8.29(b,1H),7.67-7.64(m,2H),7.47(d,1H),7.1 5(s,1H),5.50-5.44(m,1H),2.22(s,3H),1.83-1.78(m,1H),1.42(d,6H),0.78-0.75(m,2H),0.67-0.65(m,2H).

[0271] LC-MS, M / Z (ESI): 447.4 [M+H] + .

[0272] Example 9: Preparation of target compound I-9

[0273]

[0274] The synthetic route of target compound I-9 is as follows:

[0275]

[0276] Step 1: Synthesis of methyl 2-fluoro-5-nitro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoate (I-9B)

[0277] 2,6-Diazaspiro[3.4]octan-7-one hydrochloride (1 g, 6.15 mmol) was added to a solution of methyl 2,4-difluoro-5-nitrobenzoate (1.26 g, 5.8 mmol) and diisopropylethylamine (0.83 g, 6.4 mmol) in tetrahydrofuran (15 mL) at 0°C. The mixture was stirred at room temperature for 20 h. After completion of the reaction, the mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine (20 mL x 2), separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column to obtain methyl 2-fluoro-5-nitro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoate (1.5 g, 80% yield) as a yellow solid.

[0278] LC-MS, M / Z(ESI):324.3[M+H] + .

[0279] Step 2: Synthesis of methyl 5-amino-2-fluoro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoate (I-9C)

[0280] Palladium on carbon (0.6 g, 10%) was carefully added to a solution of methyl 2-fluoro-5-nitro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoate (1.5 g, 4.64 mmol) in ethanol (20 mL). The mixture was allowed to react at room temperature under a hydrogen atmosphere for 16 hours. After the reaction was complete, the reaction solution was filtered and concentrated directly. The residue was purified on a silica gel column to afford methyl 5-amino-2-fluoro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoate (0.32 g, 23.5% yield) as a yellow solid.

[0281] LC-MS, M / Z(ESI):294.3[M+H] + .

[0282] Step 3: Synthesis of methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoate (I-9D)

[0283] 2-Bromo-1-cyclopropylethanone (0.27 g, 1.48 mmol) was added dropwise to a DMF (10 mL) solution containing methyl 5-amino-2-fluoro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoate (0.29 g, 0.99 mmol) and potassium carbonate (0.18 g, 1.29 mmol). The reaction was continued at 70°C for 16 h. After the reaction, water (50 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (20 mL), separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column to give a yellow solid 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoic acid methyl ester (0.25 g, yield 67.4%).

[0284] LC-MS, M / Z(ESI):376.4[M+H] + .

[0285] Step 4: Synthesis of methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoate (I-9E)

[0286] Methyl 5-((2-cyclopropyl-2-oxoethyl)amino)-2-fluoro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoate (200 mg, 0.53 mmol) was dissolved in formamide (2 mL), heated to 180°C in a microwave oven, and stirred for 60 min. After the reaction was completed, distilled water (20 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column purification (petroleum ether: ethyl acetate (V / V) = 1:1) to give a yellow solid 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoic acid methyl ester (70 mg, 0.18 mmol, yield 34.4%).

[0287] Step 5: Synthesis of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzamide (I-9)

[0288] Potassium tert-butoxide (80 mg, 0.72 mmol) was added to a DMF (1 mL) solution containing methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzoate (70 mg, 0.18 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (37 mg, 0.18 mmol), and the mixture was stirred at room temperature for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (dichloromethane: methanol (V / V) = 10:1) to obtain 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(7-oxo-2,6-diazaspiro[3.4]octan-2-yl)benzamide.

[0289] 1 H NMR(400MHz,DMSO-d6)δ10.32(d,1H),8.85(s,1H),8.15(d,1H),7.98(t,1H),7.82(d,2H),7.59(d,1H),7.50(d,1H),7.05(s,1H),6.55( d,1H),5.59-5.52(m,1H),3.57(dd,4H),3.34(s,2H),2.36(s,2H),1.84-1.80(m,1H),1.43(d,6H),0.79-0.77(m,2H),0.68-0.65(m,2H).

[0290] LC-MS, M / Z(ESI):556.3[M+H] + .

