Benzopyrazole compound as well as preparation method and application thereof

Abstract

The invention relates to a benzopyrazole compound as well as a preparation method and application thereof. The benzopyrazole compound is a compound as shown in a formula I, an optical isomer or a pharmaceutically available salt, wherein R1 is selected from hydrogen, C1-C8 linear or branched alkyl, aryl, substituted aryl, C3-C8 aliphatic ring group, substituted C3-C8 aliphatic ring group, C3-C8 alicyclic ring group, substituted C3-C8 alicyclic ring group and-(CH2) nNR4R5, and R2 is selected from hydrogen, C1-C8 linear or branched alkyl, aryl, substituted aryl, C3-C8 alicyclic ring group, substituted C3-C8 alicyclic ring group, substituted C3-C8 alicyclic ring group, substituted C3-C8 alicyclic ring group and-(CH2) nNR4R5; substituent groups on the substituted aryl group, the substituted C3-C8 alicyclic group and the substituted C3-C8 alicyclic ring group are independently selected from C1-C8 linear chain or branched chain alkyl groups. The benzopyrazole compound containing thiazolidine-2, 4-diketone is synthesized for the first time, and the benzopyrazole compound is applied to an AMPK activator and a medicine for treating acute kidney injury and shows a good effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and relates to a benzopyrazole compound containing thiazolidine-2,4-dione and a preparation method and application thereof. BACKGROUND

[0002] Acute kidney injury (AKI) is a common clinical syndrome characterized by abnormal elevation of serum creatinine and blood urea nitrogen and decreased renal function, affecting about 13.3 million patients per year. After kidney injury, tubular cells produce FGF2 through autophagy, leading to fibroblast activation and kidney fibrosis. Further leading to abnormal proliferation of renal cells, thereby leading to fibrosis (Cell Death Dis. 2021 Oct 9; 12(10): 925.), ultimately leading to patient death. There is currently no effective drug treatment method.

[0003] Adenosine monophosphate activated protein kinase (AMPK) is a highly conserved serine / threonine protein kinase that can act as a metabolic fuel meter to sense the energy status of cells and the entire organism. Activation of AMPK can play a role in inhibiting fibrosis by regulating various downstream signaling pathways such as energy metabolism, oxidative stress, mitochondrial function, autophagy, inflammatory response, and endoplasmic reticulum stress.

[0004] The prior art CN118359575A discloses a heteroterpene compound with the structures of formula (I) and formula (II) separated from a plant of the genus Rhododendron:

[0005]

[0006] The compound has a significant protective effect on lipopolysaccharide and ischemia-reperfusion-induced acute kidney injury in mice. However, its effect still needs to be improved.

[0007] The prior art CN114028378A discloses the use of a migratory abietane diterpenoid compound in the preparation of a drug for kidney protection:

[0008]

[0009] The compound as shown in formula I has a better effect on kidney ischemia-induced kidney injury than cyclosporine, tanshinone IIa, dexamethasone, and amifostine, and has a significant protective effect on kidney injury. SUMMARY

[0010] The purpose of the present application is to provide a benzopyrazole compound and a preparation method and its application in the treatment of acute kidney injury.

[0011] To achieve the above object, the technical scheme adopted by the present application is:

[0012] A benzopyrazole compound is a compound shown in formula I, an optical isomer or a pharmaceutically acceptable salt:

[0013]

[0014] R1 is selected from hydrogen, C1-C8 straight chain or branched alkyl, aryl, substituted aryl, C3-C8 aliphatic ring group, substituted C3-C8 aliphatic ring group, C3-C8 aliphatic heterocyclic group, substituted C3-C8 aliphatic heterocyclic group, -(CH2)nNR4R5;

[0015] The substituents on the substituted aryl, substituted C3-C8 aliphatic ring group, and substituted C3-C8 aliphatic heterocyclic group are independently selected from C1-C8 straight chain or branched alkyl.

[0016] n is an integer of 0-4, and R4 and R5 are independently selected from C1-C5 straight chain or branched alkyl.

[0017] R2 is X is nitrogen or CH;

[0018] R6 is selected from hydrogen, C3-C8 nitrogen-containing aliphatic heterocyclic group,

[0019] R7 is selected from hydrogen and C1-C3 alkyl.

[0020] R8 is selected from hydrogen, C1-C8 straight chain or branched alkylcarbonyl, and C1-C8 straight chain or branched alkyl ester.

[0021] R3 is selected from hydrogen and halogen.

[0022] According to the embodiments of the present application, the present application can be further optimized, and the following is the technical scheme formed after optimization:

[0023] In one preferred embodiment, the aryl group is a phenyl group.

[0024] In one preferred embodiment, R1 is selected from C1-C5 straight chain or branched alkyl, aryl, substituted aryl, C3-C6 aliphatic ring group, substituted C3-C6 aliphatic ring group, C3-C6 aliphatic heterocyclic group, and substituted C3-C6 aliphatic heterocyclic group.

[0025] The substituents on the substituted aryl, substituted C3-C6 aliphatic ring group, and substituted C3-C6 aliphatic heterocyclic group are independently selected from C1-C3 straight chain or branched alkyl.

[0026] In one preferred embodiment, R1 is selected from substituted morpholinyl; the substituents are selected from C1-C3 linear or branched alkyl.

[0027] In one preferred embodiment, R1 is selected from

[0028] In one preferred embodiment, R4, R5 are independently selected from C1-C3 linear or branched alkyl.

[0029] In one preferred embodiment, R2 is selected from:

[0030] In one preferred embodiment, R8 is selected from C1-C4 linear or branched alkylcarbonyl, C1-C4 linear or branched alkyl ester.

[0031] In one preferred embodiment, R8 is selected from tert-butyloxycarbonyl, tert-butyloxycarbonyl.

[0032] In one preferred embodiment, the benzopyrazole compound is a compound, an optical isomer or a pharmaceutically acceptable salt as shown in the following formula:

[0033]

[0034]

[0035] Based on the same inventive concept, the present application also claims a preparation method of the benzopyrazole compound, comprising the following steps:

[0036] 5-bromo-6-halo-1H-indole-3-carboxaldehyde is subjected to ring-opening reaction and ring-closing reaction in sequence to obtain 5-bromo-6-halo-1H-indazole-3-carboxaldehyde; then subjected to Suzuki reaction with borate to introduce a heterocyclic fragment of pyridine bis-piperazine or benzene bis-piperazine at the 5-position; then subjected to Grignard reaction to introduce a thiazolidine dione functional group at the 3-position to obtain the benzopyrazole compound.

[0037] Based on the same inventive concept, the present application also claims a synthetic route of the benzopyrazole compound, as shown below:

[0038]

[0039] Based on the same inventive concept, the present application also claims an application of the benzopyrazole compound in preparing a drug for treating or preventing acute kidney injury.

[0040] In one preferred embodiment, the acute kidney injury is ischemic acute kidney injury, drug toxicity induced acute kidney injury or bacteria infection induced acute kidney injury.

[0041] In one preferred embodiment, the drug toxicity induced acute kidney injury is cisplatin induced acute kidney injury.

[0042] In one preferred embodiment, the bacteria infection induced acute kidney injury is endotoxin lipopolysaccharide of bacteria induced acute kidney injury.

[0043] Based on the same inventive concept, the application also claims the use of the benzopyrazole compound in the preparation of an AMPK activator.

[0044] Based on the same inventive concept, the application also claims a pharmaceutical composition for treating or preventing acute kidney injury, which comprises the benzopyrazole compound.

[0045] Compared with the prior art, the application has the following beneficial effects:

[0046] The application first synthesizes a benzopyrazole compound containing thiazolidine-2,4-dione, and uses it as an activator of AMPK and a drug for treating acute kidney injury, which shows good effects and is expected to become a new type of activator of AMPK and a drug for treating acute kidney injury. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 is the effect of 24f and 991 of different concentrations on the activity of AMPK alpha1 beta1 gamma1 enzyme;

[0048] Figure 2 is the effect of 24f and 991 on AMPK and its downstream proteins; wherein, Figure 2 A is the Western blot detection result graph of the expression of AMPK and its downstream proteins after treatment with different concentrations of compound 24f; Figure 2 B is a column chart of quantitative analysis of Western blot data of p-AMPK protein; Figure 2 C is a column chart of quantitative analysis of Western blot data of p-ACC protein;

[0049] Figure 3 is the HE staining graph (100 μm) of the kidney tissues of each group of mice after treatment with compound 24f. DETAILED DESCRIPTION

[0050] The application is not limited to the following detailed description, and those skilled in the art can use other various embodiments to implement the application according to the disclosure of the application, or any simple changes or modifications made by using the design structure and ideas of the application, all fall within the protection scope of the application. It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other without conflict.

[0051] The term "unsubstituted" as used herein means no substituents or only hydrogen.

[0052] Some of the terms used herein are defined below:

[0053] "Halogen" means fluorine, chlorine, bromine and iodine.

[0054] "=0" means oxo.

[0055] "-CO-NH-" is "-amide-".

[0056] "Carbonyl" means

[0057] "Alkyl" when used as a group or part of a group means a straight chain or branched chain aliphatic group. Alkyl groups are preferably C1-C14alkyl groups; more preferably C1-C10alkyl groups; most preferably C1-C6, unless otherwise indicated. Examples of straight chain or branched chain C1-C8alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, t-butyl, hexyl, pentyl, heptyl, octyl and the like. Examples of straight chain or branched chain C1-C6alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, isobutyl, t-butyl, hexyl, pentyl and the like. Examples of C1-C3alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 2-propyl.

[0058] "Alicyclic" means a saturated or partially saturated carbocyclic ring which is monocyclic, fused ring or spiro. Rings of 3-9 carbon atoms are preferred. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

[0059] "Heteroalicyclic" means a group in which one or more (preferably 1, 2 or 3) carbon atoms of an "alicyclic" group as defined above are replaced by oxygen, sulfur or nitrogen atoms (preferably oxygen, sulfur or nitrogen). The heteroalicyclic group includes the alkyl portion as defined herein. Preferably, 1-3 heteroatoms are present. Preferably, the ring is a 3-14 membered ring (i.e., 3-14 membered heteroalicyclic) and more preferably, the ring is a 4-7 membered ring (i.e., 4-7 membered heteroalicyclic). Heteroalicyclic groups include, but are not limited to, pyrrolidinyl, dihydropyrrolyl, tetrahydropyrrolyl, dihydropyrazolyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, oxiranyl, aziridinyl or 2-pyrazolinyl, as well as lactams, lactones, cyclic imides and cyclic anhydrides and the like. The heteroalicyclic group can be substituted with one or more substituents.

[0060] "Bicyclic" means that at least two atoms of the ring system of an "alicyclic" or "heteroalicyclic" group as defined above are shared by two rings.

[0061] Bicyclic (tricyclic) groups refer to ring systems defined above as "cycloalkyl" or "heterocycloalkyl" wherein at least one atom is a bridge atom.

[0062] Spiro groups refer to ring systems defined above as "cycloalkyl" or "heterocycloalkyl" wherein at least one atom is a common atom of the bicyclic ring.

[0063] "Heterocycloalkylalkyl" refers to groups of the formula (heterocycloalkyl-alkyl)- wherein the heterocycloalkyl and alkyl moieties are as defined herein. Heterocycloalkylalkyl groups include, but are not limited to, (2-tetrahydrofuranyl)methyl, (2-tetrahydrothiofuranyl)methyl, and the like.

[0064] "Arylamino" includes both mono- and di- arylamino groups unless otherwise indicated. Mono- arylamino groups are of the formula (aryl-)NH-; di- arylamino groups are of the formula (aryl)2N-. Aryl groups are as defined herein.

[0065] "Heteroalkylamino" refers to both mono- and di- heteroalkylamino groups unless otherwise indicated. Mono- heteroalkylamino groups are of the formula (heteroalkyl-)NH-; di- heteroalkylamino groups are of the formula (heteroalkyl)2N-. The "heteroalkyl" portion is as defined herein.

[0066] "Aminoalkyl" refers to groups of the formula (amino-alkyl)- wherein the "alkyl" portion is as defined herein. Preferred aminoalkyl groups are amino C1-C6 alkyl groups. It is noted that "amino-C1-C6 alkyl" refers to C1-C6 alkyl groups substituted with an "amino" group. Examples, include but are not limited to, aminoethyl, 1-aminopropyl, 2-aminopropyl, and the like.

[0067] "Arylamino" includes both mono- and di- arylamino groups unless otherwise indicated. Mono- arylamino groups are of the formula (aryl-)NH-; di- arylamino groups are of the formula (aryl)2N-. Aryl groups are as defined herein.

[0068] "Arylamino" includes both mono- and di- arylamino groups unless otherwise indicated. Mono- arylamino groups are of the formula (aryl-)NH-; di- arylamino groups are of the formula (aryl)2N-. Aryl groups are as defined herein.

[0069] "Amide group" includes (alkyl-CONH)- groups and (aryl-CONH)- groups, unless otherwise specified. Alkyl or aryl groups are defined herein. Examples of amide groups include, but are not limited to, acetamido, propionamido, butyramido, isobutyramido, benzamide, etc.

[0070] When "alkenyl" is used as a group or part of a group, it refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, which can be straight-chain or branched. C2-C14 alkenyl groups are preferred, C2-C12 are even better, and C2-C6 alkenyl groups are the most preferred. This group can contain multiple double bonds in its main chain, and their conformations can each be E or Z. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, etc.

[0071] When "alkynyl" is used as a group or part of a group, it refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond, which can be straight-chain or branched. Preferably, it is a C2-C14 alkynyl, more preferably a C2-C12 alkynyl, and most preferably a C2-C6 alkynyl. Examples of the alkynyl group include, but are not limited to: ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl, but-1-yn-1-yl, but-3-yn-1-yl, 1-methylprop-2-yn-1-yl, pent-1-yn-1-yl, pent-4-yn-1-yl, hex-1-yn-1-yl, hex-5-yn-1-yl, etc.

[0072] "Alkoxy" refers to a (alkyl-O)- group. The "alkyl" portion is defined herein. The alkoxy group is preferably C1-C8 alkoxy, more preferably C1-C6 alkoxy. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, 1-methylethoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, neopentoxy, 1-methylbutoxy, 1-ethylpropoxy, n-hexoxy, isohexoxy, 3-methylpentoxy, 2-methylpentoxy, 1-methylpentoxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,3-dimethylbutoxy, 1-ethylbutoxy, 2-ethylbutoxy, etc. Additionally, "alkoxycarbonyl" refers to a group in which the "alkoxy" group defined above is bonded to a carbonyl group, such as methoxycarbonyl and ethoxycarbonyl.

[0073] "Alkenyloxy group" refers to the (alkenyl-O)- group. The alkenyl group is defined in the relevant section of this document. C1-C6 alkenyloxy groups are preferred.

[0074] "Alkyne group" refers to the (alkynyl-O)- group. The alkynyl group is defined in the relevant section of this document. C1-C6 alkynyl groups are preferred.

[0075] "Carbonyl" or "carbonl" means a (-C(O)-) group. Preferred carbonyl groups are C1-C6carbonyl groups. Examples include, but are not limited to, acyl, formyl and the like.

[0076] "Aryloxy" means an (aryl-O-) group. Aryl is defined herein. Preferred aryl groups are C6-C12aryl groups. Examples include, but are not limited to, phenyloxy, naphthyloxy and the like.

[0077] "Aryloxy" means an (aryl-O-) group. Aryl is defined herein. Preferred aryl groups are C6-C12aryl groups. Examples include, but are not limited to, phenyloxy, naphthyloxy and the like.

[0078] "Aryloxy" means an (aryl-O-) group. Aryl is defined herein. Preferred aryl groups are C6-C12aryl groups. Examples include, but are not limited to, phenyloxy, naphthyloxy and the like.

