CN112174945A

CN112174945A - Indazole compound with anticancer effect and preparation method and application thereof

Info

Publication number: CN112174945A
Application number: CN201910589317.6A
Authority: CN
Inventors: 余洛汀
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-07-02
Filing date: 2019-07-02
Publication date: 2021-01-05
Anticipated expiration: 2039-07-02
Also published as: CN112174945B

Abstract

The invention relates to an indazole compound with an anticancer effect, a preparation method and application thereof, belonging to the field of medicines. The invention provides a compound shown as a formula I or a pharmaceutically acceptable salt thereof. Biological experiments show that the compound of the invention has obvious inhibition effect on BRD4 protein and is a potential cancer treatment drug targeting BRD 4. And, partIC for inhibiting proliferation of acute myeloid leukemia cells by differential compounds₅₀The value can reach below 2 mu M, and the cell can be blocked at G₀/G₁And effectively inhibit the transcription and expression of downstream genes. The application of the invention can provide a new drug selection for the clinical treatment of cancer.

。

Description

Indazole compound with anticancer effect and preparation method and application thereof

Technical Field

The invention relates to an indazole compound with an anticancer effect, a preparation method and application thereof, belonging to the field of medicines.

Background

Cancer is one of the major public health problems recognized in the world, and the traditional means for treating cancer include surgical resection, radiotherapy, chemotherapy and the like. Epigenetic targeted therapy has achieved significant efficacy in the treatment of certain types of cancer starting in the late 90 s, as effective as chemotherapy, but with much reduced side effects compared to chemotherapy, and is currently being investigated for fire fever and a very promising area. Epigenetic refers to the phenomenon that changes in gene expression do not involve changes in DNA sequence, but can be stably inherited as cells divide and proliferate. When affected by external environment, the normal epigenetic state of the cell may be disrupted, resulting in abnormal activation of the oncogene or inactivation of the tumor suppressor gene, promoting tumor formation. The posttranslational modification of histone is an important component of epigenetic regulation, wherein, lysine acetylation modification on histone is an important mechanism for regulating chromatin structure and activating transcription, and is one of important action mechanisms for survival and growth of tumor cells. The acetylation level of histone is commonly regulated by multiple enzyme families and proteins, and the whole regulation process is completed through a ' write ', read and erase ' mechanism. The key to this process is the recognition of specific histone post-translational modifications by the reader bromodomain protein. Abnormal acetylation levels are closely associated with the occurrence of various diseases, such as: inflammation, immune and metabolic diseases, various malignant tumors, and the like. Thus, the bromodomain and terminal domain (BET) subfamilies have become a popular anti-tumor target as proteins that play an important role in epigenetic regulation of gene transcription. Bromodomain-containing protein 4(BRD4) is one of the important members of the BET family and has been considered as the most attractive candidate target for targeted gene transcription therapy in several cancers.

Disclosure of Invention

The invention aims to provide an indazole compound with an anticancer effect, and a preparation method and application thereof.

The present invention provides a compound of formula i or a pharmaceutically acceptable salt thereof:

R₁、R₂、R₃、R₄among which only one is selected from

The remaining three are independently selected from-H, halogen, alkyl, alkoxy, -NH₂、-NO₂or-CN, wherein R₁₀、R₁₁、R₁₂、R₁₃、R₁₄Independently selected from-H or alkyl;

R₅is selected from-H or-X-R₇Wherein X is-CH₂-or is absent, R₇Selected from aryl, cycloalkyl, acyl or alkyl;

R₆is selected from-H or-NH-R₈Wherein R is₈Is an aryl group.

Further, R₁、R₂、R₃、R₄Among which only one is selected from

The other three are independently selected from-H, halogen, unsubstituted C1-C6 alkyl, halogen substituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, halogen substituted C1-C6 alkyl, -NH₂、-NO₂or-CN, wherein R₁₀、R₁₁、R₁₂、R₁₃、R₁₄Independently selected from-H or unsubstituted C1-C6 alkyl.

Preferably, R₁、R₂、R₃、R₄Among which only one is selected from

The other three are independently selected from-H, halogen, -OCH₃or-CH₃Wherein R is₁₀、R₁₁、R₁₂、R₁₃、R₁₄Independently selected from-H, -CH₃or-CH₂CH₃。

Preferably, R₁、R₂、R₃、R₄Middle R₂Or R₃Is composed of

Or R₃Is composed of

The other three are independently selected from-H or-OCH₃Wherein R is₁₀、R₁₁、R₁₄Are all-CH₃，R₁₂is-H, R₁₃is-CH₃or-CH₂CH₃。

Most preferably, R₃Is composed of

R₁、R₂、R₄Independently selected from-H or-OCH₃。

Further, R₅Is selected from-H or-X-R₇Wherein X is-CH₂-or is absent, R₇Selected from 5-to 14-membered aryl, 3-to 6-membered cycloalkyl,

Or C1-C6 alkyl, wherein R₉Is C1-C6 alkoxy.

Preferably, R₇Selected from 6-membered aryl, 3-to 6-membered cycloalkyl,

Or C1-C6 alkyl, wherein R₉Is C1-C6 alkoxy.

Preferably, R₇Selected from substituted or unsubstituted phenyl, 3-6 membered cycloalkyl or

Wherein R is₉is-OCH₂CH₃。

Preferably, the substituted phenyl group containsAt least one substituent selected from the group consisting of: halogen, alkyl, alkoxy, -NO₂。

Preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of: halogen, unsubstituted C1-C6 alkyl, halogen substituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, halogen substituted C1-C6 alkyl, -NO₂。

Preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of: -F, -CH₃、-CF₃、-OCH₃、-NO₂。

Preferably, the 3-6 membered cycloalkyl group is selected from

Most preferably, R₅is-X-R₇Wherein X is-CH₂-，R₇Is selected from

Further, R₆Is selected from-H or-NH-R₈Wherein R is₈Is a 5-to 14-membered aryl group.

Preferably, R₈Is 6-membered aryl.

Preferably, R₈Is a substituted or unsubstituted phenyl group.

Preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of: halogen, alkyl, alkoxy or-CN.

Preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of: halogen, unsubstituted C1-C6 alkyl, halogen substituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, halogen substituted C1-C6 alkoxy or-CN.

Preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of: -F, -Cl, -OCH₃、-CF₃、

or-CN.

Most preferably, R₆is-H.

Further, the compound is selected from:

the invention provides a preparation method of the compound or the pharmaceutically acceptable salt thereof, which comprises the following steps:

when R is₁、R₂、R₃、R₄Among them and only one is

In time, the method one is adopted: carrying out coupling reaction on the raw materials SM-1 and SM-2 to obtain a target product:

wherein R is_1a、R_2a、R_3a、R_4aOne and only one is halogen, and the other three are R at the same position of benzene ring in the product₁、R₂、R₃、R₄The groups are the same; a is

R₁₅、R₁₆Independently selected from-H or alkyl, or joined to form cycloalkyl;

or, when R is₁、R₂、R₃、R₄Among them and only one is

Then, the method II is adopted: carrying out coupling reaction on the raw materials SM-3 and SM-4 to obtain a target product:

wherein R is_1b、R_2b、R_3b、R_4bOne and only one is

The other three R are at the same position of benzene ring in the product₁、R₂、R₃、R₄The radicals R are the same₁₇、R₁₈Independently selected from-H or alkyl, or joined to form a cycloalkyl group, Y is halogen.