[0291] Example 10: Preparation of target compound I-10

[0292]

[0293] The synthetic route of the target compound is as follows:

[0294]

[0295] Step 1: Synthesis of methyl 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(2-oxa-6-azaspiro[3.3]hept-6-yl)picolinate (I-10B)

[0296] 2-Oxo-6-azaspiro[3.3]heptane hemioxalate (500 mg, 1.91 mmol) and potassium carbonate (1.32 g, 9.57 mmol) were added sequentially to a DMF (15 mL) solution containing 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoropicolinate (200 mg, 0.766 mmol) and stirred at 90 °C for 16 h. After the reaction, distilled water (50 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated brine (30 mL), separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column to obtain a yellow solid 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(2-oxa-6-azaspiro[3.3]hept-6-6-yl)picolinic acid methyl ester (0.3 g, yield 46.1%).

[0297] LC-MS, M / Z(ESI):341.4[M+H] + .

[0298] Step 2: Synthesis of 4-(4-cyclopropyl-1H-imidazol-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-5-(2-oxa-6-azaspiro[3.3]hept-6-yl)pyridinamide (I-10)

[0299]

[0300] Potassium tert-butoxide (396 mg, 3.53 mmol) was added to a DMF (2 mL) solution containing methyl 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(2-oxa-6-azaspiro[3.3]hept-6-yl)picolinate (300 mg, 0.881 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (29.7 mg, 0.146 mmol) and stirred at room temperature for 3 h. After the reaction is completed, distilled water (10 mL) is added to dilute the mixture, and the mixture is extracted with dichloromethane (20 mL × 3). The organic phases are combined, washed with saturated brine (10 mL), separated, and the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by silica gel column to obtain 4-(4-cyclopropyl-1H-imidazol-1-yl)-5-(3-hydroxy-3-methylpyrrolidin-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)picolinamide.

[0301] 1H NMR(400MHz,DMSO-d6)δ10.34(s,1H),8.90(s,1H),8.31(dd,1H),8.10(s,1 H),8.05(t,1H),7.86(dd,1H),7.82(s,1H),7.74(d,1H),7.21(d,1H),5.54 -5.38(m,1H),4.63(s,4H),3.96(s,4H),1.93-1.82(m,1H),1.51(d,6H),0.87-0.80(m,2H),0.76-0.67(m,2H).

[0302] LC-MS, M / Z(ESI):512.4[M+H] + .

[0303] Example 11: Preparation of target compound I-11

[0304]

[0305] The synthetic route of the target compound is as follows:

[0306]

[0307] Step 1: Synthesis of methyl 2-fluoro-5-nitro-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (I-11B)

[0308] 2-Oxa-6-azaspiro[3.3]heptane hemioxalate (6.6 g, 23 mmol) was added dropwise to a solution of methyl 2,4-difluoro-5-nitrobenzoate (5.0 g, 23 mmol) and DIEA (4.5 g, 34.5 mmol) in THF (15 mL) at 0°C. The mixture was stirred at 0°C for 1 h. After completion of the reaction, the mixture was diluted with distilled water (50 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (70 mL × 2), separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on a silica gel column (petroleum ether:ethyl acetate (v / v) = 3:1) to obtain methyl 2-fluoro-5-nitro-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (4.0 g, 13.5 mmol, 58.7% yield) as a yellow solid.

[0309] Step 2: Synthesis of methyl 5-amino-2-fluoro-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (I-11C)

[0310] Pd-C (400 mg) was carefully added to a solution of methyl 2-fluoro-5-nitro-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (4.0 g, 13.5 mmol) in MeOH (40 mL). The mixture was allowed to react overnight at room temperature under a hydrogen atmosphere. After the reaction was complete, the mixture was filtered and the filtrate was concentrated. The residue was purified on a silica gel column (petroleum ether:ethyl acetate (v / v) = 1:1) to afford methyl 5-amino-2-fluoro-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (3.7 g, 13.9 mmol) as a yellow solid.