[0079] "Aryloxy" means an (aryl-O-) group. Aryl is defined herein. Preferred aryl groups are C6-C12aryl groups. Examples include, but are not limited to, phenyloxy, naphthyloxy and the like.

[0080] "Aryloxy" means an (aryl-O-) group. Aryl is defined herein. Preferred aryl groups are C6-C12aryl groups. Examples include, but are not limited to, phenyloxy, naphthyloxy and the like.

[0081] "Aryloxy" means an (aryl-O-) group. Aryl is defined herein. Preferred aryl groups are C6-C12aryl groups. Examples include, but are not limited to, phenyloxy, naphthyloxy and the like.

[0082] "Aryloxy" means an (aryl-O-) group. Aryl is defined herein. Preferred aryl groups are C6-C12aryl groups. Examples include, but are not limited to, phenyloxy, naphthyloxy and the like.

[0083] "Aryloxy" means an (aryl-O-) group. Aryl is defined herein. Preferred aryl groups are C6-C12aryl groups. Examples include, but are not limited to, phenyloxy, naphthyloxy and the like.

[0084] "Arylalkyl" means a radical of the formula (aryl-alkyl)- where aryl and alkyl are as defined herein. Exemplary arylalkyl groups include, but are not limited to, benzyl, phenethyl, 1-napthylmethyl and the like.

[0085] "Cycloalkenyl" means a non-aromatic, mono- or polycyclic ring system. It contains at least one carbon-carbon double bond and preferably has 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include, but are not limited to, cyclopentenyl, cyclohexenyl or cycloheptenyl. The cycloalkenyl group can be substituted by one or more substituents.

[0086] "Heteroaryl" means a monocyclic or fused polycyclic aromatic heterocyclic radical, preferably it is an aromatic radical having one or more (preferably 3 to 14, more preferably 5 to 10, especially preferably 5 or 6) carbon atoms, and one or more (preferably 1, 2, 3 or 4) oxygen, nitrogen, phosphorus or sulfur ring atoms (preferably O, S or N) as ring members, preferably the aromatic radical is a 4-15 membered heteroaryl, more preferably a 5-7 membered heteroaryl. Examples of said heteroaryl groups can be listed as, for example, furanyl, thienyl, pyrrolyl, pyrazolyl, triazolyl, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl, pyridyl, imidazolyl, 3-phenylpyrrolyl, thiazolyl-oxazolyl, tetrazolyl, isoxazolyl, indazolyl, pyridazinyl, quinolinyl, purinyl, carbazolyl, acridinyl, pyrimidinyl, 2,3'-bifuranyl and isoquinolinyl.

[0087] "Heteroarylalkyl" means a radical of the formula (heteroaryl-alkyl)- where heteroaryl and alkyl are as defined herein. Exemplary heteroarylalkyl groups include, but are not limited to, 2-furane methyl, 3-furane methyl, 2-pyridine methyl and the like.

[0088] "Alkyl ether" means a radical of the formula (alkyl)-O- where "alkyl" is as defined herein.

[0089] "Cycloalkyl ether" means a radical of the formula (cycloalkyl)-O- where "cycloalkyl" is as defined herein.

[0090] "Heteroalkyl ether" means a radical of the formula (heteroalkyl)-O- where "heteroalkyl" is as defined herein.

[0091] "Aryl ether" means a radical of the formula (aryl)-O- where "aryl" is as defined herein.

[0092] "Arylalkyl ether" means a radical of the formula (aryl-alkyl)-O- where "aryl" and "alkyl" are as defined herein.

[0093] "Heteroaryl ether" means a radical of the formula (heteroaryl)-O- where "heteroaryl" is as defined herein.

[0094] "Heteroaralkyloxy" means a group of the formula: (heteroaryl-alkyl)-O-. The "heteroaryl" and "alkyl" portions of the group are as defined herein.

[0095] "Heterocycloalkyloxy" means a group of the formula: (heterocycloalkyl)-O-. The "heterocycloalkyl" portion of the group is as defined herein.

[0096] "Heterocycloalkylamino" means both mono- and di-heterocycloalkylamino, unless otherwise indicated. Mono-heterocycloalkylamino means a group of the formula: (heterocycloalkyl)-NH-; di-heterocycloalkylamino means a group of the formula: (heterocycloalkyl)2-N-. The "heterocycloalkyl" portion of the group is as defined herein.

[0097] "Arylamino" means both mono- and di-arylamino, unless otherwise indicated. Mono-arylamino means a group of the formula: (aryl)-NH-; di-arylamino means a group of the formula: (aryl)2-N-. The "aryl" portion of the group is as defined herein.

[0098] "Arylamino" means both mono- and di-arylamino, unless otherwise indicated. Mono-arylamino means a group of the formula: (aryl)-NH-; di-arylamino means a group of the formula: (aryl)2-N-. The "aryl" portion of the group is as defined herein.

[0099] "Arylamino" means both mono- and di-arylamino, unless otherwise indicated. Mono-arylamino means a group of the formula: (aryl)-NH-; di-arylamino means a group of the formula: (aryl)2-N-. The "aryl" portion of the group is as defined herein.

[0100] "Arylamino" means both mono- and di-arylamino, unless otherwise indicated. Mono-arylamino means a group of the formula: (aryl)-NH-; di-arylamino means a group of the formula: (aryl)2-N-. The "aryl" portion of the group is as defined herein.

[0101] "Arylamino" means both mono- and di-arylamino, unless otherwise indicated. Mono-arylamino means a group of the formula: (aryl)-NH-; di-arylamino means a group of the formula: (aryl)2-N-. The "aryl" portion of the group is as defined herein.

[0102] Unless otherwise stated, the subunits of this invention refer to divalent groups, that is, groups in which one hydrogen atom in a monovalent group is replaced by a chemical valence. For example, "heteroalkylene" refers to a heteroalkyl group in which one hydrogen atom is replaced by a chemical valence; "heterocyclicene" refers to a heterocyclic group in which one hydrogen atom is replaced by a chemical valence; "arylene" refers to an aryl group in which one hydrogen atom is replaced by a chemical valence; "alkylene" refers to an alkyl group in which one hydrogen atom is replaced by a chemical valence; "alkenylene" refers to an alkenyl group in which one hydrogen atom is replaced by a chemical valence; "cycloalkylene" refers to a cycloalkyl group in which one hydrogen atom is replaced by a chemical valence; "heteroarylene" refers to a heteroaryl group in which one hydrogen atom is replaced by a chemical valence; "heterocyclic alkylene" refers to a heterocyclic alkyl group in which one hydrogen atom is replaced by a chemical valence; "heterocyclic alkenylene" refers to a heterocyclic alkenyl group in which one hydrogen atom is replaced by a chemical valence; "alkoxyene" refers to an alkoxyene group in which one hydrogen atom is replaced by a chemical valence; "alkenyloxyene" refers to an alkenyloxyene group in which one hydrogen atom is replaced by a chemical valence; "alkynyloxyene" refers to an alynyloxyene group in which one hydrogen atom is replaced by a chemical valence, etc. The definitions of heterocyclic groups, aryl groups, alkyl groups, alkenyl groups, cycloalkyl groups, heteroaryl groups, heterocyclic alkyl groups, heterocyclic alkenyl groups, alkoxy groups, alkenyloxy groups, and alkynyloxy groups mentioned above are provided in the relevant definitions in this article.

[0103] This invention includes compounds represented by general formula (I) and their various possible isomers. These include: non-mirror image isomers, mirror image isomers, tautomers, and geometric isomers of "E" or "Z" configuration isomers. Any chemist with a basic understanding can isolate the above-mentioned optically pure or stereoisomerically pure compounds.

[0104] The term "pharmaceutically acceptable salt" refers to certain salts of the aforementioned compounds that retain their original biological activity and are suitable for pharmaceutical use. Pharmaceutically acceptable salts of compounds represented by general formula (I) can be formed in two forms: one is a salt formed with an acid; the other is a salt formed with a base or alkali metal. Acids that form pharmaceutically acceptable salts with compounds represented by general formula (I) include inorganic acids and organic acids. Suitable inorganic acids include hydrochloric acid, sulfuric acid, and phosphoric acid. Suitable organic acids can be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic acids, and sulfonic acids; examples include, but are not limited to, formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, glycine, arginine, citric acid, fumaric acid, alkyl sulfonic acids, and aromatic sulfonic acids. Alkali metals that form pharmaceutically acceptable salts with compounds represented by general formula (I) include lithium, sodium, potassium, magnesium, calcium, aluminum, zinc, etc.; bases that form pharmaceutically acceptable salts with compounds represented by general formula (I) include choline, diethanolamine, morpholine, etc.

[0105] The compounds of the present application can be used alone or in combination with other pharmaceutical or in combination with surgery or radiotherapy; or in combination with pharmaceutically acceptable carriers, diluents or excipients in the form of a dosage unit. The specific dosage unit will depend upon the route of administration.

[0106] The parenteral injectable pharmaceutical formulations of the present application include sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as powders for reconstitution into sterile injectable solutions at the time of use.

[0107] If desired, the compounds of the present application can be presented in a slow release or targeted delivery system such as polymeric matrices, liposomes and microspheres.

[0108] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound represented by the general formula (I) is mixed with at least one inert, pharmaceutically acceptable excipient or carrier. Such excipients or carriers include sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and salicylic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents such as paraffin; f) absoφtion accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols.

[0109] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings or shells.

[0110] The active compound can also be in micro-encapsulated form, if desired. One or more excipients can be present, as desired.

[0111] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and the like. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, stabilizers, and emulsifiers, such as ethyl alcohol, carbamic acid ethyl ester, ethyl acetate, benzoic acid alcohol, benzoic acid benzyl ester, propylene glycol, 1,3-butanediol, dimethylformamide, oils (particularly cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and the like.

[0112] In addition to inert diluents, the oral compositions can include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0113] Suspensions, in addition to active compounds, can contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

[0114] The compositions for rectal or vaginal administration are preferably suppositories which can be prepared from fatty compounds disclosed herein which are solid at room temperature. These compositions can be prepared by mixing the active compound with a suitable non-irritating excipient which is solid at room temperature and which is liquid under the use conditions. Such excipients include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

[0115] The pharmaceutical compositions of this application can be orally administered in any orally acceptable dosage form including capsules, tablets, aqueous suspensions or solutions. Such compositions contain conventional pharmaceutically-acceptable carriers and adjuvants, such as vehicles, diluents, or excipients. In addition, toothpaste, mouthwash, and chewing gum formulations can be used as vehicles for the compounds of the present application.

[0116] The synthesis routes of the compounds of the present application are as follows:

[0117] Conditions and reagents: (a) Bromide or methyl iodide, K2CO3, acetone or DMF, TBAI, 45 °C or 90 °C, 12 h, yield 33%-85%; (b) NaNO2, 2N HCl, DMF, -5 °C-rt, 12 h, yield

[0118] 55%-57%; (c) Corresponding boric acid ester, Pd(dppf)2Cl2, 1,4-Dioxane:H2O = 3:1,

[0119] N2 atmosphere, reflux, 12 h, yield 46%-52%; (d) Corresponding N-substituted

[0120] 1,3-thiazolane-2,4-dione, Piperdine, isopropanol, reflux, 5 h, yield 82%-91%; (e) CF3COOH, CH2Cl2, rt, 2 h, yield 88%-90%.

[0121]

[0122] The following detailed description:

[0123] The compound 19c-h was obtained by nucleophilic substitution reaction of 2,4-thiazolidinedione as starting material, potassium carbonate as acid binding agent, acetone or DMF as reaction solvent, TBAI as catalyst at 45 °C or 90 °C. The compound 21a-b was obtained by ring opening and ring closing reaction of compound 20a or 20b with 2N HCl as reaction solvent. The compound 22a-e was obtained by Suzuki reaction of compound 21a-b with corresponding boronate ester. The compound 23a-r was obtained by Grignard reaction of compound 22a-e with corresponding thiazolidine dione. The compound 24a-l, 24o-q was obtained by reaction of 23a-r with trifluoroacetic acid in dichloromethane.

[0124]

[0125] Conditions and reagents: 3,3-dimethylbutanoyl chloride, triethylamine, CH2Cl2, room temperature, yield 65%.

[0126] To prepare compound 24r, 24k was used as starting material, reacted with 3,3-dimethylbutanoyl chloride in dichloromethane with triethylamine as catalyst at room temperature to give compound 24r.

[0127] To synthesize boronate ester 26, 5-bromo-2-pyridinecarboxaldehyde and morpholine were used as starting material to give intermediate 25 by aldol condensation reaction, then reacted with bis(pinacolato)diboron with bis(tricyclohexylphosphine)palladium dichloride (29934-17-6) as catalyst to give boronate ester 26. Compound 20a was used as starting material, reacted with corresponding boronate ester to give compound 27a-g, then reacted with 3-methylthiazolidine-2,4-dione to introduce 3-methylthiazolidine-2,4-dione functional group at 3 position, to give target compounds 28a-g.

[0128] Conditions and reagents: (a) STAB, CH2Cl2, rt, 12 h, yield 90%; (b) 29934-17-6, Bis(pinacolato)diboron, 1,4-Dioxane, KOAc, 12 h; (c) Corresponding boric acid ester, Pd(dppf)2Cl2, 1, 4-Dioxane: H2O = 3:1, N2 atmosphere, reflux, 12 h, yield 46%-52%; (d) 3-methyl-1,3-thiazolane-2,4-dione, Piperdine, isopropanol, reflux, 5 h, yield 82%-91%.

[0129] Preparation of 3-propylthiazolidine-2,4-dione (compound 19c)

[0130] 2,4-Thiazolidinedione (2.0 g, 17.0 mmol) was added to 30 mL of acetone, then 1-bromopropane (2.5 g, 20.4 mmol) and potassium carbonate (2.8 g, 20.4 mmol) were added, the temperature of the oil bath pot was set to 90 °C, and the reaction was stirred for 10 h. After the reaction was completed, the temperature of the reaction bottle was allowed to drop to room temperature, the potassium carbonate was removed by filtration, the filtrate was collected, and the organic solvent was rotary evaporated to obtain a yellow oil. After being dissolved in 60 mL of ethyl acetate, 20 mL of saturated brine was added, washed 3 times, the organic phase was combined, anhydrous sodium sulfate was added and dried overnight, then the anhydrous sodium sulfate was removed by filtration, and the organic solvent was rotary evaporated to obtain a light yellow oil, which was weighed as 1.5 g, with a yield of 55.56%. The product was not purified and was directly used in the next step.

[0131] Preparation of 3-benzylthiazolidine-2,4-dione (compound 19d)

[0132] Using the above method for preparing compound 19c, compound 19d was prepared from 2,4-thiazolidinedione (2.0 g, 17.0 mmol) and benzyl bromide (2.9 g, 17.0 mmol) as raw materials, and was obtained as a white solid powder with a yield of 80.12%. 1 H NMR (400 MHz, Chloroform-d) δ 7.50 - 7.11 (m, 5H), 4.69 (d, J = 2.1 Hz, 2H), 3.93 - 3.66 (m, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 171.88, 171.35, 135.29, 128.89, 128.73, 128.28, 45.20, 33.81.