Preferably, R₁₅、R₁₆Independently selected from-H or unsubstituted C1-C6 alkyl, or joined to form a 5-membered cycloalkyl group.

Most preferably, the halogen is-Br and A is

Preferably, R₁₇、R₁₈Independently selected from-H or unsubstituted C1-C6 alkyl, or joined to form a 5-membered cycloalkyl group.

Most preferably, R_1b、R_2b、R_3b、R_4bOne and only one is

Y is-Br.

Further, the preparation method meets at least one of the following conditions:

in the first method and the second method, a palladium catalyst and alkali are added into a reaction system;

preferably, the palladium catalyst is Pd (dppf) Cl₂；

Preferably, the base is sodium carbonate;

preferably, SM-2: SM-1: pd (C)dppf)Cl₂: the molar ratio of sodium carbonate is 1: 1.5: 0.05: 3;

preferably, SM-3: SM-4: pd (dppf) Cl₂: the molar ratio of sodium carbonate is 1: 1.5: 0.05: 3;

the reaction solvent is a mixed solvent of dioxane and water;

preferably, the ratio of dioxane: the volume ratio of water is 2: 1;

carrying out reaction under a protective atmosphere;

the reaction temperature is 90 ℃;

the invention provides application of the compound or the pharmaceutically acceptable salt thereof in preparing BRD4 inhibitor medicines.

The invention provides application of the compound or the pharmaceutically acceptable salt thereof in preparing a medicament for treating and/or preventing cancer.

Further, the medicament is a medicament for treating and/or preventing leukemia.

Preferably, the leukemia is acute myeloid leukemia.

The invention provides a pharmaceutical composition for preventing and treating cancer, which is a preparation prepared by taking the compound or pharmaceutically acceptable salt thereof as an active ingredient and adding acceptable auxiliary materials or auxiliary ingredients.

Further, the agent is an agent for treating and/or preventing leukemia.

Preferably, the leukemia is acute myeloid leukemia.

Definition of terms:

the compounds and derivatives provided by the present invention may be named according to the IUPAC (international union of pure and applied chemistry) or CAS (chemical abstracts service, Columbus, OH) naming system.

The term "alkyl" is a radical of a straight or branched chain saturated hydrocarbon group. C₁～C₆Examples of alkyl groups include, but are not limited to, methyl (C)₁) Ethyl (C)₂) N-propyl (C)₃) Isopropyl (C)₃) N-butyl (C)₄) Tert-butyl (C)₄) Sec-butyl (C)₄) Isobutyl (C)₄) N-pentyl group (C)₅) 3-pentyl radical (C)₅) Pentyl group (C)₅) Neopentyl (C)₅) 3-methyl-2-butyl (C)₅) Tert-amyl (C)₅) And n-hexyl (C)₆). Unless otherwise indicated, each instance of alkyl is independently optionally substituted, i.e., unsubstituted or substituted with one or more substituents. "substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule. In some embodiments, said C₁～C₆Alkyl is C substituted by halogen (fluorine, chlorine, bromine, iodine)₁～C₆An alkyl group. At C₁～C₆In the case where an alkyl group is substituted with a substituent, the number of carbon atoms of the substituent is not counted in.

The term "alkoxy" refers to the group-OR, wherein R is alkyl as defined above. C₁～C₆Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1, 2-dimethylbutoxy. Said C is₁～C₆Alkoxy in the case where R is substituted with a substituent, the number of carbon atoms of the substituent is not counted in.

The term "aryl" refers to a radical of a 4n +2 aromatic ring system with or without heteroatoms in the aromatic ring system, wherein the heteroatoms are selected from nitrogen, oxygen and/or sulfur, among others. Unless otherwise indicated, each instance of an aryl group is independently optionally substituted, i.e., unsubstituted or substituted with one or more substituents.

The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, with or without heteroatoms, which may be a single ring structure or two or more rings, wherein the heteroatoms are selected from boron, phosphorus, sulfur, oxygen and/or nitrogen, and the like. Unless otherwise indicated, each instance of a cycloalkyl group is independently optionally substituted, i.e., unsubstituted or substituted with one or more substituents.

The term "acyl" refers to R '-c (o) -, where R' is an optional group.

The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.

The term "pharmaceutically acceptable salts" refers to acid and/or base salts of the compounds of the present invention with inorganic and/or organic acids and bases, and also includes zwitterionic salts (inner salts), and also includes quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound may be obtained by appropriately (e.g., equivalent) mixing the above compound with a certain amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization. The salt in the invention can be hydrochloride, sulfate, citrate, benzene sulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.

The pharmaceutically acceptable auxiliary material of the invention refers to a substance contained in a dosage form except for an active ingredient.

The pharmaceutically acceptable auxiliary components have certain physiological activity, but the addition of the components does not change the dominant position of the pharmaceutical composition in the disease treatment process, but only plays auxiliary effects, and the auxiliary effects are only the utilization of the known activity of the components and are auxiliary treatment modes which are commonly used in the field of medicine. If the auxiliary components are used in combination with the pharmaceutical composition of the present invention, the protection scope of the present invention should still be included.

The invention provides an indazole derivative with novel structure. Biological experiments show that the compounds have obvious inhibition effect on BRD4 protein and are potential cancer treatment drugs targeting BRD4. Furthermore, part of the compounds inhibit the IC of acute myeloid leukemia cell proliferation₅₀The value can reach below 2 mu M, and the cell can be blocked at G₀/G₁And effectively inhibit the transcription and expression of downstream genes. The application of the invention can provide a new drug selection for the clinical treatment of cancer.

Drawings

FIG. 1 is a graph showing the results of the cytotoxicity test in test example 1;

FIG. 2 is a graph showing the results of cell cycle measurements in test example 2;

FIG. 3 is a graph showing the results of Western blot analysis in test example 3.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 compound 5 a: preparation of 6- (3, 5-dimethyllisoxazol-4-yl) -N- (4-fluorophenyl) -1H-indazol-3-amine

Dissolving compound 1(1.0eq) and potassium hydroxide solid (2.0eq) in DMF, stirring at room temperature, and slowly adding elementary iodine I after complete dissolution₂(1.0eq), and the reaction is continued for 1-2 hours to obtain an intermediate 2. The yield was 85%.

Weighing the intermediate 2(1.0eq) and cesium carbonate (2.0eq) in a dry reaction flask, using dry DMF as a reaction solvent, and adding para-fluoroaniline liquid (1.2eq) into the reaction flask by using a liquid transfer gun. N is a radical of₂Replacement protection is carried out by pumping out water and O in the reaction flask₂And the anhydrous and oxygen-free environment is kept. Adding catalyst and ligand Pd (AcO)₂(0.1eq), Xantphos (0.1 eq). Then nitrogen replacement is carried out for many times. Transferring to an oil bath at 60 ℃ for reaction, detecting the reaction progress by TLC after 6-7 hours, and supplementing Pd (AcO) if the reaction is not completely carried out₂The reaction was continued with Xantphos. After the reaction is completed, filtering with diatomite, removing solvent by rotary evaporation, and using EA/H₂Extracting with O/saturated saline solution, drying with anhydrous magnesium sulfate, and performing column chromatography purification on the crude product obtained after rotary evaporation. The purified product is put into the next reaction.