[0311] Step 3: Synthesis of methyl 5-bromo-2-fluoro-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (I-11D)

[0312] Methyl 5-amino-2-fluoro-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (500 mg, 1.9 mmol) was dissolved in tetrahydrofuran (10 mL), and tert-butyl nitrite (290 mg, 2.8 mmol) was slowly added dropwise at 0°C. After completion of the addition, the mixture was stirred steadily for 30 minutes. Then, copper bromide (629 mg, 2.8 mmol) was added to the system, and the mixture was stirred at room temperature overnight. The mixture was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give methyl 5-bromo-2-fluoro-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (370 mg, 1.1 mmol, yield 59%).

[0313] Step 4: Synthesis of methyl 2-fluoro-5-(1-isopropyl-1H-pyrazol-4-yl)-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (I-11E)

[0314] Methyl 5-bromo-2-fluoro-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (370 mg, 1.1 mmol) was dissolved in 1,4-dioxane (5 mL), and 1-isopropyl-1H-pyrazole-4-boronic acid pinacol ester (312 mg, 1.3 mmol), Pd(dppf)Cl2 (80 mg, 0.11 mmol) and potassium carbonate (228 mg, 1.7 mmol) were added. The mixture was heated to 100 ° C in a microwave and stirred for 30 min. After completion of the reaction, the reaction mixture was concentrated and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give solid methyl 2-fluoro-5-(1-isopropyl-1H-pyrazol-4-yl)-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzoate (345 mg, 0.96 mmol, yield 87.3%).

[0315] Step 5: Synthesis of 2-fluoro-5-(1-isopropyl-1H-pyrazol-4-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzamide (I-11)

[0316] Potassium tert-butoxide (187 mg, 1.7 mmol) was added to a DMF (3 mL) solution containing methyl 2-fluoro-5-(1-isopropyl-1H-pyrazol-4-yl)-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)benzoate (150 mg, 0.42 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (85 mg, 0.420 mmol), and the mixture was stirred at room temperature for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (dichloromethane: methanol (V / V) = 10:1) to obtain 2-fluoro-5-(1-isopropyl-1H-pyrazol-4-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-(2-oxa-6-azaspiro[3.3]heptane-6-yl)benzamide.

[0317] 1 H NMR(400MHz,DMSO-d6)δ10.22(d,1H),8.84(s,1H),8.16(d,1H),7.98(t,1H),7.86(s,1H),7.82(d,1H),7.50(s ,1H),7.45(d,1H),6.44(d,1H),5.61-5.54(m,1H),4.61(s,4H),4.56-4.49(m,1H),3.81(s,4H),1.44(dd,12H).

[0318] LC-MS, M / Z(ESI):531.4[M+H] + .

[0319] Example 12: Preparation of target compound II-1

[0320]

[0321] The synthetic route of target compound II-1 is as follows:

[0322]

[0323] Step 1: Synthesis of 4-bromopicolinic acid (II-1B)

[0324] To a mixture of methyl 4-bromopicolinate (2 g, 9.26 mmol) in tetrahydrofuran (30 mL), methanol (10 mL), and water (10 mL) was added lithium hydroxide monohydrate (1.554 g, 37 mmol) and stirred at room temperature overnight. After the reaction was complete, the mixture was diluted with water (50 mL) and extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to yield 4-bromopicolinic acid (1.6 g, 87% yield) as a white solid.

[0325] Step 2: Synthesis of 4-chloro-N-(6-(5-methyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)pyridin-2-yl)pyridineamide (II-1C)

[0326] Add thionyl chloride (20 mL) into a flask containing 4-bromopicolinic acid (366 g, 1.81 mmol) and react at 85°C for 2 h. Subsequently, the reaction solution was directly concentrated, and tetrahydrofuran (10 mL), 6-(5-methyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)pyridin-2-amine (0.3 g, 1.39 mmol) and pyridine (2 mL) were added in sequence and reacted at 45 ° C overnight. After the reaction, distilled water (30 mL) was added for dilution, and the mixture was extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with saturated brine (20 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column to obtain a yellow solid 4-chloro-N-(6-(5-methyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)pyridin-2-yl)picolinamide (260 mg, yield 46.7%).

[0327] LC-MS m / z:400.2[M+H] + .