[0133] Preparation of 3-(2-(dimethylamino)ethyl)thiazolidine-2,4-dione (Compound 19g)

[0134] To a solution of 2,4-thiazolidinedione (2.0 g, 17.0 mmol) in 30 mL of acetone, 1-bromopropane (2.5 g, 20.4 mmol), potassium carbonate (2.8 g, 20.4 mmol) and TBAI (1.9 g, 5.1 mmol) were added. The oil bath was set to 90 °C and the reaction was stirred for 10 h. After the reaction was completed, the reaction flask was allowed to cool to room temperature. The potassium carbonate was removed by filtration and the filtrate was collected. The yellow oil was obtained after rotary evaporation. The oil was dissolved in 60 mL of ethyl acetate and washed with 20 mL of saturated brine three times. The organic phase was collected and dried over anhydrous sodium sulfate overnight. The anhydrous sodium sulfate was removed by filtration and the organic solvent was rotary evaporated to give a light yellow oil. The oil was purified by column chromatography (petroleum ether: ethyl acetate = 5: 1) to give 1.1 g of yellow oil with a yield of 34.98%. 1 H NMR (400 MHz, Chloroform-d) δ 4.51 (p, J = 8.5 Hz, 1H), 3.83 (s, 2H), 2.09 - 1.59 (m, 6H), 1.63 - 1.30 (m, 2H). 13 C NMR (126 MHz, Chloroform-d) δ 171.84, 171.64, 54.77, 33.33, 28.30, 25.20.

[0135] Preparation of 3-(2-(dimethylamino)ethyl)thiazolidine-2,4-dione (Compound 19g)

[0136] Compound 19f was prepared using the method described above for the preparation of compound 19c, using 2,4-thiazolidinedione (2.0 g, 17.0 mmol) and bromomethylcyclohexane (2.9 g, 17.0 mmol) as starting materials. Compound 19f was obtained as a white solid powder with a yield of 30.12%. 1 H NMR (400 MHz, Chloroform-d) δ 3.83 (s, 2H), 3.28 (d, J = 7.4 Hz, 2H), 1.66 - 1.35 (m, 6H), 1.03 (h, J = 11.6 Hz, 3H), 0.93 - 0.70 (m, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 172.02, 171.76, 48.00, 36.06, 33.65, 30.53, 26.12, 25.55.

[0137] Preparation of 3-(2-(dimethylamino)ethyl)thiazolidine-2,4-dione (Compound 19g)

[0138] Using the method described above for the preparation of compound 19c, starting from 2,4-thiazolidinedione (2.0 g, 17.0 mmol) and 1-bromo-3-ethylpentane (3.0 g, 17.0 mmol), compound 19g was prepared as a light yellow solid powder in 45.17% yield. 1 H NMR (500 MHz, Chloroform-d) δ 3.91 (d, J = 2.2 Hz, 2H), 3.66 (td, J = 7.1, 2.2 Hz, 2H), 2.58 (td, J = 7.0, 2.1 Hz, 2H), 2.49 (td, J = 7.1, 2.3 Hz, 4H), 0.95 (td, J = 7.2, 2.2 Hz, 6H). 13 C NMR (126 MHz, Chloroform-d) δ 171.74, 171.47, 49.22, 47.10, 39.89, 33.66, 12.00.

[0139] Preparation of 3-(2-morpholinoethyl)thiazolidine-2,4-dione (compound 19h)

[0140] Using the method described above for the preparation of compound 19c, starting from 2,4-thiazolidinedione (2.0 g, 17.0 mmol) and 2-(4-morpholinyl)ethyl bromide (3.3 g, 17.0 mmol), compound 19h was prepared without purification for the next step.

[0141] Preparation of 5-bromo-6-chloro-1H-indole (compound 21a)

[0142] Sodium nitrite (14.4 g, 208.7 mmol) was added into a 500 mL three-necked flask, followed by the addition of 104.4 mL of water and 78.3 mL of DMF. The flask was placed in a cold bath, and the temperature was set to -10 °C. 33 mL of concentrated hydrochloric acid was diluted 6 times for use. The whole reaction system was vacuumed, and when the temperature was reduced to -5 °C, 35.2 mL of 2N hydrochloric acid was slowly added into the reaction system under argon protection, and the temperature was controlled at -5 °C.

[0143] After the drop was completed, 5-bromo-6-chloro-1H-indole (6.0 g, 26.0 mmol) was dissolved in 78.3 mL of DMF solution for use. The temperature of the whole reaction system was controlled at -5 °C, and the DMF solution of 5-bromo-6-chloro-1H-indole was slowly added into the reaction system under argon protection. The reaction solution changed from colorless transparent solution to brown red transparent solution, and after the drop was completed, the reaction was stirred at room temperature overnight.

[0144] The next day, a large amount of red-brown solid was produced in the reaction solution, and was suction filtered to obtain about 4.1 g of a crude product. The filtrate was collected, and 100 mL of ethyl acetate was added to the filtrate three times, and the organic phases were combined. The organic phase was dried over anhydrous Na2SO4and was left to stand for 4 h. The solid obtained by suction filtration was dissolved in an appropriate amount of ethyl acetate, and was combined with the filtrate, and was rotary evaporated to obtain a red-brown oily substance, which was further separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain 5.1 g of a red-brown solid, with a yield of 76.12%. 1 H NMR (500 MHz, DMSO-d6) δ 14.36 (s, 1H), 10.17 (s, 1H), 8.43 (s, 1H), 8.04 (s, 1H). 13 C NMR (126 MHz, DMSO-d6) δ 187.44, 143.12, 140.90, 132.39, 125.44, 120.86, 117.23, 113.41. HRMS (ESI) m / z calcd for [C8H4BrClN2O + H] + : 258.9196; found: 258.9253 [M+H] + .

[0145] Example 1

[0146] Preparation of tert-butyl-4-(5-(6-chloro-3-formyl-1H-indazol-5-yl)pyridin-2-yl)piperazine-1-carboxylate (Compound 22a)

[0147] Compound 21a (0.2 g, 0.8 mmol), 6-(4-Boc-1-piperazinyl)pyridine-3-boronic acid pinacol ester (0.7 g, 2.0 mmol), [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride (0.07 g, 0.09 mmol), cesium carbonate (1.6 g, 4.9 mmol) were sequentially added into a 100 mL round bottom flask, 30 mL of 1,4-dioxane and 10 mL of water were added into the round bottom flask, the whole device was vacuumed, and was heated to reflux under nitrogen protection overnight. Then, the reaction bottle was left to cool naturally at room temperature, and the 1,4-dioxane was rotary evaporated, 40 mL of water and 20 mL of ethyl acetate were added into the reaction solution, and was extracted three times, and the ethyl acetate phases were combined. The organic phase was washed with 20 mL of saturated brine three times, and the organic phases were combined. The organic phase was dried over anhydrous Na2SO4for 24 h. Filtration by suction and rotary evaporation of the organic solvent were performed to obtain a red-black oily substance, which was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 0.2 g of a yellow solid, with a yield of 56.66%. 1H NMR (400 MHz, DMSO-d6) δ 14.31 (s, 1H), 10.18 (s, 1H), 8.19 (d, J = 2.5 Hz, 1H), 8.02 (s, 1H), 7.93 (s, 1H), 7.66 (dd, J = 8.8, 2.5 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 3.56 (dd, J = 6.9, 3.7 Hz, 4H), 3.45 (dd, J = 6.7, 3.8 Hz, 4H), 1.43 (s, 10H). 13 C NMR (101 MHz, DMSO-d6) δ 187.65, 158.34, 154.41, 148.11, 143.89, 141.03, 139.27, 133.65, 132.29, 124.40, 122.74, 120.09, 112.53, 106.66, 79.49, 44.73, 28.52. HRMS (ESI) m / z calcd for [C 22 H 24 ClN5O3+H] + : 442.1640; found: 442.1656 [M+H] + .

[0148] Example 2

[0149] Preparation of tert-butyl-4-(4-(6-chloro-3-formyl-lH-indazol-5-yl)phenyl)piperazine- 1-carboxylate (Compound 22b)

[0150] Using compound 21a (0.2 g, 0.8 mmol) and 4-(4-Boc-l-piperazinyl)benzeneboronic acid pinacol ester (0.3 g, 0.8 mmol) as starting materials, compound 22b was prepared as off-white solid powder in 51.25% yield using the method described in Example 1 above. 1 H NMR (500 MHz, Chloroform-d) δ 11.70 (s, 1H), 10.29 (d, J = 2.0 Hz, 1H), 8.29 (d, J = 1.9 Hz, 1H), 7.71 (d, J = 1.9 Hz, 1H), 7.41 (dd, J = 8.6, 2.1 Hz, 2H), 7.01 (d, J = 8.2 Hz, 2H), 3.67 (t, J = 5.1 Hz, 4H), 3.26 (t, J = 5.2 Hz, 4H), 1.55 (s, 9H). 13C NMR (126 MHz, Chloroform-d) δ 187.25, 155.01, 150.48, 144.69, 140.61, 137.12, 133.64, 130.78, 123.58, 120.14, 115.70, 111.02, 49.00, 28.51. HRMS (ESI) m / z calcd for [C 23 H 25 ClN4O3+H] + :441.1688; found: 441.1699 [M+H] + .

[0151] Example 3

[0152] Preparation of tert-butyl-4-(4-(6-chloro-3-formyl-lH-indazol-5-yl)phenyl)piperazine- 1-carboxylate (Compound 22c)

[0153] Compound 22c was prepared using the procedure described in Example 1 above, starting from compound 21b (0.18 g, 0.8 mmol) and 6-(4-Boc-l-piperazinyl)pyridine-3- boronic acid pinacol ester (0.3 g, 0.8 mmol) as a light yellow solid powder in 61.40% yield. 1 H NMR (500 MHz, DMSO-d6) δ 14.20 (s, 1H), 10.22 (s, 1H), 8.48 (d, J = 2.4 Hz, 1H), 8.25 (s, 1H), 7.90 (dt, J = 8.7, 2.0 Hz, 1H), 7.75 (s, 2H), 6.94 (d, J = 8.9 Hz, 1H), 3.55 (t, J = 5.1 Hz, 4H), 3.45 (t, J = 5.2 Hz, 4H), 1.43 (s, 10H). 13 C NMR (126 MHz, DMSO-d6) δ 187.77, 158.44, 154.42, 146.16, 144.08, 140.86, 136.67, 134.23, 126.79, 125.70, 121.72, 117.40, 112.23, 107.71, 79.47, 67.47, 44.92, 28.53, 25.58. HRMS (ESI) m / z calcd for [C 22 H 25 N5O3+H] + :408.2030; found: 408.2040 [M+H] + .

[0154] Example 4

[0155] Preparation of tert-butyl-4-(4-(3-formyl-lH-indazol-5-yl)phenyl)piperazine-l- carboxylate (Compound 22d)

[0156] Using the procedure described in Example 1 above, starting from compound 21b (0.18 g, 0.8 mmol) and 4-(4-Boc-l-piperazinyl)benzeneboronic acid pinacol ester (0.3 g, 0.8 mmol), compound 22d was prepared as an off-white solid powder in 41.40% yield. 1 H NMR (500 MHz, DMSO-d6) δ 14.20 (s, 1H), 10.22 (s, 1H), 8.26 (s, 1H), 7.75 (s, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.2 Hz, 2H), 3.48 (t, J = 5.2 Hz, 4H), 3.16 (t, J = 5.2 Hz, 4H), 1.43 (s, 10H). 13 C NMR (101 MHz, DMSO-d6) δ 187.90, 154.35, 150.66, 144.09, 140.79, 136.75, 131.21, 128.01, 127.08, 121.74, 117.36, 116.66, 112.08, 79.50, 48.52, 28.52. HRMS (ESI) m / z calcd for [C 23 H 26 N4O3+H] + : 407.2078; found: 407.2086 [M+H] + .

[0157] Example 5

[0158] Preparation of tert-butyl-4-(4-(6-chloro-3-formyl-lH-indazol-5-yl)phenyl)piperazine-l- carboxylate (Compound 22e)

[0159] Using the procedure described in Example 1 above, starting from compound 21a (0.2 g, 0.8 mmol) and (R)-tert-butyl 2-methyl-4-(5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)pyridin-2-yl)piperazine-l-carboxylate (0.3 g, 0.8 mmol), compound 22e was prepared as a white solid powder in 51.25% yield. 1H NMR (400 MHz, DMSO-d6) δ 14.32 (s, 1H), 10.19 (s, 1H), 8.17 (d, J = 2.4 Hz, 1H), 8.04 (s, 1H), 7.94 (s, 1H), 7.66 (dd, J = 8.8, 2.5 Hz, 1H), 6.91 (d, J = 8.9 Hz, 1H), 4.41 - 4.03 (m, 3H), 3.82 (dt, J = 13.3, 3.3 Hz, 1H), 3.24 - 3.08 (m, 2H), 2.92 (td, J = 12.1, 3.8 Hz, 1H), 1.43 (s, 9H), 1.13 (d, J = 6.6 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 187.69, 158.71, 154.35, 148.07, 143.90, 141.02, 139.24, 133.73, 132.34, 124.01, 122.72, 120.12, 112.53, 106.23, 79.38, 48.34, 44.64, 28.54, 16.28. HRMS (ESI) m / z calcd for [C 23 H 26 ClN5O3+H] + : 456.1797; found: 456.1809 [M+H] + .

[0160] Example 6

[0161] Preparation of tert-butyl (Z)-4-(5-(3-((2,4-dioxothiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)pyridin-2-yl)piperazine-l-carboxylate (Compound 23a)

[0162] 0.4 g of compound 22c (0.9 mmol), 2,4-thiazolidinedione (0.12 g, 1 mmol), 20 mL of isopropyl alcohol were added to a 50 mL round bottom flask respectively, 3 drops of piperidine were added dropwise, heated to reflux for 5 h, the reaction liquid precipitated yellow solid. Stop heating, wait for the reaction bottle temperature to room temperature, suction filtration, get 0.2 g of yellow solid, yield 43.95%. 1 H NMR (500 MHz, DMSO-d6) δ 13.96 (s, 1H), 12.41 (s, 1H), 8.58 (s, 1H), 8.45 (s, 1H), 8.27 (s, 1H), 7.99 (d, J = 8.7 Hz, 1H), 7.81 - 7.56 (m, 2H), 6.92 (d, J = 8.7 Hz, 1H), 3.54 (s, 5H), 3.45 (s, 5H), 1.43 (s, 9H). 13CNMR (126 MHz, DMSO-d6) δ 170.87, 167.82, 158.31, 154.42, 146.12, 140.41, 139.84, 136.50, 132.17, 126.17, 125.71, 124.36, 123.52, 120.78, 116.64, 111.70, 107.60, 79.48, 44.95, 28.53. HRMS (ESI) m / z calcd for [C 25 H 26 N6O4S+H] + : 507.1809; found: 507.1828 [M+H] + .

[0163] Example 7

[0164] Preparation of tert-butyl (Z)-4-(5-(3-((3-methyl-2,4-dioxothiazolidin-5- yl)methyl)-lH-indazol-5-yl)pyridin-2-yl)piperazine-l-carboxylate (Compound 23b)

[0165] Compound 23b was prepared using the procedure described in Example 6 above, starting from compound 22c (0.4 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol) as a yellow solid powder in 75.42% yield. 1 H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 8.58 (d, J = 2.6 Hz, 1H), 8.48 (s, 1H), 8.37 (s, 1H), 8.01 (dd, J = 8.9, 2.6 Hz, 1H), 7.82 - 7.70 (m, 1H), 7.65 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 8.9 Hz, 1H), 3.54 (dd, J = 6.9, 3.6 Hz, 4H), 3.45 (s, 4H), 3.10 (s, 3H), 1.43 (d, J = 4.3 Hz, 9H). 13 C NMR (101 MHz, DMSO-d6) δ 170.20, 166.28, 158.32, 154.43, 146.12, 140.39, 139.84, 136.52, 132.23, 126.20, 125.67, 124.35, 121.50, 121.46, 116.67, 111.73, 107.62, 79.51, 44.96, 28.53, 27.84. HRMS (ESI) m / z calcd for [C 26 H 28 N6O4S+H]+ 521.1966; found: 521.1985 [M+H] + .