The product obtained above (1.0eq) and 3, 5-dimethylisoxazoleboronic acid (1.5eq) were dissolved in a mixture of dioxane and water (dioxane: water 2:1) and stirred at room temperature. Sodium carbonate solids (3.0eq) were then added and stirred well. Finally weighing Pd (dppf) Cl₂(0.05eq) is added into the mixed solution, nitrogen is replaced and protected, and the mixed solution is transferred into an oil bath at the temperature of 90 ℃ for heating reaction for 6 to 7 hours. After TLC detection reaction is completed, filtering by using diatomite, and extracting by using water/ethyl acetate/saturated saline water; dried over anhydrous magnesium sulfate. And (3) distilling under reduced pressure to remove the solvent, and purifying the obtained crude product by column chromatography to obtain the target compound 5 a. The yield was 50%.

¹HNMR(400MHz,Chloroform-d)9.55(s,1H),7.60(dd,J＝8.3,0.8Hz,1H),7.41–7.35(m,2H),7.23(t,J＝1.1Hz,1H),7.04–6.99(m,2H),6.97(dd,J＝8.3,1.3Hz,1H),6.33(s,1H),2.43(s,3H),2.30(s,3H).

¹³CNMR(101MHz,Chloroform-d)165.56,158.77,156.40,145.81,141.85,138.46,138.44,129.85,121.13,119.90,118.36,118.28,116.79,115.81,115.58,114.44,110.38,11.61,10.84.

ESI-HRMS:323.1311,[M+1]⁺.

Example 2 compound 5 b: preparation of 6- (3,5-dimethylisoxazol-4-yl) -N- (4-fluoro-3-methoxy phenyl) -1- (tetrahydro-2H-pyran-2-yl) -1H-indazol-3-amine

1.0eq of intermediate 2 was dissolved in dichloromethane solvent and stirred at room temperature. 3, 4-dihydropyran (2.0eq) and p-toluenesulfonic acid (0.03eq) were then added slowly and left at room temperature overnight. After TLC detection reaction is completed, extracting with water/ethyl acetate/saturated saline; dried over anhydrous magnesium sulfate. And carrying out reduced pressure distillation to obtain a crude product, and carrying out column chromatography purification to obtain the target intermediate 3 a. The yield was 60%.

Putting the intermediate 3a (1.0eq) and cesium carbonate (2.0eq) in a dry reaction flask, using dry DMF as a reaction solvent, and adding m-methoxy-p-fluoroaniline (1.2eq) into the reaction flask by using a liquid-transferring gun. N is a radical of₂Replacement protection is carried out by pumping out water and O in the reaction flask₂And the anhydrous and oxygen-free environment is kept. Adding catalyst and ligand Pd (AcO)₂(0.1eq), Xantphos (0.1 eq). Then nitrogen replacement is carried out for many times. Transferring to an oil bath at 60 ℃ for reaction, detecting the reaction progress by TLC after 6-7 hours, and supplementing Pd (AcO) if the reaction is not completely carried out₂The reaction was continued with Xantphos. After the reaction is completed, filtering with diatomite, removing solvent by rotary evaporation, and using EA/H₂Extracting with O/saturated saline solution, drying with anhydrous magnesium sulfate, and performing column chromatography purification on the crude product obtained after rotary evaporation.

The resulting purified product and 3, 5-dimethylisoxazoleboronic acid (1.5eq) were dissolved in a mixture of dioxane and water (dioxane: water: 2:1) and stirred at room temperature. Sodium carbonate solids (3.0eq) were then added and stirred well. Finally weighing Pd (dppf) Cl₂(0.05eq) is added into the mixed solution, nitrogen is replaced and protected, and the mixed solution is transferred into an oil bath at the temperature of 90 ℃ for heating reaction for 6 to 7 hours. TLC detection reaction is completeThen, filtering by using diatomite, and extracting by using water/ethyl acetate/saturated saline; dried over anhydrous magnesium sulfate. And (3) distilling under reduced pressure to remove the solvent, and purifying the obtained crude product by column chromatography to obtain the target compound 5 b. The yield was 50%.

¹HNMR(400MHz,Chloroform-d)8.15(dd,J＝8.7,6.0Hz,1H),7.66(d,J＝8.2Hz,1H),7.33(s,1H),6.98(dd,J＝8.2,1.3Hz,1H),6.82(s,1H),6.73–6.64(m,2H),5.61(dd,J＝9.3,2.7Hz,1H),4.05(d,J＝11.4Hz,1H),3.95(s,3H),3.73(t,J＝9.5Hz,1H),2.62(q,J＝9.2,8.5Hz,1H),2.44(s,3H),2.30(s,3H),2.16(dd,J＝28.9,12.3Hz,2H),1.81–1.62(m,3H).

¹³CNMR(101MHz,Chloroform-d)165.39,165.27,158.61,158.51,135.94,133.55,130.21,129.46,128.81,127.71,123.71,122.28,121.69,116.82,116.08,109.62,67.37,56.00,52.86,29.38,25.21,22.72,11.55,11.53,10.77.

ESI-HRMS:437.1980,[M+1]⁺.

Example 3 compound 5 c: preparation of 6- (3,5-dimethylisoxazol-4-yl) -N- (4-fluorophenyl) -1- (tetrahydro-2H-pyran-2-yl) -1H-indazol-3-amine

Compound 5c was prepared according to a similar procedure as example 2.

¹HNMR(400MHz,Chloroform-d)7.54(dd,J＝8.3,0.8Hz,1H),7.41–7.36(m,2H),7.34(d,J＝1.1Hz,1H),7.04–6.98(m,2H),6.96(dd,J＝8.3,1.3Hz,1H),6.21(s,1H),5.61(dd,J＝9.5,2.6Hz,1H),4.06(d,J＝11.8Hz,1H),3.77–3.72(m,1H),2.58(d,J＝12.0Hz,1H),2.44(s,3H),2.30(s,3H),2.17(s,1H),2.11(d,J＝14.3Hz,1H),1.76(d,J＝7.9Hz,1H),1.67(s,1H).

¹³CNMR(101MHz,Chloroform-d)165.50,158.76,144.51,140.91,138.34,133.76,131.02,129.69,121.41,119.90,118.14,118.06,116.89,115.72,115.50,110.52,67.52,29.43,25.18,22.74,11.64,10.86.

ESI-HRMS:407.1883,[M+1]⁺.

Example 4 compound 5 d: preparation of 6- (3,5-dimethylisoxazol-4-yl) -N- (4-fluoro-2-methoxy phenyl) -1- (tetra-hydro-2H-pyran-2-yl) -1H-indazol-3-amine

Compound 5d was prepared according to a similar procedure as example 2.