[0328] Step 3: Synthesis of 4-(4-cyclopropyl-1H-imidazol-1-yl)-N-(6-(5-methyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)pyridin-2-yl)pyridineamide (II-1)

[0329] Under nitrogen, dimethyl sulfoxide (6 mL) was added to a flask containing cuprous iodide (10.7 mg, 0.056 mmol), 8-hydroxyquinoline (16.4 mg, 0.113 mmol), potassium carbonate (97 mg, 0.705 mmol), 4-cyclopropyl-1H-imidazole (61 mg, 0.564 mmol), and 4-chloro-N-(6-(5-methyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)pyridin-2-yl)picolinamide (1.410 g, 8.65 mmol). The mixture was reacted at 110°C overnight. After the reaction is completed, distilled water (20 mL) is added to dilute the mixture, and the mixture is extracted with ethyl acetate (20 mL × 3). The organic phases are combined, washed with saturated brine (20 mL), separated, and the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by silica gel plate to obtain 4-(4-cyclopropyl-1H-imidazol-1-yl)-N-(6-(5-methyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)pyridin-2-yl)pyridineamide.

[0330] 1 H NMR (400MHz, CDCl3) δ10.39(s,1H),8.72(d,1H),8.45-8.40(m,1H),8.32(d,1H),8.15-8.07(m,1H),8.02(d,1H),7.92(t,1H),7.50(dd,1H),7. 24(d,1H),5.18-5.11(m,1H),3.14-2.99(m,3H),2.49-2.42(m,1H),1.9 6-1.89(m,1H),1.34-1.19(m,3H),0.97-0.91(m,2H),0.88-0.83(m,2H).

[0331] Example 13: Preparation of target compound II-2

[0332]

[0333] The synthetic route of the target compound is as follows:

[0334]

[0335] Step 1: Synthesis of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)-N-(6-(5-methyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)pyridin-2-yl)benzamide (II-2)

[0336] Potassium tert-butoxide (130 mg, 1.16 mmol) was added to a DMF (3 ml) solution containing methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)benzoate (100 mg, 0.29 mmol) and 6-(5-methyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)pyridin-2-amine (70 mg, 0.32 mmol) and stirred at room temperature for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane: methanol (V / V) = 10:1) to obtain 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(3-hydroxy-3-methylazetidin-1-yl)-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)benzamide (.

[0337] 1 H NMR(400MHz,DMSO-d6)δ10.21(d,1H),8.09(d,1H),7.93(t,1H),7.80(d, 1H),7.55(s,1H),7.47(d,1H),7.02(s,1H),6.00(d,1H),5.50(s,1H),5. 11-5.07(m,1H),3.40(dd,4H),3.78(s,4H),2.97-2.84(m,3H),2.29(t,1 H),1.81(b,1H),1.32-1.29(m,6H),0.77-0.75(m,2H),0.64-0.63(m,2H).

[0338] LC-MS, M / Z(ESI):529.4[M+H] + .

[0339] Example 14: Preparation of target compound II-3

[0340]

[0341] The synthetic route of target compound II-3 is as follows:

[0342]

[0343] Step 1: Synthesis of 1-(2-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoro-4-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N-methylpiperidine-4-carboxamide (II-3)

[0344] Potassium tert-butoxide (117 mg, 1.043 mmol) was added to a DMF (2 mL) solution containing methyl 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-(4-(methylcarbamoyl)piperidin-1-yl)benzoate (100 mg, 0.261 mmol) and 6-(5-methyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-3-yl)pyridin-2-amine (67.4 mg, 0.313 mmol) and stirred at room temperature for 2 h. After the reaction is completed, water (10 mL) is added to dilute the mixture, and the mixture is extracted with dichloromethane (20 mL × 3). The organic phases are combined, washed with saturated brine (10 mL), separated, and the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by silica gel plate to obtain 1-(2-(4-cyclopropyl-1H-imidazol-1-yl)-5-fluoro-4-((6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)carbamoyl)phenyl)-N-methylpiperidine-4-carboxamide.

[0345] 1 H NMR(400MHz,DMSO-d6)δ10.56(s,1H),8.12(d,1H),7.95(t,1H),7.867.78(m,2H),7.7 2-7.67(m,1H),7.56(d,1H),7.20(s,1H),7.07(d,1H),5.15-5.08(m,1H),4.46-4.29( m,1H),3.02-2.79(m,5H),2.59-2.48(m,4H),2.32-2.25(m,1H),2.16-2.07(m,1H),1. 87-1.79(m,1H),1.63-1.49(m,4H),1.29(d,3H),0.80-0.74(m,2H),0.69-0.62(m,2H).