[0166] Example 8

[0167] Preparation of tert-butyl (Z)-4-(4-(3-((2,4-dioxothiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)phenyl)piperazine- 1-carboxylate (Compound 23c)

[0168] Compound 23c was prepared in 79.42% yield using the procedure described in Example 6 above, starting from compound 22d (0.4 g, 0.9 mmol), 2,4-thiazolidinedione (0.12 g, 1 mmol) as a yellow solid powder. 1 H NMR (400 MHz, DMSO-d6) δ 8.34 (s, 1H), 8.07 (s, 1H), 7.79 - 7.53 (m, 4H), 7.04 (d, J = 8.3 Hz, 2H), 3.48 (t, J = 5.0 Hz, 4H), 3.15 (d, J = 10.4 Hz, 4H), 1.43 (s, 9H). 13 CNMR (101 MHz, DMSO-d6) δ 154.33, 150.42, 140.54, 140.34, 134.21, 131.55, 127.88, 126.35, 124.22, 116.58, 111.38, 79.46, 48.63, 44.16, 28.53, 22.69, 22.11. HRMS (ESI) m / z calcd for [C 26 H 27 N5O4S+H] + : 506.1857; found: 506.1867 [M+H] + .

[0169] Example 9

[0170] Preparation of tert-butyl (Z)-4-(4-(3-((3-methyl-2,4-dioxothiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)phenyl)piperazine- 1-carboxylate (Compound 23d)

[0171] Compound 23d was prepared in 79.52% yield using the procedure described in Example 6 above, starting from compound 22d (0.4 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol) as a yellow solid powder. 1H NMR (400 MHz, DMSO-d6) δ 13.97 (s, 1H), 8.44 (d, J = 26.2 Hz, 2H), 7.69 (td, J = 19.7, 8.7 Hz, 4H), 7.04 (d, J = 8.3 Hz, 2H), 3.54 - 3.43 (m, 4H), 3.16 (t, J = 5.2 Hz, 4H), 3.11 (s, 3H), 1.43 (s, 9H). 13 C NMR (126 MHz, DMSO-d6) δ 170.23, 166.29, 154.35, 150.51, 140.36, 139.87, 134.77, 131.31, 127.91, 126.59, 124.38, 121.61, 121.38, 116.71, 116.53, 111.53, 79.48, 48.61, 28.53, 27.84. HRMS (ESI) m / z calcd for [C 27 H 29 N5O4S+H] + : 520.2013; found: 520.2027 [M+H] + .

[0172] Example 10

[0173] Preparation of tert-butyl (Z)-4-(4-(6-chloro-3-((2,4-dioxothiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)phenyl)piperazine-l-carboxylate (Compound 23e)

[0174] Using compound 22a (0.4 g, 0.9 mmol), 2,4-thiazolidinedione (0.12 g, 1 mmol) as the raw material, the method described in Example 6 above was used to prepare compound 23e as a yellow solid powder in a yield of 85.42%. 1 H NMR (400 MHz, DMSO-d6) δ 13.97 (s, 1H), 8.44 (d, J = 26.2 Hz, 2H), 7.69 (td, J = 19.7, 8.7 Hz, 4H), 7.04 (d, J = 8.3 Hz, 2H), 3.54 - 3.43 (m, 4H), 3.16 (t, J = 5.2 Hz, 4H), 3.11 (s, 3H), 1.43 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 170.69, 167.62, 154.34, 150.52, 140.42, 139.95, 134.50, 131.91, 130.96, 130.18, 124.28, 122.70, 122.35, 115.59, 111.82, 79.49, 48.52, 36.28, 28.52. HRMS (ESI) m / z calcd for [C28H31CIN6O4S + H] + : 540.1467; found: 540.1479 [M+H] + .

[0175] Example 11

[0176] Preparation of tert-butyl (Z)-4-(4-(6-chloro-3-((3-methyl-2,4-dioxothiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)phenyl)piperazine-l-carboxylate (Compound 23f)

[0177] Compound 23f was prepared using the procedure described in Example 6 above, starting from compound 22a (0.4 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol) as a yellow solid powder in 88.42% yield. 1 H NMR (400 MHz, DMSO-d6) δ 14.07 (s, 1H), 8.34 (s, 1H), 8.27 (s, 1H), 7.81 (s, 1H), 7.43 - 7.30 (m, 2H), 7.14 - 6.95 (m, 2H), 3.49 (t, J = 5.0 Hz, 4H), 3.18 (dd, J = 6.4, 3.9 Hz, 4H), 3.10 (s, 3H), 1.44 (s, 8H). 13 C NMR (101 MHz, DMSO-d6) δ 170.07, 166.16, 154.33, 150.57, 140.43, 140.00, 134.59, 131.96, 130.97, 130.15, 122.73, 122.49, 122.33, 121.14, 115.59, 111.84, 79.48, 48.54, 28.54, 27.88. HRMS (ESI) m / z calcd for [C 27 H 28 ClN5O4S + H] + : 554.1623; found: 554.1641 [M+H] + .

[0178] Example 12

[0179] Preparation of tert-butyl (Z)-4-(4-(6-chloro-3-((2,4-dioxo-3-propylthiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)phenyl)piperazine- 1 -carboxylate (Compound 23g)

[0180] Using the procedure described in Example 6 above, Compound 23g was prepared in 71.23% yield as a yellow solid powder, starting from Compound 22a (0.4 g, 0.9 mmol), Compound 19c (0.16 g, 1 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.34 (s, 1H), 8.25 (s, 1H), 7.81 (s, 1H), 7.49 - 7.27 (m, 2H), 7.14 - 6.94 (m, 2H), 3.61 (dd, J = 7.9, 6.4 Hz, 2H), 3.49 (t, J = 5.1 Hz, 4H), 3.18 (dd, J = 6.2, 4.3 Hz, 4H), 1.61 (h, J = 7.4 Hz, 2H), 1.44 (s, 9H), 0.87 (t, J = 7.4 Hz, 3H). 13 CNMR (101 MHz, DMSO-d6) δ 170.04, 166.06, 154.34, 150.58, 140.49, 140.00, 134.62, 131.96, 130.96, 130.15, 122.76, 122.44, 122.00, 121.40, 115.59, 111.87, 79.48, 48.53, 43.08, 28.54, 25.96, 21.08, 11.53. HRMS (ESI) m / z calcd for [C 29 H 32 ClN5O4S+H] + : 582.1936; found: 582.1953 [M+H] + .

[0181] Example 13

[0182] Preparation of tert-butyl (Z)-4-(4-(6-chloro-3-((3-cyclopentyl-2,4-dioxothiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)phenyl)piperazine- 1 -carboxylate (Compound 23h)

[0183] Using the procedure described in Example 6 above, Compound 23h was prepared in 58.13% yield as a yellow solid powder, starting from Compound 22a (0.4 g, 0.9 mmol), Compound 19e (0.19 g, 1 mmol). 1HNMR (500 MHz, DMSO-d6) δ 14.04 (s, 1H), 8.30 (s, 1H), 8.22 (s, 1H), 7.81 (s, 1H), 7.53 - 7.27 (m, 2H), 7.13 - 6.89 (m, 2H), 4.69 (p, J = 8.4 Hz, 1H), 3.49 (t, J = 4.9 Hz, 4H), 3.18 (t, J = 5.2 Hz, 4H), 2.00 (qd, J = 13.3, 11.6, 6.5 Hz, 2H), 1.86 (qd, J = 9.9, 9.0, 5.2 Hz, 4H), 1.65 - 1.52 (m, 2H), 1.44 (s, 9H). 13 C NMR (126 MHz, DMSO-d6) δ 169.89, 166.01, 154.36, 150.58, 140.45, 140.07, 134.62, 131.99, 130.96, 130.15, 122.72, 122.38, 121.87, 121.13, 115.59, 111.84, 79.48, 54.06, 48.53, 28.89, 28.54, 25.39. HRMS (ESI) m / z calcd for [C 31 H 34 ClN5O4S+H] + :608.2093; found: 608.2110 [M+H] + .

[0184] Example 14

[0185] Preparation of tert-butyl (Z)-4-(4-(3-((3-benzyl-2,4-dioxothiazolidin-5- yl)methyl)-6-chloro-lH-indazol-5-yl)phenyl)piperazine-l-carboxylate (Compound 23i)

[0186] Compound 23i was prepared using the procedure described in Example 6 above, starting from compound 22a (0.4 g, 0.9 mmol) and compound 19d (0.21 g, 1 mmol) as a yellow solid powder in 62.43% yield. 1 HNMR (600 MHz, DMSO-d6) δ 14.02 (s, 1H), 8.39 (s, 1H), 8.25 (s, 1H), 7.82 (s, 1H), 7.44 - 7.22 (m, 7H), 7.11 - 6.91 (m, 2H), 4.84 (s, 2H), 3.49 (t, J = 5.1 Hz, 4H), 3.18 (t, J = 5.2 Hz, 4H), 1.44 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 169.97, 165.87, 154.34, 150.59, 140.51, 139.96, 136.17, 134.69, 132.00, 130.96, 130.14, 129.13, 128.21, 128.03, 126.84, 122.79, 122.44, 122.01, 121.70, 115.59, 111.91, 79.48, 48.53, 44.69, 28.54, 25.96. HRMS (ESI) m / z calcd for [C33H32CIN5O4S + H] + : 630.1936; found: 630.1953 [M+H] + .

[0187] Example 15

[0188] Preparation of tert-butyl (Z)-4-(5-(6-chloro-3-((2,4-dioxothiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)pyridin-2-yl)piperazine-l-carboxylate (Compound 23j)

[0189] Using compound 22b (0.4 g, 0.9 mmol), 2,4-thiazolidinedione (0.12 g, 1 mmol) as the starting material, the method described in Example 6 above was used to prepare compound 23j as a yellow solid powder in 62.63% yield. 1 H NMR (500 MHz, DMSO-d6) δ 14.05 (s, 1H), 12.44 (s, 1H), 8.30 - 8.17 (m, 3H), 7.82 (s, 1H), 7.71 (d, J = 8.7 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 3.56 (t, J = 4.9 Hz, 4H), 3.45 (t, J = 5.2 Hz, 4H), 1.43 (s, 8H). 13 CNMR (126 MHz, DMSO-d6) δ 170.71, 167.75, 158.25, 154.44, 148.39, 140.60, 140.02, 139.44, 132.02, 131.64, 124.60, 122.75, 122.48, 120.20, 111.94, 106.56, 79.51, 44.81, 28.54. HRMS (ESI) m / z calcd for [C 25 H 25 ClN6O4S + H] + : 541.1419; found: 541.1431 [M+H] + .

[0190] Example 16

[0191] Preparation of tert-butyl (Z)-4-(5-(6-chloro-3-((3-methyl-2,4-dioxothiazolidin-5- ylidenemethyl)-lH-indazol-5-yl)pyridin-2-yl)piperazin-l-ylcarboxylate (Compound 23k)

[0192] Compound 23k was prepared using the procedure described in Example 6 above, starting from compound 22b (0.4 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol) as a yellow solid powder in 80.42% yield. 1 H NMR (400 MHz, DMSO-d6) δ 14.08 (s, 1H), 8.30 (d, J = 2.2 Hz, 2H), 8.24 (d, J = 2.5 Hz, 1H), 7.82 (s, 1H), 7.71 (dd, J = 8.8, 2.5 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 3.56 (dd, J = 7.0, 3.7 Hz, 4H), 3.46 (t, J = 5.2 Hz, 4H), 3.09 (s, 3H), 1.44 (s, 9H). 13 CNMR (126 MHz, DMSO-d6) δ 169.94, 166.15, 158.39, 154.69, 148.33, 140.81, 140.09, 139.33, 132.25, 131.96, 124.68, 122.73, 122.68, 122.16, 120.84, 111.97, 106.58, 79.65, 44.95, 43.55, 28.60, 27.75. HRMS (ESI) m / z calcd for [C 26 H 27 ClN6O4S+H] + : 555.1576; found: 555.1548 [M+H] + .

[0193] Example 17

[0194] Preparation of tert-butyl (Z)-4-(5-(6-chloro-3-((2,4-dioxo-3-n-propylthiazolidin-5- ylidenemethyl)-lH-indazol-5-yl)pyridin-2-yl)piperazin-l-ylcarboxylate (Compound 23l)

[0195] Using compound 22b (0.4 g, 0.9 mmol), compound 19c (0.16 g, 1 mmol) as the raw material, the compound 23l was prepared by the method described in Example 6 above as a yellow solid powder, in a yield of 65.43%. 1 H NMR (500 MHz, DMSO-d6) δ 14.26 - 13.95 (m, 1H), 8.33 (d, J = 7.3 Hz, 2H), 8.25 (d, J = 2.5 Hz, 1H), 7.84 (s, 1H), 7.72 (dd, J = 8.8, 2.6 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 3.62 (t, J = 7.1 Hz, 2H), 3.57 (dd, J = 6.9, 3.8 Hz, 4H), 3.46 (t, J = 5.2 Hz, 4H), 1.61 (h, J = 7.4 Hz, 2H), 1.44 (s, 8H), 0.87 (t, J = 7.4 Hz, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 170.01, 166.07, 158.26, 154.43, 148.40, 140.61, 140.04, 139.44, 132.08, 131.76, 124.57, 122.78, 122.57, 122.17, 121.27, 111.99, 106.58, 79.51, 44.82, 43.11, 28.55, 21.08, 11.53. HRMS (ESI) m / z calcd for [C 28 H 31 ClN6O4S+H] + :583.1889; found: 583.1909 [M+H] + .

[0196] Example 18

[0197] Preparation of tert-butyl (Z)-4-(5-(6-chloro-3-((3-cyclopentyl-2,4-dioxothiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)pyridin-2-yl)piperazine-l-carboxylate (compound 23m)

[0198] Using compound 22b (0.4 g, 0.9 mmol), compound 19e (0.19 g, 1 mmol) as the raw material, the compound 23m was prepared by the method described in Example 6 above as a yellow solid powder, in a yield of 55.43%. 1H NMR (500 MHz, Chloroform-d) δ 11.76 (s, 1H), 8.31 (d, J = 2.7 Hz, 1H), 8.06 (d, J = 2.3 Hz, 1H), 7.79 (d, J = 2.3 Hz, 1H), 7.72 (s, 1H), 7.66 (d, J = 8.8 Hz, 1H), 6.87 - 6.60 (m, 1H), 4.80 (q, J = 8.4 Hz, 1H), 3.64 (d, J = 8.0 Hz, 8H), 2.20 - 2.05 (m, 2H), 2.02 - 1.89 (m, 4H), 1.64 (p, J = 8.3, 6.8 Hz, 2H), 1.53 (d, J = 2.2 Hz, 9H). 13 C NMR (126 MHz, Chloroform-d) δ 170.45, 166.46, 158.39, 155.00, 148.32, 140.66, 140.43, 139.10, 133.47, 132.52, 124.85, 123.40, 122.50, 121.03, 119.59, 111.35, 106.19, 54.53, 45.06, 28.77, 28.48, 25.26. HRMS (ESI) m / z calcd for [C 30 H 33 ClN6O4S+H] + : 609.2045; found: 609.2066 [M+H] + .