¹HNMR(400MHz,Chloroform-d)8.15(dd,J＝8.7,6.0Hz,1H),7.66(dd,J＝8.3,0.8Hz,1H),7.33(t,J＝1.1Hz,1H),6.98(dd,J＝8.3,1.3Hz,1H),6.82(s,1H),6.73–6.65(m,2H),5.61(dd,J＝9.3,2.7Hz,1H),4.05(d,J＝11.3Hz,1H),3.95(s,3H),3.76–3.68(m,1H),2.62(q,J＝9.4,8.5Hz,1H),2.44(s,3H),2.30(s,3H),2.22–2.15(m,1H),2.12(d,J＝13.0Hz,1H),1.82–1.70(m,2H),1.67(s,1H).

¹³CNMR(101MHz,Chloroform-d)165.45,144.41,140.64,129.65,121.22,119.52,116.96,116.14,115.91,115.82,110.42,106.57,106.36,98.67,98.40,85.06,67.37,56.00,31.59,29.38,25.21,22.72,22.66,14.12,11.64.

ESI-HRMS:437.1979,[M+1]⁺.

Example 5 compound 5 e: preparation of N- (4-chlorophenylyl) -6- (3,5-dimethylisoxazol-4-yl) -1- (tetrahydro-2H-pyran-2-yl) -1H-indazol-3-amine

Compound 5e was prepared in 40% yield according to a similar procedure as example 2.

¹HNMR(400MHz,Chloroform-d)7.57(d,J＝8.3Hz,1H),7.37(d,J＝2.1Hz,1H),7.35(q,J＝1.7,1.2Hz,2H),7.24(d,J＝2.1Hz,1H),6.98(dd,J＝8.3,1.3Hz,1H),6.32(s,1H),5.62(dd,J＝9.4,2.7Hz,1H),4.09–4.02(m,1H),3.78–3.70(m,1H),2.58(dtd,J＝11.4,9.3,5.7Hz,1H),2.44(s,3H),2.30(s,3H),2.20–2.07(m,2H),1.82–1.71(m,3H).

¹³CNMR(101MHz,Chloroform-d)165.52,158.75,143.89,140.87,129.75,129.01,125.21,121.56,119.81,117.69,116.86,116.00,110.57,85.08,67.50,29.41,25.17,22.69,11.64,10.86.

ESI-HRMS:423.1591,[M+1]⁺.

Example 6 compound 5 f: preparation of 6- (3, 5-dimethyllisoxazol-4-yl) -N- (4-fluorophenyl) -1- (4-methyllbenzyl) -1H-indazol-3-amine

Sodium hydride solid (1.5eq) was weighed into a parallel reaction tube, replaced with nitrogen, and water vapor and oxygen were withdrawn. 1.0eq of intermediate 2 was dissolved in DMSO solvent, added to the reaction tube and stirred for 15 minutes. P-methylbenzyl bromide (1.2eq) was then added and nitrogen substitution was again carried out a number of times, maintaining an anhydrous and oxygen-free environment. The reaction was carried out at room temperature overnight. And detecting the reaction degree by TLC, adding water for quenching after the reaction is completed, and separating out a solid. Filtering with a baffle funnel, and washing with water for multiple times. Drying to obtain intermediate 4 a-2. The yield was 85%.

Taking the intermediate 4a-2(1.0eq) and cesium carbonate (2.0eq) in a dry reaction flask, taking dry DMF as a solvent, and taking para-fluoroaniline liquid (1.2eq) by a liquid transfer gun and adding the para-fluoroaniline liquid into the reaction flask. N is a radical of₂Replacement protection, and maintenance of anhydrous and oxygen-free environment. Adding catalyst Pd (AcO)₂(0.1eq) and ligand Xantphos (0.1 eq). Carrying out oil bath reaction at 60 ℃, detecting the reaction progress by TLC after 6-7 hours, and supplementing Pd (AcO) if the reaction is not completely carried out₂The reaction was continued with Xantphos. After the reaction is completed, filtering with diatomite, removing solvent by rotary evaporation, and using EA/H₂Extracting with O/saturated saline solution, drying with anhydrous magnesium sulfate, and performing column chromatography purification on the crude product obtained after rotary evaporation. The purified product is put into the next reaction.

The product obtained above (1.0eq) and 3, 5-dimethylisoxazoleboronic acid (1.5eq) were dissolved in a mixture of dioxane and water (dioxane: water 2:1) and stirred at room temperature. Sodium carbonate solid (3.0eq) and Pd (dppf) Cl were then added₂(0.05eq), protected by nitrogen, and transferring to a 90 ℃ oil bath for heating reaction for 6-7 hours. After TLC detection reaction is completed, filtering by using diatomite, and extracting by using water/ethyl acetate/saturated saline water; dried over anhydrous magnesium sulfate. And (3) distilling under reduced pressure to remove the solvent, and purifying the obtained crude product by column chromatography to obtain the target compound 5 f. The yield was 50%.

Example 7 compound 5 g: preparation of 6- (3, 5-dimethyllisoxazol-4-yl) -1- (4-methylbesyl) -N- (4- (trifluoromethylphenyl) -1H-indazol-3-amine

Compound 5g was prepared in 50% yield according to a similar method to example 6.

¹HNMR(400MHz,Chloroform-d)7.53(d,J＝8.4Hz,1H),7.45(d,J＝8.5Hz,2H),7.34(d,J＝8.5Hz,2H),7.11(d,J＝8.0Hz,2H),7.05(d,J＝8.1Hz,2H),6.99(d,J＝1.1Hz,1H),6.88(dd,J＝8.3,1.3Hz,1H),6.47(s,1H),5.40(s,2H),2.28(s,3H),2.24(s,3H),2.14(s,3H).

¹³CNMR(101MHz,Chloroform-d)165.50,158.64,145.65,142.44,140.59,137.73,133.59,129.48,129.46,127.43,126.56,126.52,121.02,119.94,116.70,115.71,115.29,110.03,53.03,21.09,11.55,10.76.

ESI-HRMS:477.1895,[M+1]⁺.

Example 8 compound 5 h: preparation of 4- ((6- (3, 5-dimethyllisozol-4-yl) -1- (4-methylbesyl) -1H-indazol-3-yl) amino) nitrile

Compound 5h was prepared in 50% yield according to a similar procedure as example 6.

¹HNMR(400MHz,Chloroform-d)7.55(d,J＝8.3Hz,1H),7.51–7.46(m,2H),7.34(dd,J＝8.9,2.2Hz,2H),7.12–7.04(m,4H),7.00(s,1H),6.90(dd,J＝8.3,1.3Hz,1H),5.41(s,2H),2.28(s,3H),2.24(s,3H),2.14(s,3H).

¹³CNMR(101MHz,Chloroform-d)165.53,158.60,146.53,141.72,140.51,137.82,133.61,133.43,129.64,129.49,127.41,121.29,119.86,119.74,116.63,115.71,115.59,110.10,102.26,53.10,21.10,11.56,10.76.

ESI-HRMS:434.1972,[M+1]⁺.