[0346] LC-MS, M / Z(ESI):584.5[M+H] + .

[0347] Example 15: Preparation of target compound III-1

[0348]

[0349] The synthetic route of target compound III-1 is as follows:

[0350]

[0351] Step 1: Synthesis of methyl 1-(5-fluoro-4-carboxylate)-2-nitrophenyl)azetidine-2-carboxylate (III-1B): Methyl 2-azetidine-1-carboxylate hydrochloride (1.7 g, 11.0 mmol) was added dropwise to a solution of methyl 2,4-difluoro-5-nitrobenzoate (2.0 g, 9.2 mmol) and DIEA (1.8 g, 13.8 mmol) in DMF (15 mL) at 0°C. The mixture was stirred at 0°C for 1 h. After the reaction, distilled water (50 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated brine (70 mL × 2), separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (petroleum ether: ethyl acetate (V / V) = 3:1) to give solid 1-(5-fluoro-4-methylformate)-2-nitrophenyl)azetidine-2-carboxylic acid methyl ester (3.0 g, 9.6 mmol).

[0352] Step 2: Synthesis of methyl 7-fluoro-3-carbonyl-2,2a,3,4-tetrahydro-1H-azetidin[1,2-a]quinoxaline-6-carboxylate (III-1C)

[0353] Pd-C (300 mg) was carefully added to a solution of methyl 1-(5-fluoro-4-carboxylate)-2-nitrophenyl)azetidine-2-carboxylate (3.0 g, 9.6 mmol) in MeOH (30 mL). The mixture was allowed to react overnight at room temperature under a hydrogen atmosphere. After the reaction was complete, the reaction mixture was filtered and the filtrate was concentrated. The residue was purified on a silica gel column (petroleum ether:ethyl acetate (v / v) = 1:1) to afford methyl 5-amino-4-(2-carboxamidoazetidin-1-yl)-2-fluorobenzoate (1.0 g, 4.0 mmol, 41.6% yield) as a solid.

[0354] Step 3: Synthesis of methyl 7-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3-carbonyl-2,2a,3,4-tetrahydro-1H-azetidin[1,2-a]quinoxaline-6-carboxylate (III-1)

[0355] Potassium tert-butoxide (269 mg, 2.4 mmol) was added to a DMF (3 mL) solution containing methyl 7-fluoro-3-carbonyl-2,2a,3,4-tetrahydro-1H-azetidin[1,2-a]quinoxaline-6-carboxylate (150 mg, 0.60 mmol) and 6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-amine (122 mg, 0.60 mmol) and stirred at room temperature for 3 h. After the reaction, distilled water (10 mL) was added to dilute the mixture, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), separated, and dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column (dichloromethane: methanol (V / V) = 10:1) to obtain 7-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-3-carbonyl-2,2a,3,4-tetrahydro-1H-azetidin-[1,2-a]quinoxaline-6-carboxylic acid methyl ester.

[0356] 1 H NMR (400MHz, DMSO-d6) δ10.50(s,1H),10.31(d,1H),8.85(s,1H),8.17(d,1H),7.99(t,1H),7.84(d,1H),7.13(d,1H),6.60( d,1H),5.63-5.56(m,1H),4.82(t,1H),4.29(dd,1H),3.86-3.80(m,1H),2.89-2.85(m,1H),2.75-2.72(m,1H),1.42(d,6H).

[0357] LC-MS, M / Z(ESI):422.3[M+H] + .

[0358] Biological activity and related properties test examples

[0359] Test Example 1: Inhibition test of ASK1 kinase activity by compounds

[0360] The inhibition of ASK1 kinase activity by the compounds was detected using the ADP-Glo ​​luminescence assay (Promege, Cat. No. v9102 / 3).

[0361] First, prepare a 10mM stock solution of the test compound in DMSO solvent, then use DMSO gradient dilution to the required 10 concentration points three times as the compound working solution. Use a biological workstation to transfer 50nL of the compound working solution to a 384-well plate, and transfer another 50nL of DMSO to empty wells as negative and positive control wells. Repeat the well operation for each concentration point.