[0199] Example 19

[0200] Preparation of tert-butyl (Z)-4-(5-(6-chloro-3-((3-benzyl-2,4-dioxothiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)pyridin-2-yl)piperazine-l-carboxylate (Compound 23n)

[0201] Compound 23n was prepared using the procedure described in Example 6 above, starting from compound 22b (0.4 g, 0.9 mmol) and compound 19d (0.21 g, 1 mmol) as a yellow solid powder in 75.43% yield. 1H NMR (400 MHz, DMSO-d6) δ 14.12 (s, 1H), 8.33 (s, 1H), 8.28 - 8.20 (m, 2H), 7.80 (s, 1H), 7.69 (dd, J = 8.8, 2.5 Hz, 1H), 7.33 (tt, J = 11.3, 5.6 Hz, 5H), 6.90 (d, J = 8.9 Hz, 1H), 4.83 (s, 2H), 3.55 (dd, J = 7.0, 3.7 Hz, 4H), 1.43 (s, 9H). 13 C NMR (126 MHz, DMSO-d6) δ 169.91, 165.84, 158.21, 154.45, 148.30, 140.61, 139.93, 139.42, 136.12, 132.10, 131.79, 129.11, 128.20, 128.03, 124.56, 122.77, 122.42, 121.82, 121.79, 111.98, 106.57, 79.51, 74.02, 70.75, 44.82, 44.70, 32.09, 28.53, 25.39. HRMS (ESI) m / z calcd for [C 32 H 31 ClN6O4S+H] + : 631.1889; found: 631.1911 [M+H] + .

[0202] Example 20

[0203] Preparation of tert-butyl (Z)-4-(5-(6-chloro-3-((3-(2-(diethylamino)ethyl)-2,4- dioxothiazolidin-5-ylidene)methyl)-lH-indazol-5-yl)pyridin-2-yl)piperazine-l-carboxylate (Compound 23o)

[0204] Compound 23o was prepared using the procedure described in Example 6 above, starting from compound 22b (0.4 g, 0.9 mmol) and compound 19g (0.21 g, 1 mmol) as a yellow solid powder in 45.43% yield. 1H NMR (400 MHz, Chloroform-d) δ 12.91 (s, 1H), 8.18 (d, J = 2.5 Hz, 1H), 7.90 (s, 1H), 7.67 (d, J = 10.1 Hz, 2H), 7.46 (dd, J = 8.7, 2.5 Hz, 1H), 6.69 (d, J = 8.8 Hz, 1H), 3.87 (t, J = 6.7 Hz, 2H), 3.62 (d, J = 4.3 Hz, 8H), 2.80 (t, J = 6.7 Hz, 2H), 2.67 (q, J = 7.1 Hz, 4H), 1.53 (s, 9H), 1.07 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 170.73, 166.39, 158.24, 154.96, 148.29, 140.42, 140.13, 138.97, 133.04, 132.19, 124.75, 122.77, 122.52, 120.81, 119.92, 111.52, 106.07, 49.47, 49.18, 47.12, 46.96, 45.03, 39.79, 39.25, 33.75, 28.48, 11.92, 11.57. HRMS (ESI) m / z calcd for [C 31 H 38 ClN7O4S+H] + : 640.2467; found: 640.2490 [M+H] + .

[0205] Example 21

[0206] Preparation of tert-butyl (Z)-4-(5-(6-chloro-3-((3-(cyclohexylmethyl)-2,4- dioxothiazolidin-5-ylidene)methyl)-lH-indazol-5-yl)pyridin-2-yl)piperazine-l- carboxylate (Compound 23p)

[0207] Compound 23p was prepared using the procedure described in Example 6 above, starting from compound 22b (0.4 g, 0.9 mmol) and compound 19f (0.21 g, 1 mmol) as a yellow solid powder in 35.43% yield. 1H NMR (400 MHz, Chloroform-d) δ 11.57 (s, 1H), 8.31 (d, J = 2.5 Hz, 1H), 8.09 (s, 1H), 7.81 (s, 1H), 7.77 - 7.65 (m, 2H), 6.78 (d, J = 8.8 Hz, 1H), 3.65 (p, J = 4.8, 4.0 Hz, 10H), 1.84 (tt, J = 7.7, 3.5 Hz, 1H), 1.78 - 1.64 (m, 6H), 1.53 (s, 9H), 1.24 (d, J = 23.2 Hz, 4H). 13 C NMR (101 MHz, Chloroform-d) δ 170.75, 166.65, 154.94, 140.57, 140.42, 133.47, 124.81, 123.42, 122.49, 121.05, 119.95, 111.43, 80.24, 47.69, 45.12, 36.38, 30.59, 28.47, 26.17, 25.60. HRMS (ESI) m / z calcd for [C 32 H 37 ClN6O4S+H] + : 637.2358; found: 637.2379 [M+H] + .

[0208] Example 22

[0209] Preparation of tert-butyl (Z)-4-(5-(6-chloro-3-((3-(2-morpholinoethyl)-2,4-dioxothiazolidin-5- ylidene)methyl)-lH-indazol-5-yl)pyridin-2-yl)piperazine-l-carboxylate (Compound 23q)

[0210] Compound 23q was prepared using the procedure described in Example 6 above, starting from compound 22b (0.4 g, 0.9 mmol) and compound 19h (0.23 g, 1 mmol) as a yellow solid powder in 36.73% yield. 1 H NMR (400 MHz, Chloroform-d) δ 11.86 (s, 1H), 8.23 (d, J = 2.4 Hz, 1H), 8.02 (s, 1H), 7.73 (d, J = 11.1 Hz, 2H), 7.56 (dd, J = 8.8, 2.5 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H), 3.93 (t, J = 6.2 Hz, 2H), 3.70 (t, J = 4.6 Hz, 4H), 3.63 (q, J = 6.9, 5.7 Hz, 8H), 2.71 (t, J = 6.3 Hz, 2H), 2.59 (s, 4H), 1.53 (s, 10H).13 C NMR(101MHz,Chloroform-d)δ170.47,166.36,158.31,154.94,148.27,140.50,140.33,139.00,133.47,132.55,124. 66,123.29,122.47,120.97,119.88,111.36,106.09,66.91,55.48,53.50,45.02,38.34,29.72,28.48.HRMS(ESI)m / z calcd for[C 31 H 36 ClN7O5S+H] + :654.2260; found:654.2280[M+H] + .

[0211] Example 23

[0212] Preparation of tert-butyl(S,Z)-4-(5-(6-chloro-3-((3-methyl-2,4-dioxothiazolidin-5-ylidene)methyl)-1H-indazol-5-yl)pyridin-2-yl)-2-methylpiperazine-1-carboxylic acid ester (compound 23r)

[0213] Compound 23r was prepared as a yellow solid powder using compound 22e (0.4 g, 0.9 mmol) and 3-methylthiazolidin-2,4-dione (0.13 g, 1 mmol) as raw materials, following the method described in Example 6 above. The yield was 26.71%. 1 H NMR(400MHz,Chloroform-d)δ10.88(s,1H),8.30(dd,J=2.5,0.7Hz,1H),8.12(s,1H),7.84(d,J=0.6Hz,1H),7.73(d,J=0.5Hz ,1H),7.66(dd,J=8.8,2.5Hz,1H),6.72(dd,J=8.9,0.8Hz,1H),3.29(s,3H),1.62(s,7H),1.53(s,8H),1.28(d,J=4.8Hz,3H). 13C NMR (101 MHz, Chloroform-d) δ 170.20, 166.42, 158.69, 154.86, 148.32, 140.79, 140.28, 138.95, 133.82, 132.95, 124.14, 123.85, 122.52, 121.08, 119.79, 111.17, 105.59, 79.95, 48.63, 44.90, 29.71, 28.49, 27.69, 16.34. HRMS (ESI) m / z calcd for [C 27 H 29 ClN6O4S+H] + : 569.1732; found: 569.1750 [M+H] + .

[0214] Example 24

[0215] Preparation of (Z)-5-((5-(6-(piperazin-l-yl)pyridin-3-yl)-lH-indazol-3- yl)methylene)thiazolidine-2,4-dione (Compound 24a)

[0216] Compound 23a (0.3 g, 0.5 mmol) was added to a 50 mL round bottom flask, 10 mL of dichloromethane was added, then 2 mL of trifluoroacetic acid was added, and the reaction was stirred at room temperature for 2 h. After the reaction was completed, the dichloromethane and trifluoroacetic acid were evaporated, and the reaction liquid was a yellow oily substance, 10 mL of saturated sodium bicarbonate solution was added with stirring, and a light yellow solid was precipitated, and the filtrate was obtained. 0.18 g of product, yield 88.67%. 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 2.5 Hz, 1H), 8.27 (d, J = 1.6 Hz, 1H), 8.02 (dd, J = 8.8, 2.6 Hz, 1H), 7.85 (s, 1H), 7.75 - 7.56 (m, 2H), 6.96 (d, J = 8.9 Hz, 1H), 3.64 (t, J = 5.1 Hz, 4H), 3.04 (t, J = 5.1 Hz, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 180.19, 177.52, 158.16, 146.04, 141.22, 140.41, 136.62, 134.00, 131.19, 126.26, 125.82, 124.08, 116.61, 113.47, 111.47, 107.70, 44.24. HRMS (ESI) m / z calcd for [C 22 H 19 N6O2S+H] +: 407.1285; found: 407.1303 [M+H] + .

[0217] Example 25

[0218] Preparation of (Z)-3-methyl-5-((5-(6-(piperazin-l-yl)pyridin-3-yl)-lH- indazol-3-yl)methylene)thiazolidine-2,4-dione (Compound 24b)

[0219] Using the above described procedure of Example 24, starting from compound 23b (0.3 g, 0.5 mmol), compound 24b was prepared as a light yellow solid powder in 82.78% yield. 1 H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 1H), 8.34 (d, J = 5.4 Hz, 2H), 7.96 (d, J = 8.9 Hz, 1H), 7.79 - 7.42 (m, 2H), 6.88 (d, J = 8.9 Hz, 1H), 3.43 (s, 4H), 3.10 (s, 3H), 2.80 (t, J = 5.0 Hz, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 166.42, 158.77, 145.99, 139.39, 136.28, 131.40, 125.83, 125.67, 124.02, 122.70, 115.87, 113.85, 107.26, 46.40, 45.92, 27.67. HRMS (ESI) m / z calcd for [C 21 H 20 N6O2S+H] + : 421.1441; found: 421.1451 [M+H] + .

[0220] Example 26

[0221] Preparation of (Z)-5-((5-(4-(piperazin-l-yl)phenyl)-lH-indazol-3- yl)methylene)thiazolidine-2,4-dione (Compound 24c)

[0222] Using the above described procedure of Example 24, starting from compound 23c (0.3 g, 0.5 mmol), compound 24c was prepared as a light yellow solid powder in 81.58% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.21 (s, 1H), 7.81 (s, 1H), 7.68 (dd, J = 8.9, 4.3 Hz, 2H), 7.60 (d, J = 8.7 Hz, 1H), 7.04 (d, J = 8.3 Hz, 2H), 3.18 (s, 4H), 3.03 (s, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 180.93, 177.68, 150.55, 141.33, 140.30, 134.90, 133.71, 131.55, 127.85, 126.08, 124.04, 116.42, 116.23, 112.73, 111.20, 48.41, 45.06. HRMS (ESI) m / z calcd for [C 21 H 19 N5O2S+H] + : 406.1332; found: 406.1337 [M+H] + .

[0223] Example 27

[0224] Preparation of (Z)-3-methyl-5-((5-(4-(piperazin-l-yl)phenyl)-lH-indazol-3- yl)methylene)thiazolidine-2,4-dione (Compound 24d)

[0225] Compound 24d was prepared from compound 23d (0.3 g, 0.5 mmol) using the procedure described in Example 24 above as a pale yellow solid powder in 83.48% yield. 1 H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 4.7 Hz, 2H), 7.67 (d, J = 8.9 Hz, 4H), 7.01 (d, J = 8.2 Hz, 2H), 3.11 (d, J = 6.6 Hz, 7H), 2.86 (d, J = 6.1 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 170.64, 166.38, 151.19, 139.60, 134.41, 131.03, 127.79, 125.42, 125.10, 122.27, 116.21, 115.90, 112.70, 49.70, 46.08, 27.76. HRMS (ESI) m / z calcd for [C 22 H 21 N5O2S+H] + : 420.1489; found: 420.1495 [M+H] + .

[0226] Example 28

[0227] Preparation of (Z)-5-((6-chloro-5-(4-(piperazin-1-yl)phenyl)-1H-indazol-3- yl)methylene)thiazolidine-2,4-dione (Compound 24e)

[0228] Compound 24e was prepared from compound 23e (0.3 g, 0.5 mmol) using the procedure described in Example 24 above as a pale yellow solid powder in 75.78% yield. 1 H NMR (500 MHz, DMSO-d6) δ 7.92 (s, 1H), 7.73 (s, 1H), 7.59 (s, 1H), 7.35 (d, J = 8.3 Hz, 2H), 6.99 (d, J = 8.3 Hz, 2H), 3.14 (t, J = 5.0 Hz, 4H), 2.88 (t, J = 4.9 Hz, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 182.87, 178.45, 151.09, 141.75, 140.38, 137.73, 133.36, 131.26, 130.88, 129.84, 122.44, 121.89, 115.01, 111.42, 110.74, 49.33, 45.91. HRMS (ESI) m / z calcd for [C 21 H 18 ClN5O2S+H] + : 440.0942; found: 440.0951 [M+H] + .

[0229] Example 29

[0230] Preparation of (Z)-5-((6-chloro-5-(4-(piperazin-1-yl)phenyl)-1H-indazol-3- yl)methylene)-3-methylthiazolidine-2,4-dione (Compound 24f)

[0231] Compound 24f was prepared from compound 23f (0.3 g, 0.5 mmol) using the procedure described in Example 24 above as a pale yellow solid powder in 80.78% yield. 1 H NMR (500 MHz, DMSO-d6) δ 8.30 (s, 1H), 8.15 (s, 1H), 7.80 (s, 1H), 7.34 (d, J = 8.3 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 3.12 (d, J = 4.8 Hz, 6H), 2.86 (t, J = 5.0 Hz, 3H), 1.85 (s, 1H).13 C NMR (126 MHz, DMSO-d6) δ 170.60, 166.32, 151.22, 144.14, 144.13, 139.60, 130.81, 130.14, 123.72, 121.98, 121.34, 114.98, 113.42, 49.80, 46.07, 27.61. HRMS (ESI) m / z calcd for [C 22 H 20 ClN5O2S+H] + : 454.1099; found: 454.1102 [M+H] + .

[0232] Example 30

[0233] Preparation of (Z)-5-((6-chloro-5-(4-(piperazin-l-yl)phenyl)-lH-indazol-3- yl)methylene)-3-ethylthiazolidine-2,4-dione (Compound 24e)

[0234] Compound 24e was prepared from compound 23e (0.3 g, 0.5 mmol) using the procedure described in Example 24 above as a yellow solid powder in 93.18% yield. 1 H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 8.21 (s, 1H), 7.80 (s, 1H), 7.44 - 7.21 (m, 2H), 7.11 - 6.84 (m, 2H), 3.68 - 3.53 (m, 2H), 3.22 - 3.07 (m, 4H), 2.92 (t, J = 5.0 Hz, 4H), 1.60 (h, J = 7.4 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 170.06, 166.05, 151.10, 140.56, 139.92, 134.65, 131.90, 130.90, 129.63, 122.78, 122.29, 121.80, 121.39, 115.01, 111.90, 49.05, 45.69, 43.06, 21.08, 11.52. HRMS (ESI) m / z calcd for [C 24 H 24 ClN5O2S+H] + : 482.1412; found: 482.1424 [M+H] + .