Example 9 compound 5 i: preparation of 6- (3, 5-dimethyllisoxazol-4-yl) -N- (4-methoxyphenyl) -1- (4-methyllbenzyl) -1H-indazol-3-amine

Compound 5i was prepared in 50% yield according to a similar method to example 6.

¹HNMR(400MHz,Chloroform-d)7.50(d,J＝8.3Hz,1H),7.34(d,J＝8.5Hz,2H),7.17(d,J＝7.9Hz,2H),7.11(d,J＝7.9Hz,2H),6.98(s,1H),6.91–6.83(m,3H),6.12(s,1H),5.42(s,2H),3.80(s,3H),2.34(s,3H),2.31(s,3H),2.20(s,3H).

¹³CNMR(101MHz,Chloroform-d)165.38,158.69,154.33,140.82,137.47,136.06,133.98,129.37,129.11,127.38,120.33,120.21,118.78,116.83,115.21,114.56,109.77,55.67,52.79,21.09,11.54,10.76.

ESI-HRMS:439.2123,[M+1]⁺.

Example 10 compound 5 j: preparation of 6- (3, 5-dimethyllisoxazol-4-yl) -N- (4-fluorophenyl) -1- (4-methoxybenzyl) -1H-indazol-3-amine

Sodium hydride solid (1.5eq) was weighed into a parallel reaction tube, purged with nitrogen, and water and oxygen were removed. Dissolving the intermediate 2(1.0eq) in DMSO solvent, adding p-methoxybenzyl bromide (1.2eq), and performing nitrogen replacement for multiple times while maintaining anhydrous and oxygen-free environment. The reaction was carried out at room temperature overnight. And detecting the reaction degree by TLC, adding water for quenching after the reaction is completed, and separating out a solid. Filtering with a baffle funnel, and washing with water for multiple times. Drying to obtain intermediate 4 a-3. The yield was 85%.

The intermediate 4a-3(1.0eq) and cesium carbonate (2.0eq), para-fluoroaniline liquid (1.2eq) were dissolved in dry DMF solvent. N is a radical of₂Replacement protection, and maintenance of anhydrous and oxygen-free environment. Adding catalyst Pd (AcO)₂(0.1eq) and ligand Xantphos (0.1 eq). Carrying out oil bath reaction at 60 ℃, and detecting the reaction progress by TLC after 6-7 hours. After the reaction is completed, filtering by diatomite, removing solvent by rotary evaporation, and using EA/H₂Extracting with O/saturated saline solution, drying with anhydrous magnesium sulfate, and performing column chromatography purification on the crude product obtained after rotary evaporation. The purified product is put into the next reaction.

The product obtained above (1.0eq) and 3, 5-dimethylisoxazoleboronic acid (1.5eq) were dissolved in a mixture of dioxane and water (dioxane: water 2:1) and stirred at room temperature. Sodium carbonate solid (3.0eq) and Pd (dppf) Cl were then added₂(0.05eq), protected by nitrogen, and transferring to a 90 ℃ oil bath for heating reaction for 6-7 hours. After TLC detection reaction is completed, filtering by using diatomite, and extracting by using water/ethyl acetate/saturated saline water; dried over anhydrous magnesium sulfate. And (4) distilling under reduced pressure to remove the solvent, and purifying the obtained crude product by column chromatography to obtain the target compound 5 j. The yield was 50%.

¹HNMR(400MHz,Chloroform-d)7.55(dd,J＝8.3,0.8Hz,1H),7.38–7.32(m,2H),7.24–7.20(m,2H),7.04–6.97(m,3H),6.90(dd,J＝8.3,1.3Hz,1H),6.86–6.82(m,2H),5.41(s,2H),3.77(s,3H),2.36(s,3H),2.22(s,3H).

¹³CNMR(101MHz,Chloroform-d)165.43,159.26,158.66,143.94,140.64,138.73,129.30,128.95,128.75,120.51,120.05,117.81,116.79,115.78,115.56,115.32,114.12,109.84,55.30,52.57,11.58,10.80.

ESI-HRMS:443.1875,[M+1]⁺.

Example 11 compound 5 k: preparation of 6- (3, 5-dimethyllisoxazol-4-yl) -N- (3-isopropylphenyl) -1- (4-methoxybenzyl) -1H-indazol-3-amine

Compound 5k was prepared in 50% yield according to a similar method to example 10.

¹HNMR(400MHz,Chloroform-d)7.59(dd,J＝8.3,0.8Hz,1H),7.26(s,1H),7.25–7.19(m,4H),7.04(t,J＝1.1Hz,1H),6.89(dd,J＝8.3,1.3Hz,1H),6.87–6.79(m,3H),6.24(s,1H),5.42(s,2H),3.77(s,3H),2.89(p,J＝6.9Hz,1H),2.36(s,3H),2.23(s,3H),1.27(s,3H),1.25(s,3H).

¹³CNMR(101MHz,Chloroform-d)165.40,159.24,158.69,150.07,143.84,142.60,140.62,129.13,129.07,129.02,128.85,120.39,120.32,118.78,116.84,115.67,114.57,114.09,113.89,109.76,55.29,52.54,34.21,23.97,11.59,10.81.

ESI-HRMS:467.2435,[M+1]⁺.

Example 12 compound 5 l: preparation of 5- (3,5-dimethylisoxazol-4-yl) -N- (4-fluorophenyl) -1- (tetrahydro-2H-pyran-2-yl) -1H-indazol-3-amine

Compound 5l was prepared in 50% yield according to a similar method to example 2.

¹HNMR(400MHz,Chloroform-d)7.55(dd,J＝8.6,0.8Hz,1H),7.42–7.36(m,2H),7.32(t,J＝1.1Hz,1H),7.26(d,J＝1.8Hz,1H),7.24(d,J＝1.5Hz,1H),7.03–6.95(m,2H),6.31(s,1H),5.63(dd,J＝9.6,2.6Hz,1H),4.12–4.02(m,1H),3.81–3.74(m,1H),2.67–2.53(m,1H),2.36(s,3H),2.22(s,3H),2.10(dq,J＝13.4,3.3Hz,1H),1.84–1.73(m,2H),1.70–1.55(m,2H).

¹³CNMR(101MHz,Chloroform-d)165.21,158.95,144.68,140.02,128.78,122.06,120.27,118.36,116.90,116.76,115.73,115.43,111.18,110.42,102.46,85.12,67.66,29.46,25.16,22.83,11.47,10.74.

ESI-HRMS:407.1871,[M+1]⁺.

Example 13 compound 5 m: preparation of 5- (3,5-dimethylisoxazol-4-yl) -N- (4-fluoro-2-methoxyphenyl) -1- (tetra-hydro-2H-pyran-2-yl) -1H-indazol-3-amine

Compound 5m was prepared in 50% yield according to a similar method to example 2.