[0362] Prepare kinase assay buffer (20 mM HEPES, pH 7.5; 0.01% Triton X-100; 25 mM MgCl2). Prepare ASK1 kinase (eurofins, Cat. No. 14-606M) at twice the final concentration (15 nM) in kinase assay buffer as the ASK1 enzyme working solution. Transfer 2.5 μL of the ASK1 kinase working solution to each well of a 384-well plate. Replace the negative control well with 2.5 μL of kinase assay buffer. Mix the 384-well plate thoroughly.

[0363] Use kinase assay buffer to prepare MBP (Active Motif, Cat. No. 31314) and ATP (Sigma, Cat. No. A7699-1G, final concentration 218 μM) at a concentration of 4 times the final concentration as the substrate working solution; transfer 2.5 μL of the substrate working solution to each reaction well of a 384-well plate, mix the 384-well plate, and start the reaction.

[0364] After incubation at 37°C for 60 minutes, add 5 μL of ADP-Glo ​​reagent and continue incubation at 37°C for 180 minutes. After incubation, add 5 μL of kinase detection reagent and equilibrate at room temperature for 30 minutes. Relative light units (RLU) data were collected using Envision and converted to percent inhibition. Percent inhibition = (positive control value - sample control value) / (positive control value - negative control value) * 100.

[0365] IC was calculated using Excel's built-in curve fitting tool XLFit (version 5.4.0.8) 50 value.

[0366] Table 1 Inhibition results of ASK1 kinase activity by test compounds

[0367]

[0368]

[0369] The experimental results show that the compound of the present invention has a good inhibitory effect on ASK1 kinase; compared with the control compound, it shows better ASK1 kinase inhibitory activity.

[0370] Test Example 2: Inhibition test of compound on α-SMA and COL1A1 gene expression

[0371] Hepatic stellate cells (HSC) were cultured and collected. 5Cells were seeded into type I collagen-coated 96-well plates at a density of 100 μL per well. A negative control well was set up with 100 μL of culture medium without cells. The plates were incubated overnight at 37°C in a 5% CO2 incubator. After overnight, the plates were removed, replaced with fresh serum-free culture medium, and incubated for another 4 hours.

[0372] The test compound was prepared into a 10 mM stock solution in DMSO solvent, diluted to an appropriate concentration with DMSO, and then diluted to the required concentration with cell culture medium (DMSO content not exceeding 0.3%) to serve as the compound working solution.

[0373] Remove the culture medium from the plate and add 50 μL of compound working solution. Replace the negative control with DMSO-containing medium. Incubate the plate in triplicate for 30 minutes at 37°C in a 5% CO2 incubator. After 30 minutes, add 50 μL of a 20 ng / mL TGF-β solution diluted in culture medium to each well. Final compound concentrations are 10 nM and 20 nM.

[0374] After 48 hours of culture, wash the plates with cold PBS. Add 50 μL of a 1:100 dilution of DNase I lysis buffer to each well and mix five times. Incubate the plates at room temperature (19-25°C) for 5 minutes. Add 5 μL of stop buffer to each well and mix five times. Incubate the plates again at room temperature for 2 minutes.

[0375] Reverse transcription working solution was prepared on ice at a ratio of 35 μL reverse transcription mixture and 15 μL cell lysate. The solution was placed in a PCR instrument (Biometra TAdvanced 96SG) and the experiment was performed according to the program of 37°C for 1 hour, 95°C for 5 minutes, and 4°C for infinite cycles. After the reaction, the reverse transcription product was stored at -20°C for further detection.

[0376] On wet ice, use Gene Expression Cells-to-CT TM Prepare a real-time PCR reaction mixture containing a GAPDH gene probe (Invitrogen, 4448489) and an α-SMA gene probe (Invitrogen, 4331182) using the kit. Perform three replicates per sample for each gene, gently shaking. Place the reaction mixture in a PCR instrument and perform the reaction according to the kit instructions. Detection of the COL1A1 gene (Invitrogen, 4331182) is the same as for the α-SMA gene.