[0235] Example 31

[0236] Preparation of (Z)-5-((6-chloro-5-(4-(piperazin-l-yl)phenyl)-lH-indazol-3- yl)methylene)-3-cyclopentylthiazolidine-2,4-dione (Compound 24h)

[0237] Using the above procedure in Example 24, Compound 24h was prepared in 82.16% yield as a yellow solid powder, starting from Compound 23h (0.3 g, 0.5 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (s, 1H), 8.18 (s, 1H), 7.79 (s, 1H), 7.40 - 7.21 (m, 2H), 7.04 - 6.92 (m, 2H), 4.69 (p, J = 8.3 Hz, 2H), 3.18 - 3.06 (m, 4H), 2.88 (dd, J = 6.3, 3.6 Hz, 4H), 1.99 (p, J = 8.9, 7.4 Hz, 2H), 1.90 - 1.76 (m, 4H), 1.66 - 1.50 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 169.95, 166.02, 151.23, 140.68, 139.97, 134.62, 131.83, 130.88, 129.55, 122.80, 122.20, 121.44, 121.19, 114.94, 111.95, 54.01, 49.36, 45.93, 28.88, 25.40. HRMS (ESI) m / z calcd for [C 26 H 26 ClN5O2S+H] + : 508.1568; found: 508.1584 [M+H] + .

[0238] Example 32

[0239] Preparation of (Z)-3-phenyl-5-((6-chloro-5-(4-(piperazin-l-yl)phenyl)-lH-indazol-3- yl)methylene)thiazolidine-2,4-dione (Compound 24i)

[0240] Using the above procedure in Example 24, Compound 24i was prepared in 72.56% yield as a yellow solid powder, starting from Compound 23i (0.3 g, 0.5 mmol). 1H NMR (500 MHz, DMSO-d6) δ 8.39 (s, 1H), 8.24 (s, 1H), 7.82 (s, 1H), 7.51 - 7.19 (m, 7H), 7.00 (d, J = 8.2 Hz, 2H), 4.84 (s, 2H), 3.16 (t, J = 5.1 Hz, 4H), 2.92 (t, J = 5.2 Hz, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 170.01, 165.88, 151.06, 140.60, 139.91, 136.17, 134.71, 131.93, 130.91, 129.66, 129.13, 128.20, 128.01, 122.83, 122.38, 122.05, 121.52, 115.05, 111.97, 48.91, 45.60, 44.66. HRMS (ESI) m / z calcd for [C 28 H 24 ClN5O2S+H] + : 530.1412; found: 530.1421 [M+H] + .

[0241] Example 32

[0242] Preparation of (Z)-5-((6-chloro-5-(6-(piperazin-l-yl)pyridin-3-yl)-lH-indazol-3- yl)methylene)thiazolidine-2,4-dione (Compound 24j)

[0243] Compound 23j (0.3 g, 0.5 mmol) was added to a 50 mL round bottom flask, 10 mL of dichloromethane was added, then 2 mL of trifluoroacetic acid was added, and the reaction was stirred at room temperature for 2 h. After the reaction was completed, the dichloromethane and trifluoroacetic acid were evaporated to obtain a yellow oil, 10 mL of saturated sodium carbonate solution was added with stirring, and a yellow solid was precipitated, which was filtered to obtain 0.2 g of yellow solid powder with a yield of 83.33%. 1 H NMR (500 MHz, DMSO-d6) δ 8.39 (s, 1H), 8.24 (s, 1H), 7.82 (s, 1H), 7.51 - 7.19 (m, 7H), 7.00 (d, J = 8.2 Hz, 2H), 4.84 (s, 2H), 3.16 (t, J = 5.1 Hz, 4H), 2.92 (t, J = 5.2 Hz, 4H). 13C NMR (126 MHz, DMSO-d6) δ 181.20, 177.75, 158.16, 148.34, 141.57, 140.58, 139.53, 131.42, 130.49, 124.95, 122.51, 122.21, 111.95, 111.62, 106.59, 44.45, 44.23. HRMS (ESI) m / z calcd for [C 20 H 17 ClN6O2S + H + : 441.0895; found: 441.0914 [M+H] + .

[0244] Example 33

[0245] Preparation of (Z)-5-((6-chloro-5-(6-(piperazin-l-yl)pyridin-3-yl)-lH-indazol-3- yl)methylene)-3-methylthiazolidine-2,4-dione (Compound 24k)

[0246] Compound 24k was prepared from compound 23k (0.3 g, 0.5 mmol) using the procedure described in Example 32 above as a pale yellow solid powder in 70.12% yield. 1 H NMR (500 MHz, DMSO-d6) δ 14.17 (s, 1H), 8.95 (s, 1H), 8.35 (d, J = 7.2 Hz, 1H), 8.30 (d, J = 2.5 Hz, 1H), 7.87 (s, 1H), 7.80 (dd, J = 8.8, 2.5 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H), 3.81 (t, J = 5.2 Hz, 4H), 3.24 (q, J = 4.8 Hz, 4H), 3.11 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 170.03, 166.18, 157.49, 148.16, 140.70, 140.06, 139.88, 131.97, 131.43, 125.42, 122.75, 122.71, 122.53, 120.97, 112.07, 107.08, 42.99, 42.23, 27.90. HRMS (ESI) m / z calcd for [C 21 H 19 ClN6O2S + H + : 455.1051; found: 455.1060 [M+H] + .

[0247] Example 34

[0248] Preparation of (Z)-5-((6-chloro-5-(6-(piperazin-l-yl)pyridin-3-yl)-lH-indazol-3- yl)methylene)-3-(2-(dimethylamino)ethyl)thiazolidine-2,4-dione (Compound 24n)

[0249] Using the method described in Example 32 above, Compound 24n was prepared from Compound 23n (0.3 g, 0.5 mmol) as a yellow solid powder in 82.78% yield. 1 H NMR (500 MHz, DMSO-d6) δ 8.34 - 8.19 (m, 3H), 7.84 (s, 1H), 7.74 (d, J = 8.6 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 3.11 (t, J = 5.3 Hz, 5H), 1.59 (h, J = 7.3 Hz, 2H), 0.86 (t, J = 7.4 Hz, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 170.03, 166.09, 157.97, 148.34, 140.68, 139.99, 139.60, 132.01, 131.61, 125.03, 122.72, 122.48, 122.13, 121.19, 112.05, 106.77, 43.84, 43.34, 43.10, 21.06, 11.49. HRMS (ESI) m / z calcd for [C 23 H 23 ClN6O2S+H] + : 483.1364; found: 483.1377 [M+H] + .

[0250] Example 35

[0251] Preparation of (Z)-5-((6-chloro-5-(6-(piperazin-l-yl)pyridin-3-yl)-lH-indazol-3- yl)methylene)-3-(2-(dimethylamino)ethyl)thiazolidine-2,4-dione (Compound 24n)

[0252] Using the method described in Example 32 above, Compound 24n was prepared from Compound 23n (0.3 g, 0.5 mmol) as a yellow solid powder in 82.78% yield. 1H NMR (400 MHz, Chloroform-d) δ 8.27 (d, J = 2.5 Hz, 1H), 8.03 (s, 1H), 7.77 (s, 1H), 7.71 (s, 1H), 7.59 (dd, J = 8.8, 2.5 Hz, 1H), 6.74 (d, J = 8.8 Hz, 1H), 3.88 (t, J = 6.7 Hz, 2H), 3.75 (q, J = 7.0 Hz, 2H), 3.63 (t, J = 5.1 Hz, 3H), 3.07 (dd, J = 6.1, 3.9 Hz, 4H), 2.76 (t, J = 6.8 Hz, 2H), 2.62 (d, J = 7.1 Hz, 2H), 1.05 (t, J = 7.1 Hz, 5H). 13 C NMR (126 MHz, DMSO-d6) δ 169.84, 166.03, 158.26, 148.39, 140.70, 140.04, 139.46, 132.06, 131.74, 124.69, 122.80, 122.60, 122.18, 121.16, 112.04, 106.56, 52.51, 49.28, 47.14, 44.49, 44.28, 12.54, 7.64. HRMS (ESI) m / z calcd for [C 26 H 30 ClN7O2S+H] + : 540.1943; found: 540.1960 [M+H] + .

[0253] Example 36

[0254] Preparation of (Z)-5-((6-chloro-5-(6-(piperazin-l-yl)pyridin-3-yl)-lH-indazol-3- yl)methylene)-3-(cyclohexylmethyl)thiazolidine-2,4-dione (Compound 24p)

[0255] Compound 24p was prepared from compound 23p (0.3 g, 0.5 mmol) using the procedure described in Example 32 above as a pale yellow solid powder in 92.78% yield. 1H NMR (600 MHz, DMSO-d6) δ 8.28 (s, 1H), 8.22 (s, 1H), 8.15 (s, 1H), 7.83 (s, 1H), 7.67 (d, J = 8.9 Hz, 1H), 6.87 (d, J = 8.9 Hz, 1H), 3.46 (t, J = 13.9 Hz, 6H), 2.81 (s, 2H), 1.67 (d, J = 11.2 Hz, 2H), 1.60 (d, J = 12.7 Hz, 3H), 1.13 (dq, J = 26.6, 13.7 Hz, 3H), 0.95 (q, J = 12.2 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 170.74, 166.35, 158.69, 148.33, 144.16, 139.56, 139.27, 130.85, 130.08, 124.61, 123.79, 122.31, 121.73, 118.01, 113.63, 106.20, 47.13, 46.22, 45.91, 36.36, 31.01, 30.58, 26.23, 25.97, 25.59. HRMS (ESI) m / z calcd for [C 27 H 29 ClN6O2S+H] + : 537.1834; found: 537.1850 [M+H] + .

[0256] Example 37

[0257] Preparation of (Z)-5-((6-chloro-5-(6-(piperazin-l-yl)pyridin-3-yl)-lH-indazol-3- yl)methylene)-3-(2-morpholinoethyl)thiazolidine-2,4-dione (Compound 24q)

[0258] Compound 24q was prepared from compound 23q (0.3 g, 0.5 mmol) using the procedure described in Example 32 above as a pale yellow solid powder in 72.78% yield. 1 H NMR (500 MHz, DMSO-d6) δ 8.35 (d, J = 15.3 Hz, 2H), 8.24 (d, J = 2.5 Hz, 1H), 7.86 (s, 1H), 7.70 (dd, J = 8.7, 2.5 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 3.79 (t, J = 6.3 Hz, 2H), 3.51 (d, J = 4.1 Hz, 8H), 2.86 (t, J = 4.9 Hz, 4H), 2.54 (d, J = 6.3 Hz, 2H), 2.41 (t, J = 4.7 Hz, 4H). 13C NMR (126 MHz, DMSO-d6) δ 169.90, 166.00, 158.68, 148.39, 140.77, 139.30, 132.05, 131.87, 122.86, 122.53, 121.91, 121.41, 112.08, 106.30, 66.69, 55.29, 53.57, 45.75, 45.58, 38.64. HRMS (ESI) m / z calcd for [C 26 H 28 ClN7O3S+H] + : 554.1736; found: 554.1756 [M+H] + .

[0259] Example 38

[0260] (Z)-5-((6-chloro-5-(6-(4-(3,3-dimethylbutanoic anhydride)piperazin-1-yl)pyridin-3-yl)-1H-indazol-3-yl)methylene)-3-methylthiazolidine-2,4-dione (Compound 24r)

[0261] Compound 24k (0.3 g, 0.5 mmol) was added to a 50 mL round bottom flask, 10 mL of dichloromethane was added, 2 drops of triethylamine was added as a catalyst, stirred uniformly at room temperature, slowly added a solution of tert-butyl acetyl chloride (1 mmol) in dichloromethane, half an hour later, the dropwise addition was completed, and the reaction was carried out at room temperature overnight. The next day, stop stirring, spin dry the reaction liquid under reduced pressure to obtain a yellow oil, further separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain a red-brown solid 0.2 g, yield 76.12%. 1 H NMR (500 MHz, DMSO-d6) δ 14.10 (s, 1H), 8.34 (d, J = 1.7 Hz, 2H), 8.26 (d, J = 2.5 Hz, 1H), 7.85 (s, 1H), 7.73 (dd, J = 8.7, 2.5 Hz, 1H), 6.95 (d, J = 8.9 Hz, 1H), 3.72 - 3.51 (m, 8H), 3.11 (s, 3H), 2.30 (s, 2H), 1.02 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 170.14, 170.06, 166.18, 158.24, 148.40, 140.62, 140.05, 139.47, 132.08, 131.76, 124.58, 122.77, 122.59, 122.49, 121.02, 111.99, 106.58, 45.95, 45.25, 45.02, 44.19, 31.49, 30.23, 27.90. HRMS (ESI) m / z calcd for [C 27 H 29 ClN6O3S+H] + :553.1783; found: 553.1798 [M+H] + .

[0262] Example 39

[0263] 4-((5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyridin-2-yl)methyl)morpholine (Compound 26)

[0264] 5-bromopyridine-2-carboxaldehyde (1.0 g, 5.4 mmol), morpholine (0.9 g, 10.0 mmol), sodium triacetoxyborohydride (2.1 g, 10.0 mmol), 20 mL dichloromethane were added into a 50 mL round bottom flask in turn, the reaction was stirred at room temperature overnight. After the reaction was completed, the reaction solution was washed with 20 mL saturated brine twice, the organic phase was combined, the organic layer was dried over anhydrous Na2S04and placed for 4 h. The anhydrous Na2S04was removed by filtration, and the organic phase was rotary evaporated to a yellow oil, which was weighed as 0.9 g, with a yield of 69.23%. 1 H NMR (400 MHz, Chloroform-d) δ 8.60 (d, J = 2.4 Hz, 1H), 7.77 (dd, J = 8.3, 2.4 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H), 3.76 - 3.68 (m, 4H), 3.59 (s, 2H), 2.64 - 2.41 (m, 4H). 13 C NMR (126 MHz, Chloroform-d) δ 156.55, 150.34, 139.09, 124.65, 119.19, 66.81, 64.11, 53.65.

[0265] Into a 50 mL round bottom flask, was added sequentially intermediate 25 (0.9 g, 3.5 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(l,3,2-dioxaborolane) (1.2 g, 4.7 mmol), potassium acetate (0.6 g, 5.8 mmol), [l,l-bis(diphenylphosphino)ferrocene]dichloropalladium (0.06 g, 0.08 mmol), 30 mL 1,4-dioxane. Vacuum, nitrogen protection, heated to reflux for 12 h, the reaction was completed. Lift the reaction bottle, the reaction bottle temperature dropped to room temperature, spin dry reaction, add 15 mL of methanol, a large amount of potassium acetate precipitated, filtered to remove potassium acetate, the filtrate was collected. Spin dry methanol, oil, weighing 1.2 g. Without purification, directly into the next step.

[0266] Example 40

[0267] 6-chloro-5-(pyridin-3-yl)-lH-indazole-3-carbaldehyde (compound 27a)

[0268] Using the method described in Example 1 above, compound 27a was prepared from compound 21a (0.2 g, 0.8 mmol) and 3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyridine (0.2 g, 0.8 mmol) as an off-white solid powder in 81.25% yield. 1 H NMR (500 MHz, DMSO-d6) δ 14.41 (s, 1H), 10.21 (s, 1H), 8.73 - 8.60 (m, 2H), 8.11 (s, 1H), 8.03 (s, 1H), 7.93 (dt, J = 7.8, 2.0 Hz, 1H), 7.53 (dd, J = 7.8, 4.8 Hz, 1H). 13 C NMR (126 MHz, DMSO-d6) δ 187.75, 150.19, 149.33, 144.05, 141.41, 137.65, 135.11, 133.15, 131.78, 123.73, 123.40, 119.97, 112.76. HRMS (ESI) m / z calcd for [C 13 H8ClN3O+H] + : 258.0429; found: 258.0432 [M+H] + .