¹HNMR(400MHz,Chloroform-d)8.14–8.08(m,1H),7.55(dd,J＝8.7,0.8Hz,1H),7.42(dd,J＝1.5,0.8Hz,1H),7.27(d,J＝1.7Hz,1H),7.24(d,J＝1.6Hz,1H),6.77(s,1H),6.72–6.63(m,2H),5.64(dd,J＝9.4,2.7Hz,1H),4.08(d,J＝11.0Hz,1H),3.94(s,3H),3.74(dd,J＝11.3,2.8Hz,1H),2.69–2.57(m,1H),2.39(s,3H),2.25(s,3H),2.21(dd,J＝8.5,4.1Hz,1H),2.15–2.07(m,1H),1.84–1.72(m,2H),1.70–1.61(m,1H).

¹³CNMR(101MHz,Chloroform-d)165.18,158.98,144.36,139.74,128.82,121.94,119.80,117.19,116.82,115.94,115.86,110.35,106.52,106.31,98.71,98.43,85.09,67.53,56.00,53.42,29.40,25.20,22.81,11.48,10.75.

ESI-HRMS:437.1977,[M+1]⁺.

Example 14 compound 5 n: preparation of 5- (3, 5-dimethyllisozol-4-yl) -N- (4-fluoro-2-methoxyphenyl) -1- (4-methoxybenzyl) -1H-indazol-3-amine

Compound 5n was prepared in 50% yield according to a similar method to example 10.

¹HNMR(400MHz,Chloroform-d)8.02(dd,J＝9.6,5.9Hz,1H),7.42(dd,J＝1.5,0.8Hz,1H),7.29(dd,J＝8.7,0.8Hz,1H),7.24(d,J＝2.1Hz,1H),7.18(dd,J＝8.6,1.5Hz,1H),6.89–6.82(m,2H),6.75–6.62(m,3H),5.43(s,2H),3.94(s,3H),3.78(s,3H),2.38(s,3H),2.24(s,3H).

¹³CNMR(101MHz,Chloroform-d)165.14,159.20,143.92,129.14,128.73,128.67,127.93,121.15,120.06,116.76,116.35,115.82,115.73,114.08,109.65,106.55,106.34,98.85,98.58,56.00,55.27,52.33,11.52,10.78.

ESI-HRMS:473.1974,[M+1]⁺.

Example 15 compound 5 o: preparation of 4- (1- (4-fluorobenzyl) -5-methoxy-1H-indazol-6-yl) -3, 5-dimethylisoxazole

The 6-bromo-5 methoxy-1H-indazole starting material was purchased, 1.0eq and sodium hydride (1.5eq) were dissolved in DMSO solvent, added to a reaction tube and then replaced with nitrogen, appropriately heated with a hair dryer, and water and O in the reaction tube were removed₂And stirred for 15 minutes. P-fluorobenzyl bromide (1.2eq) was then added and nitrogen substitution was again carried out several times, maintaining an anhydrous and oxygen-free environment. At room temperature overnight. And detecting the reaction degree by TLC, adding water for quenching after the reaction is completed, separating out solids, filtering by using a clapboard funnel, and washing by water for multiple times. The crude product obtained is purified by column chromatography and then put into the next reaction.

The product (1.0eq) and 3, 5-dimethylisoxazoleboronic acid (1.5eq) were dissolved in a mixture of dioxane and water (dioxane: water: 2:1) and stirred at room temperature. Then adding carbonic acidSodium solid (3.0eq) and Pd (dppf) Cl₂(0.05eq), protected by nitrogen, and transferring to a 90 ℃ oil bath for heating reaction for 6-7 hours. After TLC detection reaction is completed, filtering by using diatomite, and extracting by using water/ethyl acetate/saturated saline water; dried over anhydrous magnesium sulfate. And (4) distilling under reduced pressure to remove the solvent, and purifying the obtained crude product by column chromatography to obtain the target compound 5 o. The yield was 60%.

¹HNMR(400MHz,Chloroform-d)7.96(s,1H),7.44(s,1H),7.25–7.19(m,2H),7.08–6.96(m,2H),6.70(s,1H),5.54(s,2H),3.79(s,3H),2.29(s,3H),2.14(s,3H).

¹³CNMR(101MHz,Chloroform-d)165.90,160.06,157.50,140.43,133.60,132.49,132.45,128.98,128.90,123.79,118.69,115.85,115.64,115.35,113.42,89.63,55.48,52.20,11.52,10.68.

ESI-HRMS:352.1446,[M+1]⁺.

EXAMPLE 16 preparation of Compound 11a 5- (1- (4-fluorobenzyl) -1H-indazol-6-yl) -1-methyl-3- (methylamino) pyridine-2(1H) -one

Dissolving compound 1(1.0eq) and sodium hydride solid (1.5eq) in DMSO solvent, adding into a reaction tube, placing in a parallel reaction tube, replacing with nitrogen, and extracting water vapor and O in the reaction tube₂Then, p-fluorobenzyl bromide (1.2eq) was added thereto, and nitrogen substitution was performed again a plurality of times while maintaining an anhydrous and oxygen-free atmosphere. At room temperature overnight. And detecting the reaction degree by TLC, adding water for quenching after the reaction is completed, and separating out a solid. Filtering with a baffle funnel, washing with water for several times, and drying. The obtained product is put into the next reaction.

The product obtained above (1.0eq) and intermediate 9a (1.5eq), sodium carbonate solid (3.0eq) and Pd (dppf) Cl₂(0.05eq) was dissolved in a mixed solvent of dioxane and water (dioxane: water ═ water)2:1), transferring to a 90 ℃ oil bath for heating and stirring under the protection of nitrogen, and continuing the reaction for 6-7 hours. After TLC detection reaction is completed, filtering by using diatomite, and extracting by using water/ethyl acetate/saturated saline water; dried over anhydrous magnesium sulfate. The solvent was removed by distillation under reduced pressure, and the obtained crude product was purified by column chromatography to obtain the target compound 11 a. The yield was 70%.

¹HNMR(400MHz,Chloroform-d)8.04(d,J＝1.0Hz,1H),7.76(dd,J＝8.4,0.8Hz,1H),7.32(q,J＝1.0Hz,1H),7.27(d,J＝1.4Hz,1H),7.25(d,J＝1.4Hz,1H),7.22–7.17(m,2H),7.02–6.97(m,2H),6.84(d,J＝2.2Hz,1H),6.36(d,J＝2.3Hz,1H),5.60(s,2H),5.12(s,1H),3.64(s,3H),2.90(d,J＝4.9Hz,3H).

¹³CNMR(101MHz,Chloroform-d)140.09,139.65,137.18,133.56,130.95,128.97,128.89,123.49,121.88,121.60,121.34,120.31,115.79,115.58,106.14,105.75.

ESI-HRMS:363.1608,[M+1]⁺.

EXAMPLE 17 preparation of Compound 11b 5- (1- (4-methoxybenzyl) -1H-indazol-6-yl) -1-methyl-3- (methylamino) pyridine-2(1H) -one

Compound 11b was prepared in 70% yield according to a similar method to example 16.

¹HNMR(400MHz,Chloroform-d)8.03(d,J＝0.9Hz,1H),7.75(dd,J＝8.4,0.8Hz,1H),7.34(q,J＝1.1Hz,1H),7.25(dd,J＝8.4,1.5Hz,1H),7.21–7.16(m,2H),6.87–6.82(m,3H),6.39(d,J＝2.2Hz,1H),5.57(s,2H),3.76(s,3H),3.65(s,3H),2.90(s,3H).