[0377] The CT value was automatically calculated according to the default settings of Quant Studio 5 software. The relative expression of genes was calculated using the following formula:

[0378] ΔCt=Ct(target gene)-Ct(GAPDH)

[0379] Related gene expression = 2 -ΔCt

[0380] Expression fold = sample group 2 ( -ΔCt ) / negative control group 2( -ΔCt )

[0381] The inhibition rate was calculated based on the relative expression level of the target gene.

[0382] Table 2 Inhibition rate of test compounds on α-SMA and COL1A1 gene expression

[0383]

[0384] The experimental results show that compared with the control compound, the compound of the present invention has a better inhibitory effect on the expression of α-SMA and COL1A1 genes in HSC cells.

[0385] Test Example 3: Human liver microsome stability test

[0386] The stability test for human liver microsomes was performed by incubating the compound with human liver microsomes in vitro. The test compound was first prepared as a 10 mM stock solution in DMSO solvent, and then diluted to 0.5 mM with acetonitrile. Human liver microsomes (Corning) were diluted with PBS to form a microsome / buffer solution, and this solution was used to dilute 0.5 mM of the compound to form a working solution. The working solution contained 1.5 μM compound and 0.75 mg / ml human liver microsomes. A deep-well plate was prepared, and 30 μL of the working solution was added to each well. The reaction was then initiated by adding 15 μL of preheated 6 mM NADPH solution and incubated at 37°C. The reaction was terminated by adding 135 μL of acetonitrile to the corresponding wells at 0, 5, 15, 30, and 45 minutes of incubation. After terminating the reaction with acetonitrile at the final 45-minute time point, the deep-well plate was vortexed for 10 minutes (600 rpm / min) and then centrifuged for 15 minutes. After centrifugation, the supernatant was collected and purified water was added in a 1:1 ratio. LC-MS / MS was then performed to obtain the ratio of the compound peak area to the internal standard peak area at each time point. The peak area ratios of the compound at 5, 15, 30, and 45 minutes were compared with the peak area ratio at 0 minute. The remaining percentage of the compound at each time point was calculated. T was calculated using Excel. 1 / 2 .

[0387] Table 3 Human liver microsome stability test results

[0388]

[0389] Note: ∞ represents infinity, indicating that the compound is very poorly metabolized in human liver microsomes.

[0390] The experimental results show that compared with the control compound, the compound of the present invention exhibits better liver metabolic stability, is metabolized more slowly in the human body, and has a higher exposure amount.

[0391] Test Example 4: Inhibition test of compounds on cytochrome P450

[0392] Compounds were tested for their inhibitory potential against the cytochrome P450 (CYP450) isoforms CYP2C9, CYP2D6, and CYP3A4 (two substrates, midazolam and testosterone). Test compounds were prepared in DMSO to a 10 mM stock solution. The CYP2C9 inhibitor sulfaphenazole, the CYP2D6 inhibitor quinidine, and the CYP3A4 inhibitor ketoconazole were prepared in DMSO to 10 mM, 2.5 mM, and 2.5 mM stock solutions, respectively. Test compounds and ketoconazole were diluted in acetonitrile to a 400-fold final concentration (compound: 10 μM, ketoconazole: 2.5 μM). Sulfaphenazole and quinidine were diluted in DMSO to a 400-fold final concentration (sulfaphenazole: 10 μM, quinidine: 2.5 μM).

[0393] Potassium phosphate buffer (0.1 M, pH 7.4) was used to prepare 4 times the final concentration of NADPH cofactor (66.7 mg NADPH was added to 10 mL potassium phosphate buffer) and substrate. The final concentration of the CYP2C9 substrate diclofenac was 40 μM, the final concentration of the CYP2D6 substrate bufuralol was 40 μM, the final concentration of the CYP3A4 substrate midazolam was 320 μM, and the final concentration of the CYP3A4 substrate testosterone was 20 μM.