[0269] Example 41

[0270] 6-chloro-5-(6-morpholinopyridin-3-yl)-lH-indazole-3-carbaldehyde (compound 27b)

[0271] Using the above described method of Example 1, compound 27b was prepared in 71.25% yield as a white solid powder, starting from compound 21a (0.2 g, 0.8 mmol) and 6-(morpholin-4- yl)pyridine-3-boronic acid pinacol ester (0.2 g, 0.8 mmol). 1 H NMR (500 MHz, DMSO-d6) δ 14.25 (s, 1H), 10.14 (s, 1H), 8.17 (d, J = 2.5 Hz, 1H), 7.98 (s, 1H), 7.86 (s, 1H), 7.62 (dd, J = 8.7, 2.6 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 3.76 - 3.65 (m, 4H), 3.48 (d, J = 5.2 Hz, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 187.49, 158.70, 148.10, 143.86, 140.98, 139.13, 133.54, 132.21, 124.59, 122.69, 120.04, 112.40, 106.37, 74.03, 66.42, 45.42, 25.35. HRMS (ESI) m / z calcd for [C 17 H 15 ClN4O2+H] + : 343.0956; found: 343.0957 [M+H] + .

[0272] Example 42

[0273] 6-chloro-5-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-1H-indazole-3-carbaldehyde (compound 27c)

[0274] Using the above described method of Example 1, compound 27c was prepared in 81.25% yield as a white solid powder, starting from compound 21a (0.2 g, 0.8 mmol) and 6-(4-methyl-1- piperazinyl)pyridine-3-boronic acid pinacol ester (0.3 g, 0.8 mmol). 1 H NMR (500 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.18 (d, J = 2.5 Hz, 1H), 8.03 (s, 1H), 7.95 (s, 1H), 7.65 (dd, J = 8.7, 2.5 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 3.56 (d, J = 5.0 Hz, 4H), 2.43 (t, J = 5.0 Hz, 4H), 2.24 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 187.67, 158.61, 148.11, 143.88, 141.14, 139.18, 133.74, 132.29, 124.14, 122.71, 120.14, 112.60, 106.54, 54.81, 46.21, 44.86. HRMS (ESI) m / z calcd for [C 18 H 18 ClN5O+H] + :356.1273; found: 356.1277 [M+H] + .

[0275] Example 43

[0276] 6-chloro-5-(6-(morpholinomethyl)pyridin-3-yl)-lH-indazole-3-carbaldehyde (Compound 27d)

[0277] Using the method described in Example 1 above, starting from compound 21a (0.2 g, 0.8 mmol) and 4-((5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyridin-2- yl)methyl)morpholine (0.3 g, 0.8 mmol), compound 27d was prepared as an off-white solid powder in 41.25% yield. 1 H NMR (500 MHz, DMSO-d6) δ 10.17 (s, 1H), 8.56 (d, J = 2.4 Hz, 1H), 8.06 (s, 1H), 7.96 (s, 1H), 7.86 (dd, J = 8.0, 2.4 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H), 3.66 (s, 2H), 3.60 (t, J = 4.5 Hz, 4H), 2.45 (d, J = 9.6 Hz, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 187.59, 157.85, 149.36, 143.94, 141.34, 137.99, 133.42, 133.01, 131.75, 123.29, 122.56, 119.93, 112.69, 66.66, 64.27, 53.80. HRMS (ESI) m / z calcd for [C 18 H 17 ClN4O2+H] + :357.1113; found: 357.1122 [M+H] + .

[0278] Example 44

[0279] 6-chloro-5-(6-(piperidin-l-yl)pyridin-3-yl)-lH-indazole-3-carbaldehyde (Compound 27e)

[0280] Using the above described method of Example 1, starting from compound 21a (0.2 g, 0.8 mmol) and 2-(piperidin-l-yl)pyridine-5-boronic acid pinacol ester (0.2 g, 0.8 mmol), compound 27e was prepared as a white solid in 76.89% yield. 1 HNMR (600 MHz, DMSO-d6) δ 14.31 (s, 1H), 10.19 (s, 1H), 8.17 (d, J = 2.5 Hz, 1H), 8.04 (s, 1H), 7.95 (s, 1H), 7.62 (dd, J = 8.8, 2.6 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 3.58 (t, J = 5.3 Hz, 4H), 1.60 (dq, J = 34.2, 5.7, 5.2 Hz, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 187.70, 158.54, 148.18, 143.91, 141.00, 139.09, 133.92, 132.38, 123.29, 122.65, 120.17, 112.54, 106.28, 45.91, 25.52, 24.78. HRMS (ESI) m / z calcd for [C18H17CIN4O + H] + : 341.1164; found: 341.1168 [M+H] + .

[0281] Example 45

[0282] 6-chloro-5-(6-(piperidin-l-yl)pyridin-3-yl)-lH-indazole-3-carbaldehyde (Compound 27e)

[0283] Using the above described method of Example 1, starting from compound 21a (0.2 g, 0.8 mmol) and 2-(piperidin-l-yl)pyridine-5-boronic acid pinacol ester (0.2 g, 0.8 mmol), compound 27e was prepared as a white solid in 76.89% yield. 1H NMR (500 MHz, DMSO-d6) δ 14.27 (s, 1H), 10.16 (s, 1H), 8.12 (d, J = 2.6 Hz, 1H), 8.00 (s, 1H), 7.88 (s, 1H), 7.55 (dd, J = 8.6, 2.6 Hz, 1H), 6.47 (d, J = 8.7 Hz, 1H), 3.40 (d, J = 6.8 Hz, 4H), 1.93 (d, J = 6.4 Hz, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 187.56, 156.53, 148.44, 143.82, 140.93, 138.54, 134.16, 132.43, 122.52, 122.34, 120.12, 112.42, 105.85, 46.86, 25.48. HRMS (ESI) m / z calcd for [C 17 H 15 ClN4O+H] + : 327.1007; found: 327.1009 [M+H] + .

[0284] Example 46

[0285] 6-chloro-5-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-1H-indazole-3-carbaldehyde (Compound 27g)

[0286] Using the method described in Example 1 above, starting from compound 21a (0.2 g, 0.8 mmol) and 1-methyl-4-((4-(4,4,5,5--tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)piperazine (0.3 g, 0.8 mmol), compound 27g was prepared as a white solid in 81.25% yield. 1 H NMR (500 MHz, DMSO-d6) δ 14.39 (s, 1H), 10.20 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 7.9 Hz, 2H), 2.38 (t, J = 4.9 Hz, 4H), 2.14 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 187.69, 144.05, 143.92, 141.42, 134.84, 134.50, 131.25, 131.10, 127.95, 123.25, 119.89, 112.87, 53.89, 46.26, 45.72. HRMS (ESI) m / z calcd for [C19 H 19 ClN4O3S+H] + :419.0939;found:419.0944[M+H] + .

[0287] Example 47

[0288] (Z)-5-((6-chloro-5-(pyridin-3-yl)-lH-indazol-3-yl)methylene)-3-methylthiazolidine- 2,4-dione (Compound 28a)

[0289] Using the method described in Example 6 above, starting from compound 27a (0.23 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol), compound 28a was prepared as a yellow solid in 71.75% yield. 1 H NMR (400 MHz, DMSO-d6) δ 14.18 (s, 1H), 8.71 (d, J = 2.3 Hz, 1H), 8.64 (dd, J = 4.8, 1.7 Hz, 1H), 8.49 (s, 1H), 8.39 (s, 1H), 8.02 - 7.88 (m, 2H), 7.53 (dd, J = 7.9, 4.8 Hz, 1H), 3.11 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 170.01, 166.17, 150.46, 149.05, 140.96, 140.32, 137.81, 135.20, 131.53, 131.18, 123.55, 122.78, 122.66, 120.94, 112.16, 27.93. HRMS (ESI) m / z calcd for [C 17 H 11 ClN4O2S+H] + :371.0364; found: 371.0367 [M+H] + .

[0290] Example 48

[0291] (Z)-5-((6-chloro-5-(6-morpholinopyridin-3-yl)-lH-indazol-3-yl)methylene)-3- methylthiazolidine-2,4-dione (Compound 28b)

[0292] Using the method described in Example 6 above, starting from compound 27b (0.3 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol), compound 28b was prepared as a yellow solid in 61.25% yield. 1H NMR (400 MHz, DMSO-d6) δ 14.09 (s, 1H), 8.36 (d, J = 2.9 Hz, 2H), 8.27 (d, J = 2.5 Hz, 1H), 7.86 (s, 1H), 7.73 (dd, J = 8.8, 2.6 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 3.74 (t, J = 4.7 Hz, 4H), 3.52 (t, J = 4.8 Hz, 4H), 3.11 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 170.06, 166.18, 158.68, 148.40, 140.62, 140.05, 139.40, 132.07, 131.75, 124.79, 122.78, 122.65, 122.47, 121.04, 111.99, 106.42, 66.81, 66.45, 45.52, 27.91. HRMS (ESI) m / z calcd for [C 21 H 18 ClN5O3S+H] + : 456.0892; found: 456.0899 [M+H] + .

[0293] Example 49

[0294] (Z)-5-((6-chloro-5-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-1H-indazol-3- yl)methylene)-3-methylthiazolidine-2,4-dione (Compound 28c)

[0295] Using the method described in Example 6 above, starting from compound 27c (0.3 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol), compound 28c was prepared as a yellow solid in 70.75% yield. 1 H NMR (400 MHz, DMSO-d6) δ 14.09 (s, 1H), 8.36 (d, J = 2.9 Hz, 2H), 8.27 (d, J = 2.5 Hz, 1H), 7.86 (s, 1H), 7.73 (dd, J = 8.8, 2.6 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 3.74 (t, J = 4.7 Hz, 4H), 3.52 (t, J = 4.8 Hz, 4H), 3.11 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 170.05, 166.19, 158.55, 148.41, 140.62, 140.07, 139.32, 132.12, 131.87, 124.28, 122.81, 122.61, 122.51, 121.06, 111.99, 106.42, 54.86, 46.27, 44.99, 27.91, 25.96. HRMS (ESI) m / z calcd for [C 22 H 21 ClN6O2S+H] + : 469.1208; found: 469.1215 [M+H] + .

[0296] Example 50

[0297] (Z)-5-((6-chloro-5-(6-(morpholinomethyl)pyridin-3-yl)-1 H-indazol-3- yl)methylene)-3-methylthiazolidine-2,4-dione (Compound 28d)

[0298] Using the method described in Example 6 above, starting from compound 27d (0.3 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol), compound 28d was prepared as a yellow solid in 71.25% yield. 1 H NMR (500 MHz, DMSO-d6) δ 14.14 (s, 1 H), 8.62 (s, 1 H), 8.46 (s, 1 H), 8.36 (s, 1 H), 8.01 - 7.82 (m, 2H), 7.56 (d, J = 8.0 Hz, 1 H), 3.67 (s, 2H), 3.62 (t, J = 4.7 Hz, 4H), 3.10 (s, 3H), 2.47 (d, J = 5.2 Hz, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 170.01, 166.16, 157.62, 149.64, 140.90, 140.25, 138.18, 133.55, 131.57, 131.12, 123.46, 122.69, 122.47, 120.92, 112.13, 66.70, 64.32, 62.47, 53.82, 27.91, 25.96. HRMS (ESI) m / z calcd for [C 22 H 20 ClN5O3S+H] + : 470.1048; found: 470.1055 [M+H] + .

[0299] Example 51

[0300] (Z)-5-((6-chloro-5-(6-(piperidin-l-yl)pyridin-3-yl)-lH-indazol-3-yl)methylene)- 3-methylthiazolidine-2,4-dione (Compound 28e)

[0301] Using the method described in Example 6 above, starting from compound 27e (0.3 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol), compound 28e was prepared as a yellow solid in 85.25% yield. 1 H NMR (400 MHz, DMSO-d6) δ 14.08 (s, 1H), 8.36 (d, J = 4.4 Hz, 2H), 8.24 - 8.19 (m, 1H), 7.85 (s, 1H), 7.67 (dd, J = 8.8, 2.6 Hz, 1H), 6.91 (d, J = 8.9 Hz, 1H), 3.59 (t, J = 5.3 Hz, 4H), 3.11 (s, 3H), 1.74 - 1.47 (m, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 170.07, 166.19, 158.43, 148.47, 140.56, 140.03, 139.27, 132.13, 131.98, 123.45, 122.83, 122.52, 122.43, 121.09, 111.97, 106.17, 45.98, 27.91, 25.50, 24.81. HRMS (ESI) m / z calcd for [C 22 H 20 ClN5O2S+H] + : 454.1099; found: 454.1106 [M+H] + .

[0302] Example 52

[0303] (Z)-5-((6-chloro-5-(6-(piperidin-l-yl)pyridin-3-yl)-lH-indazol-3-yl)methylene)- 3-methylthiazolidine-2,4-dione (Compound 28e)

[0304] Using the method described in Example 6 above, starting from compound 27e (0.3 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol), compound 28e was prepared as a yellow solid in 85.25% yield. 1H NMR (500 MHz, DMSO-d6) δ 14.08 (s, 1H), 8.35 (s, 1H), 8.32 (s, 1H), 8.19 (d, J = 2.5 Hz, 1H), 7.83 (s, 1H), 7.65 (dd, J = 8.6, 2.5 Hz, 1H), 6.53 (d, J = 8.7 Hz, 1H), 3.49 - 3.39 (m, 4H), 3.11 (s, 3H), 2.03 - 1.92 (m, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 170.07, 166.18, 156.48, 148.74, 140.50, 139.98, 138.79, 132.33, 132.26, 122.82, 122.50, 122.38, 121.11, 111.92, 105.77, 46.92, 27.90, 25.52. HRMS (ESI) m / z calcd for [C 21 H 18 ClN5O2S+H] + : 440.0942; found: 440.0949 [M+H] + .

[0305] Example 53

[0306] (Z)-5-((6-chloro-5-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-1H-indazol-3- yl)methylene)-3-methylthiazolidine-2,4-dione (Compound 28g)

[0307] Using the method described in Example 6 above, starting from compound 27g (0.4 g, 0.9 mmol), 3-methylthiazolidine-2,4-dione (0.13 g, 1 mmol), compound 28g was prepared as a yellow solid in 81.75% yield. 1 H NMR (500 MHz, DMSO-d6) δ 14.08 (s, 1H), 8.35 (s, 1H), 8.32 (s, 1H), 8.19 (d, J = 2.5 Hz, 1H), 7.83 (s, 1H), 7.65 (dd, J = 8.6, 2.5 Hz, 1H), 6.53 (d, J = 8.7 Hz, 1H), 3.49 - 3.39 (m, 4H), 3.11 (s, 3H), 2.03 - 1.92 (m, 4H). 13C NMR (126 MHz, DMSO-d6) d 169.82, 166.09, 144.17, 141.17, 140.48, 135.47, 133.12, 131.21, 131.16, 127.62, 123.12, 123.04, 122.62, 120.78, 112.17, 54.09, 46.22, 45.66, 27.79. HRMS (ESI) m / z calcd for [C 23 H 22 ClN5O4S2+H] + : 532.0874; found: 532.0886 [M+H] + .

[0308] Example 54

[0309] After acute kidney injury, tubular cells produce FGF2 through autophagy, leading to fibroblast activation and kidney fibrosis, so that rat kidney fibroblasts (NRK-49F) are generated in vivo.

[0310] The synthesized compound was subjected to anti-rat kidney fibroblast (NRK-49F) proliferation experiment, as follows:

[0311] 1. Experimental materials

[0312] (1) Cell strain: NRK-49F cells

[0313] (2) Reagents and instruments: 96-well plate (Corning); DMEM medium (BI); biosafety cabinet, carbon dioxide incubator (ESCO); Counting Kit-8 (CCK-8) cell viability detection kit (Nanjing Enzhi Biotechnology Co., Ltd.); Cytation 5 multifunctional imager (Bio-Tek).

[0314] 2. Experimental method

[0315] (1) According to the CCK-8 staining method, cells with a viable cell ratio of more than 90% were used for the experiment, and the CCK-8 cell viability detection kit was used for cell proliferation inhibition test.