¹³CNMR(101MHz,Chloroform-d)159.25,157.40,140.05,139.63,136.93,133.26,128.87,128.66,123.49,121.86,121.49,121.35,120.10,114.16,106.37,105.76,55.27,52.55,37.62,29.87.

ESI-HRMS:375.1808,[M+1]⁺.

EXAMPLE 18 preparation of the compound 11c 1-methyl-3- (methylamino) -5- (1- (4-methylbenzyl) -1H-indazol-6-yl) pyridine-2(1H) -one

Compound 11c was prepared in 70% yield according to a similar method to example 16.

¹HNMR(400MHz,Chloroform-d)8.04(d,J＝0.9Hz,1H),7.75(dd,J＝8.3,0.8Hz,1H),7.35(q,J＝1.1Hz,1H),7.24(d,J＝1.5Hz,1H),7.12(s,4H),6.85(d,J＝2.3Hz,1H),6.39(d,J＝2.3Hz,1H),5.60(s,2H),3.64(s,3H),2.90(s,3H),2.30(s,3H).

¹³CNMR(101MHz,Chloroform-d)157.40,140.14,139.62,137.54,136.94,133.78,133.28,129.43,127.24,123.48,121.85,121.49,121.36,120.10,106.36,105.77,52.81,37.61,29.87,21.09.

ESI-HRMS:359.1861,[M+1]⁺.

EXAMPLE 19 preparation of Compound 11d 5- (1- (3,5-dimethoxybenzyl) -1H-indazol-6-yl) -1-methyl-3- (methylimino) pyridine-2(1H) -one

Compound 11d was prepared in 70% yield according to a similar method to example 16.

¹HNMR(400MHz,Chloroform-d)8.04(d,J＝1.0Hz,1H),7.75(dd,J＝8.4,0.8Hz,1H),7.36(q,J＝1.1Hz,1H),7.24(d,J＝1.4Hz,1H),6.85(d,J＝2.2Hz,1H),6.39(d,J＝2.2Hz,1H),6.35(s,3H),5.56(s,2H),3.72(s,6H),3.64(s,3H),2.90(s,3H).

¹³CNMR(101MHz,Chloroform-d)161.13,157.40,140.26,139.63,139.19,137.05,133.41,123.47,121.82,121.50,121.39,120.21,106.33,105.75,105.35,99.36,55.31,53.01,37.61,29.85.

ESI-HRMS:405.1919,[M+1]⁺.

EXAMPLE 20 preparation of Compound 11e 5- (1- (3,4-difluorobenzyl) -1H-indazol-6-yl) -1-methyl-3- (methylimino) pyridine-2(1H) -one

Compound 11e was prepared in 70% yield according to a similar procedure as example 16.

¹HNMR(400MHz,Chloroform-d)8.06(d,J＝0.9Hz,1H),7.77(dd,J＝8.4,0.8Hz,1H),7.32(q,J＝1.1Hz,1H),7.28(dd,J＝8.4,1.4Hz,1H),7.10(dt,J＝10.1,8.2Hz,1H),6.98(d,J＝10.8Hz,2H),6.84(d,J＝2.2Hz,1H),6.37(d,J＝2.3Hz,1H),5.60–5.55(m,2H),3.64(s,3H),2.90(d,J＝5.3Hz,3H).

¹³CNMR(101MHz,Chloroform-d)157.41,140.14,139.68,137.49,133.90,123.51,123.20,123.16,123.13,123.10,121.69,121.40,120.53,117.68,117.50,116.36,116.18,105.90,105.57,51.74,37.62,29.84.

ESI-HRMS:381.1528,[M+1]⁺.

EXAMPLE 21 preparation of Compound 11f 5- (1- (4-fluoro-3-nitrobenzyl) -1H-indazol-6-yl) -1-methyl-3- (methyl-amino) pyridine-2(1H) -one

Compound 11f was prepared in 70% yield according to a similar procedure as example 16.

¹HNMR(400MHz,Chloroform-d)8.10–8.06(m,1H),7.96(dd,J＝7.0,2.3Hz,1H),7.78(d,J＝8.4Hz,1H),7.45(ddd,J＝8.7,4.2,2.4Hz,1H),7.37(s,1H),7.31(dd,J＝8.4,1.4Hz,1H),7.23(dd,J＝10.4,8.6Hz,1H),6.87(d,J＝2.3Hz,1H),6.39(d,J＝2.2Hz,1H),5.66(s,2H),3.65(s,3H),2.90(d,J＝3.4Hz,3H).

¹³CNMR(101MHz,CDCl₃)157.39,140.14,139.72,137.85,134.41,134.27,134.18,134.01,124.80,123.56,121.85,121.61,121.44,120.80,119.11,118.90,105.60,105.46,51.17,37.65,29.85.

ESI-HRMS:408.1459,[M+1]⁺.

EXAMPLE 22 preparation of 11g of the compound 1-methyl-3- (methylamino) -5- (1- (4- (trifluoromethyl) benzyl) -1H-indazol-6-yl) pyridin-2(1H) -one

Compound 11g was obtained in 70% yield according to a similar manner to example 16.

¹HNMR(400MHz,Chloroform-d)8.07(d,J＝0.9Hz,1H),7.78(d,J＝8.3Hz,1H),7.56(d,J＝8.1Hz,2H),7.27(s,4H),6.83(d,J＝2.2Hz,1H),6.36(d,J＝2.2Hz,1H),5.69(s,2H),3.63(s,3H),2.88(s,3H).

¹³CNMR(101MHz,Chloroform-d)157.35,140.82,140.23,139.65,137.43,133.90,127.44,127.37,126.80,125.79,125.75,123.51,121.86,121.71,121.36,120.52,105.96,105.74,52.28,37.65,29.81.

ESI-HRMS:413.1579,[M+1]⁺.

EXAMPLE 23 preparation of Compound 11H 5- (1- (3-fluoro-4-methoxybenzyl) -1H-indazol-6-yl) -1-methyl-3- (methyl-amino) pyridine-2(1H) -one

Compound 11h was prepared according to a similar procedure to example 16. The yield was 70%.

¹HNMR(400MHz,Chloroform-d)8.04(d,J＝1.0Hz,1H),7.75(dd,J＝8.4,0.8Hz,1H),7.23–7.14(m,3H),6.95(d,J＝1.3Hz,1H),6.93–6.88(m,2H),6.77(s,1H),5.54(s,2H),3.87(s,3H),3.84(s,3H),3.67(s,3H).

¹³CNMR(101MHz,Chloroform-d)158.94,141.64,140.04,136.53,133.57,127.02,124.25,124.22,123.50,123.10,123.06,121.73,120.27,120.12,119.57,113.57,113.11,106.02,56.31,56.29,38.56,38.25.

ESI-HRMS:393.1727,[M+1]⁺.