[0394] Prepare a 0.2 mg / mL human liver microsomal solution in potassium phosphate buffer on ice. Prepare the test compound and control inhibitor at 2x the final concentration in the human liver microsomal solution on ice. Add 30 μL of the test compound and control inhibitor solution to each well, along with 15 μL of substrate, in duplicate. Incubate the 96-well assay plate and NADPH solution at 37°C for 5 minutes. Add 15 μL of preheated 8 mM NADPH solution to the assay plate to initiate the reaction. Preincubate the CYP2C9 and CYP2D6 assay plates at 37°C for 10 minutes, and the CYP3A4 assay plate at 37°C for 5 minutes. Terminate the reaction by adding 120 μL of acetonitrile. After quenching, shake the plate on a shaker (IKA, MTS2 / 4) for 10 minutes (600 rpm / min) and then centrifuge for 15 minutes. After centrifugation, the supernatant was collected and purified water was added in a 1:1 ratio. LC-MS / MS was then performed to obtain the ratio of the compound peak area to the internal standard peak area. The peak area ratio of the compound was compared with the peak area ratio of the control inhibitor to calculate the inhibition rate.

[0395] Table 4 Results of CYP450 enzyme inhibition test of test compounds

[0396]

[0397] The experimental results show that compared with the control compound, the compound of the present invention exhibits weaker CYP450 enzyme inhibitory activity and better drugability.

Claims

1. A compound, which is a compound represented by formula (I), or a pharmaceutically acceptable salt of a compound represented by formula (I): in: X is selected from -N=, -C(R a )=;the R a selected from halogen; R 1 independently selected from hydrogen, unsubstituted or optionally substituted with one or more R b Substituted by the following groups: C1-C6 alkyl, C3-C6 cycloalkyl; the R b Selected from halogen, C1-C3 alkyl; R 2 Selected from one or more -C(O)-NH-R d substituted 6-membered nitrogen-containing heterocyclic group, unsubstituted or optionally substituted with one or more R c The following groups substituted: 4-5 membered nitrogen-containing heterocyclic group, 7-10 membered nitrogen-containing spirocyclic group; wherein the 4-5 membered nitrogen-containing heterocyclic group, 7-10 membered nitrogen-containing spirocyclic group, substituted by one or more -C(O)-NH-R d The substituted 6-membered nitrogen-containing heterocyclic group is connected to the aryl or heteroaryl group where X is located via N; The R c is selected from hydrogen, halogen, hydroxy, C1-C3 alkyl, oxo; R d Selected from hydrogen, C1-C3 alkyl; R 3 Selected from hydrogen, C1-C6 alkyl.

2. The compound according to claim 1, wherein: X is selected from -N=, -C(F)=; R 1 Independently selected from isopropyl, cyclopropyl; R 2 unsubstituted or optionally substituted with one or more R c The following groups substituted: 4-membered N-containing heterocyclic group, morpholinyl group, 7-10-membered nitrogen-containing spirocyclic group; the R c is selected from hydrogen, halogen, hydroxy, methyl, oxo; or, R 2 is selected from piperidinyl substituted with one or more -C(O)-NH2 and -C(O)-NH-CH3; R 3 Selected from isopropyl.

3. The compound according to claim 1, wherein: R 2 Selected from R 3 Selected from 4. A compound represented by the following formula, or a pharmaceutically acceptable salt thereof:

5. A pharmaceutical composition, characterized in that The method comprises the compound according to any one of claims 1 to 4.

6. Use of the compound according to any one of claims 1 to 4, or the pharmaceutical composition according to claim 5, in the preparation of a medicament for treating ASK1-related diseases.

7. The use according to claim 6, wherein the ASK1-related disease is selected from cardiovascular and cerebrovascular diseases, chronic kidney diseases, lung diseases, chronic liver diseases, multiple sclerosis, metabolic diseases, stones, neurodegenerative diseases and cancer.

8. The use according to claim 6, wherein the ASK1-related disease is selected from hyperproliferative diseases.

9. The use according to claim 6, wherein the ASK1-related disease is selected from myocardial infarction, stroke, thrombosis, diabetic nephropathy, end-stage renal disease, renal fibrosis, cholesterol stones, cholelithiasis, pulmonary hypertension, pulmonary fibrosis, chronic obstructive pulmonary disease, acute lung injury, fatty liver hepatitis, liver fibrosis, bile acid disorders, primary sclerosing cholangitis, diabetes, Alzheimer's disease, Parkinson's disease, gastric cancer, liver cancer, polyposis, colon cancer, breast cancer, pancreatic cancer, and esophageal cancer.

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