[0316] (2) Cell digestion, counting, and preparation of cell suspension with a concentration of 1 x 10 5 μL of cell suspension was added to each well of the 96-well plate (1 x 10 4 cells per well), and 10 ng / μL of TGF-β was added.

[0317] (3) 96-well plate was placed in 37℃, 5% CO2 incubator for 24 hours; 100 μL of the corresponding drug-containing medium was added to each well, and negative control group, solvent control group and positive control group were set up, 5 replicates for each group.

[0318] (4) 96-well plate was placed in 37℃, 5% CO2 incubator for 48 hours; 10 μL of CCK-8 solution was added to each well, and the plate was incubated in the incubator for 4 hours, and the culture solution in the well was carefully aspirated.

[0319] (5) The OD value at 450 nm was measured by the microplate reader, and the inhibition rate and IC 50 value of the compound on NRK-49F cells were calculated, respectively.

[0320] (6) Dose setting: dose 1 group: 100 μM; dose 2 group: 50 μM; dose 3 group: 25 μM; dose 4 group: 12.5 μM; dose 5 group: 6.25 μM; dose 6 group: 3.125 μM; dose 7 group: 1.5625 μM; dose 8 group: 0.78125 μM; dose 9 group: 0.390625 μM; dose 10 group: 0.1953125 μM.

[0321] (7) Data processing: data were expressed as mean and SD, and GraphPad Prism 5.0 statistical software was used, t-test was used for comparison between two groups. The IC 50 value of the compound was calculated by fitting the curve with concentration as the abscissa and inhibition rate as the ordinate. The absorbance OD value of each well was measured on the microplate reader at 450 nm, and the cell growth inhibition rate was calculated according to the following formula:

[0322] Inhibition rate (%) = [(control well average OD value-experimental well average OD) / control well average OD value] * 100%.

[0323] The results are shown in the following table.

[0324] Table 1 Result data table of compound anti-NRK-49F abnormal proliferation activity

[0325]

[0326]

[0327] As shown in Table 1, the compound of the present application has good anti-proliferation effect on NRK-49F cells, and the anti-proliferation effect is better than that of positive drug 991.

[0328] The structure of positive drug 991 is:

[0329] Example 55

[0330] Activation activity experiment of AMPK on the synthesized compounds

[0331] 1. Experimental materials and instruments

[0332] ATP, MgCl2, DTT used in the experiment were purchased from Sigma company; human recombinant phosphorylated AMPK a1b1g1 and AMPK a2b1g1 were purchased from Carna company, HTRF kit was purchased from Cisbio company, multifunctional enzyme marker was purchased from Bio Tek company, and 384 well plate was purchased from Thermo Fisher company.

[0333] 2. Experimental method

[0334] The first step is to configure the working solution, and the preparation ratio is 1x Buffer: MgCl2: DTT = 97: 2: 1. Then the compound is prepared at a concentration of 2.5 μM, and the corresponding working concentration is 1 μM. ATP and STK-S1 are diluted 20 times and 10 times with the working solution respectively.

[0335] The second step is to explore the enzyme concentration, and the enzyme is set to different concentrations (0.002, 0.02, 0.2, 0.5, 2, 20 ng / μL). Add 4 μL of working solution, 2 μL of diluted ATP and STK-S1, and 2 μL of enzyme with different concentrations to each well of 384 well plate respectively. After incubation at 37℃ for 1 h, the enzyme standard curve is made to determine the optimal enzyme concentration.

[0336] The third step is to add 4 μL of drug with a concentration of 1 μM to 200 μL EP tube. Then add 2 μL of diluted ATP and STK-S1 and enzyme with specific concentration to the wall of the tube. After centrifugation and mixing, put it into 37℃ oven for 1 hour.

[0337] The fourth step is to dilute Streptavidin-XL665 33.3 times with working solution, and mix the diluted Streptavidin-XL665 and STK-antibody in a ratio of 1:1 to get the detection solution. Add 10 μL of detection solution to the wall of the EP tube, and mix it well after centrifugation. Keep it away from light at room temperature for 1 h. Transfer the liquid in the EP tube to the 384 well plate, and detect the OD value at 665 nm and 620 nm wavelength with the enzyme marker. Calculate the ratio of the two OD values (Ratio):

[0338]

[0339] Calculate the activation rate of the compound at a concentration of 1 μM:

[0340] Activation Efficacy = [Ratio (Sample) - Ratio (Standard)] / Ratio (Standard) x 100%.

[0341] The compound 24f with good selectivity and selectivity was selected as the positive control compound 991. The activation rates of the test compound on AMPKα1β1γ1 and AMPKα2β1γ1 at different concentrations, respectively, the Efficacy at each concentration, and the activation curve were fitted using Graphpad software.

[0342] As shown in Figure 1 A, the activation activity of 24f on AMPKα1β1γ1 was 35.1±1.2nM, respectively. As shown in Figure 1 B, the activation activity of 991 on AMPKα1β1γ1 was 60.2±2.3nM, respectively. Compared with the positive control drug 991, the activity of compound 24f was doubled.

[0343] Example 56

[0344] Effect of compound on downstream proteins of AMPK

[0345] 1. Experimental materials

[0346] (1) Sample: NRK-52E cells

[0347] (2) Antibodies: AMPK, p-AMPK, ACC, p-ACC were purchased from CST company.

[0348] (3) Drugs and reagents: hypersensitive ECL chemiluminescence reagent was purchased from Abbkine company; goat anti-rabbit first antibody was purchased from Cell Signaling Technology company, goat anti-rabbit second antibody was purchased from Abbkine company; developing solution, fixing solution, transfer buffer were purchased from Abbkine company, blocking solution was purchased from Abbkine company, RIPA lysis buffer, BSA, BCA protein concentration detection kit, pre-stained protein marker were purchased from Nanjing Enzhi Biotechnology Co., Ltd.

[0349] (4) Instruments and consumables: micro vertical electrophoresis tank (Bio-RAD); transfer electrophoresis tank (Bio-RAD); electrophoresis instrument (Junyi Limited Company); decolorization shaker (Huali Da Limited Company).

[0350] 2. Experimental method

[0351] (1) Preparation of protein sample

[0352] The treated cells were discarded, and PBS was used to wash the cells three times. Then, 100 μL of RIPA lysis solution was added to lyse the cells for 30 min. The supernatant was transferred to a 0.5 mL centrifuge tube, and the protein concentration was determined using a protein quantification kit. The supernatant was stored at -20°C, and the sample protein was denatured before electrophoresis.

[0353] (2) SDS-PAGE electrophoresis

[0354] The separation gel was slowly poured along the glass, and then a layer of anhydrous ethanol was added for liquid sealing. After the gel was solidified, the upper layer of water was discarded, and the water was absorbed with a water-absorbing paper. The concentrated gel was inserted into the comb. After the concentrated gel was solidified, the comb was gently pulled out vertically upward, and then the sample was loaded. After sufficient electrophoresis buffer was added, the sample was loaded. The electrophoresis was performed at 70 V for 20 min, and then the voltage was changed to 130 V when the protein was aggregated to the boundary of the separation gel. The electrophoresis was terminated when the bromophenol blue was just run out, and then the membrane was transferred.

[0355] (3) Membrane transfer

[0356] The soaked PVDF membrane was attached to the desired target band, and after the air bubbles were carefully removed, the PVDF membrane could not be moved. A cotton pad was placed on the membrane transfer plate. The membrane transfer plate was placed in the membrane transfer tank in the manner of PVDF membrane on the positive electrode and negative electrode. The membrane transfer buffer was added to the membrane transfer tank, and the membrane transfer was performed at 80 V for 45 min. After the membrane transfer was completed, the PVDF membrane was taken out, soaked in TBS, and then placed in the blocking solution for overnight blocking at 4°C. After the blocking was completed, the first antibody and the second antibody were incubated, and then the chemiluminescence development was performed.

[0357] 3. Experimental results

[0358] The results are shown in the following table. Figure 2 A is the expression of AMPK and its downstream proteins after treatment with different concentrations of compound 24f, which was detected by Western blot; Figure 2 B and C are column charts of the quantitative analysis of the Western blot data of p-AMPK and p-ACC proteins, respectively. Figure 2 Figure 2 **** indicates p<0.0001 compared with the positive control group (0 / +), *** indicates p<0.001 compared with the positive control group (0 / +), and ** indicates p<0.01 compared with the positive control group (0 / +). As can be seen from the figure, the target compound can effectively regulate the level of the downstream protein of AMPK. Compared with 991, the compound has better AMPK activation effect.

[0359] Example 57

[0360] ​In vivo pharmacodynamic study of compounds on acute kidney injury in mice

[0361] 1. Experimental materials

[0362] Male healthy C57BL / 6 mice (16-18 g) required for the experiment were purchased from Hunan Slike Jingda Experimental Animal Co., Ltd. The needle suture, forceps, and scissors required for the operation were purchased from Henan Baihe Medical Instrument Sales Co., Ltd. The positive control drug 991 was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. The protease inhibitors and phosphatase inhibitors required for the experiment were purchased from Beijing Solaybao Technology Co., Ltd. The RIPA lysis buffer was purchased from Biyun Tian Biological Technology Co., Ltd.

[0363] 2. Experimental method

[0364] First, prepare the drug solution required for the animal experiment. Take 0.25 g of sodium carboxymethyl cellulose, add 50 mL of ddH2O, and place it on a room temperature shaker overnight to obtain a uniform 0.5% sodium carboxymethyl cellulose solution. This prevents the rapid settling of drug particles and ensures uniformity and accuracy of the dose. Take 44.1 mg of 24f, add 7.05 mL of 0.5% sodium carboxymethyl cellulose solution, and grind it evenly in a mortar to obtain a 6.25 mg / mL drug solution. Take 86.9 mg of 24f, add 6.952 mL of 0.5% sodium carboxymethyl cellulose solution, and grind it evenly in a mortar to obtain a 12.5 mg / mL drug solution. Take 77.9 mg of 991, add 6.232 mL of 0.5% sodium carboxymethyl cellulose solution, and grind it evenly in a mortar to obtain a 12.5 mg / mL drug solution of 991.

[0365] Second, the mice were raised in an environment with a temperature of 20-25°C and a relative humidity of 50-60%, and were provided with standard food and drinking water. After two weeks of adaptive feeding, 30 male healthy C57BL / 6 mice were randomly divided into 5 groups for weighing and numbering. The groups were: sham operation group (both renal pedicles were exposed under the lateral incision without clamping, and the mice were returned to the abdominal cavity and the wound was sutured), operation group (the renal pedicles were exposed under the lateral incision, and the left and right kidneys were removed and clamped for 30 minutes before being returned to the abdominal cavity of the mice and the wound was sutured), operation + 100 mg / kg 991 treatment group, operation + 50 mg / kg 24f treatment group, and operation + 100 mg / kg 24f treatment group. The drugs were administered orally by gavage. Each mouse was given the corresponding drug solution according to the group and body weight, and the administration was performed for 4 days.

[0366] The third step involved anesthetizing the mice sequentially. Sodium pentobarbital was injected intraperitoneally at a dose of 6 mg / kg. After anesthesia, the fur on the mice's abdomen was removed, and any remaining fur was wiped away with an alcohol swab. Both renal pedicles were exposed under the lateral incision. Simultaneously, the left and right kidneys were removed, clamped for 30 minutes, and then returned to the mouse's abdomen. The wound was then sutured. In the sham surgery group, both renal pedicles were exposed under the lateral incision, not clamped, and returned to the mouse's abdomen; the wound was then sutured.

[0367] Fourth, 24 hours after reperfusion, the mice were euthanized under anesthesia. Both kidneys were removed and stored at room temperature in 4% paraformaldehyde solution for H&E staining to assess the degree of kidney tissue damage.

[0368] 3. Experimental Results

[0369] like Figure 3 As shown, the model group tissues exhibited characteristic pathological changes of ischemia-reperfusion injury, and the degree of damage (e.g., the proportion of necrotic cells and the lesion extent score) was significantly more severe than that of the sham-operated group, indicating successful modeling. In the sham-operated group, kidney cells were intact and the histological appearance was normal; however, in the surgical group, histological damage and immune cell infiltration increased, indicating that the model of ischemic acute kidney injury was successfully established surgically. Compared with the surgical group, the 991 group alleviated surgery-induced kidney injury. 24f also dose-dependently alleviated surgery-induced kidney injury, and at the same dose, the 24f group showed a more significant reduction.

[0370] 4. Conclusion

[0371] The benzopyrazole compounds of this invention can alleviate ischemic acute kidney injury.

[0372] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A benzopyrazole compound, characterized in that, It is a compound, optical isomer, or pharmaceutically usable salt represented by Formula I: R1 is selected from hydrogen, C1-C8 straight-chain or branched alkyl, aryl, substituted aryl, C3-C8 alicyclic group, substituted C3-C8 alicyclic group, C3-C8 alicyclic group, substituted C3-C8 alicyclic group, and -(CH2)nNR4R5. Substituents on aryl groups, C3-C8 aliphatic cyclic groups, and C3-C8 heterocyclic cyclic groups are independently selected from C1-C8 straight-chain or branched alkyl groups; n is an integer from 0 to 4, and R4 and R5 are independently selected from C1-C5 straight-chain or branched alkyl groups; R2 is X is nitrogen or CH; R6 is selected from hydrogen, C3-C8 nitrogen-containing aliphatic heterocyclic groups, R7 is selected from hydrogen and C1-C3 alkyl groups; R8 is selected from hydrogen, C1-C8 straight-chain or branched alkyl carbonyl, and C1-C8 straight-chain or branched alkyl ester. R3 is selected from hydrogen or halogens.

2. The benzopyrazole compound according to claim 1, characterized in that, The aryl group is phenyl.

3. The benzopyrazole compound according to claim 1, characterized in that, R1 is selected from C1-C5 straight-chain or branched alkyl, aryl, substituted aryl, C3-C6 alicyclic, substituted C3-C6 alicyclic, C3-C6 heterocyclic, and substituted C3-C6 heterocyclic; the substituents on the substituted aryl, substituted C3-C6 alicyclic, and substituted C3-C6 heterocyclic groups are independently selected from C1-C3 straight-chain or branched alkyl.

4. The benzopyrazole compound according to claim 1, characterized in that, R1 is selected from substituted morpholino groups; The substituents are selected from C1-C3 straight-chain or branched alkyl groups.

5. The benzopyrazole compound according to claim 1, characterized in that, R1 is selected from 6. The benzopyrazole compound according to claim 1, characterized in that, R4 and R5 are independently selected from C1-C3 straight-chain or branched alkyl groups; R2 is selected from: R8 is selected from C1-C4 straight-chain or branched alkyl carbonyl groups and C1-C4 straight-chain or branched alkyl ester groups.

7. The benzopyrazole compound according to any one of claims 1-6, characterized in that, The benzopyrazole compound is a compound, optical isomer, or pharmaceutically usable salt of the following formula:

8. The method for preparing benzopyrazole compounds according to any one of claims 1-7, characterized in that, Includes the following steps: 5-Bromo-6-halo-1H-indole-3-carboxaldehyde undergoes ring-opening and ring-closing reactions sequentially to give 5-bromo-6-halo-1H-indazole-3-carboxaldehyde; then, it undergoes a Suzuki reaction with a borate ester to introduce a heterocyclic fragment of pyridine-piperazine or phenyl-piperazine at the 5-position; then, through a Knauwenger reaction, a thiazolidinedione functional group is introduced at the 3-position to give benzopyrazole compounds.

9. The use of the benzopyrazole compounds according to any one of claims 1-7 in the preparation of medicaments for treating or preventing acute kidney injury or in the preparation of AMPK activators.

10. A pharmaceutical composition for treating or preventing acute kidney injury, characterized in that, The pharmaceutical composition comprises a benzopyrazole compound according to any one of claims 1-7.

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