EXAMPLE 24 preparation of the compound 11i 3- (ethyllamino) -5- (1- (4-fluorobenzyl) -1H-indazol-6-yl) -1-methylpyridin-2(1H) -one

The product obtained above (1.0eq) and intermediate 9b (1.5eq), sodium carbonate solid (3.0eq) and Pd (dppf) Cl₂(0.05eq) is dissolved in a mixed solvent of dioxane and water (dioxane: water: 2:1), the mixture is transferred to a 90 ℃ oil bath for heating and stirring under the protection of nitrogen, and the reaction lasts for 6-7 hours. After TLC detection reaction is completed, filtering by using diatomite, and extracting by using water/ethyl acetate/saturated saline water; dried over anhydrous magnesium sulfate. And (3) distilling under reduced pressure to remove the solvent, and purifying the obtained crude product by column chromatography to obtain the target compound 11 i. The yield was 70%.

¹HNMR(400MHz,Chloroform-d)8.04(d,J＝1.0Hz,1H),7.75(dd,J＝8.4,0.8Hz,1H),7.30(q,J＝1.1Hz,1H),7.26–7.23(m,1H),7.22–7.17(m,2H),7.03–6.96(m,2H),6.83(d,J＝2.2Hz,1H),6.37(d,J＝2.2Hz,1H),5.59(s,2H),3.64(s,3H),3.18(dt,J＝7.5,4.7Hz,2H),1.32(t,J＝7.2Hz,3H).

¹³CNMR(101MHz,Chloroform-d)157.37,140.09,138.64,137.24,133.56,129.00,128.91,123.49,121.77,121.59,121.24,120.32,115.81,115.59,106.14,105.93,52.25,37.82,37.70,14.17.

ESI-HRMS:377.1778,[M+1]⁺.

EXAMPLE 25 preparation of the Compound 11j 5- (1- (3,5-dimethoxybenzyl) -1H-indazol-6-yl) -3- (ethyllamino) -1-methyl-pyridine-2 (1H) -one

Compound 11j was obtained in 70% yield according to a similar manner to example 24.

¹HNMR(400MHz,Chloroform-d)8.04(d,J＝1.0Hz,1H),7.74(dd,J＝8.4,0.8Hz,1H),7.34(q,J＝1.0Hz,1H),7.24(dd,J＝8.4,1.4Hz,1H),6.83(d,J＝2.2Hz,1H),6.40(d,J＝2.3Hz,1H),6.36–6.35(m,2H),5.55(s,2H),3.71(s,6H),3.64(s,3H),3.17(d,J＝5.6Hz,2H),1.32(t,J＝7.2Hz,3H).

¹³CNMR(101MHz,Chloroform-d)161.13,140.25,139.18,138.60,137.08,133.41,123.46,121.81,121.49,121.27,120.21,106.32,106.04,105.39,105.34,99.37,55.31,53.01,37.81,37.68,14.19.

ESI-HRMS:419.2071,[M+1]⁺.

EXAMPLE 26 preparation of the compound 11k 5- (1- (cyclohexymethyl) -1H-indazol-6-yl) -1-methyl-3- (methylimino) pyri-din-2(1H) -one

Compound 11k was prepared in 70% yield according to a similar procedure as example 16.

¹HNMR(400MHz,Chloroform-d)7.99(d,J＝0.9Hz,1H),7.74(dd,J＝8.4,0.8Hz,1H),7.38(q,J＝1.1Hz,1H),7.24(dd,J＝8.4,1.4Hz,1H),6.89(d,J＝2.3Hz,1H),6.45(d,J＝2.2Hz,1H),5.14(d,J＝5.6Hz,1H),4.23(d,J＝7.2Hz,2H),3.67(s,3H),2.93(d,J＝5.3Hz,3H),1.75–1.63(m,4H),1.30–0.98(m,10H).

¹³CNMR(101MHz,Chloroform-d)157.38,140.44,139.64,136.71,132.63,122.94,122.21,121.42,121.33,119.94,106.25,106.02,55.15,38.81,30.98,30.93,29.90,29.69,26.28,25.71,25.66.

ESI-HRMS:351.2177,[M+1]⁺.

EXAMPLE 27 preparation of 11l of the compound ethyl2- (6- (1-methyl-5- (methylamino) -6-oxo-1,6-dihydropyridin-3-yl) -1H-indazol-1-yl) acetate

Compound 11l was prepared in 70% yield according to a similar method to example 16.

ESI-HRMS:341.1601,[M+1]⁺.

EXAMPLE 28 preparation of the compound 11m 5- (1- (cyclopropropylethyl) -1H-indazol-6-yl) -1-methyl-3- (methylimino) pyridine-2(1H) -one

Compound 11m was prepared in 70% yield according to a similar method to example 16.

¹HNMR(400MHz,Chloroform-d)8.00(d,J＝1.0Hz,1H),7.75(dd,J＝8.4,0.8Hz,1H),7.44(t,J＝1.1Hz,1H),7.24(d,J＝1.4Hz,1H),6.90(d,J＝2.3Hz,1H),6.46(d,J＝2.2Hz,1H),4.31(d,J＝6.8Hz,2H),3.67(s,3H),2.93(s,3H),1.38(dddd,J＝11.2,8.1,4.9,1.2Hz,1H),0.64–0.58(m,2H),0.45(dt,J＝6.2,4.8Hz,2H).

¹³CNMR(101MHz,CDCl₃)157.41,139.92,139.67,136.78,132.76,123.23,122.01,121.46,121.32,119.95,106.15,105.77,53.47,37.62,29.91,11.30,4.00.

ESI-HRMS:309.1719,[M+1]⁺.

The beneficial effects of the invention are demonstrated by biological experiments below.

Test example 1BRD4(1, 2) protein Activity test and cell proliferation inhibition test

1.1BRD4(1, 2) protein Activity assay

The compounds of the present invention were tested by Shanghai Ruizi chemical company for BRD4 protein. The compounds tested inhibited BRD4(1, 2) protein activity using the HTRF method. The test method is as follows:

the compounds were diluted 5-fold from 25. mu.M, 5 concentration gradients were set, and 5. mu.l of the prepared BRD4(1, 2) protein solution, GST-tagged substrate, and 5. mu.l of [ Lys (5,8,12,16) AC were added to each well plate]H4(1-21) -biotin, SA acceptor, anti-GST donor solution, 5. mu.l of blank buffer was added to the control group. Incubate at room temperature for 3 hours under closed and dark conditions. Detecting the fluorescence emission at 665nm and 620nm by using a tester compatible with HTRF, calculating the inhibition rate of the compounds at different concentrations on BRD4(1, 2) protein, fitting by using Excel software to obtain an inhibition value, and fitting and calculating by using GraphPad to obtain IC₅₀The value is obtained. The experimental results are shown in tables 1 and 2.

1.2 cell proliferation inhibition assay

1.2.1 Experimental materials

Human acute myeloid leukemia cell MV4-11 and normal human stem cell LO2, thiazole blue tetrazolium bromide (MTT) were purchased from Sigma; DMEM medium and RPMI1640 medium, fetal bovine serum and trypsin were purchased from GibcoBRL; MV4-11 cell line was cultured in complete medium composed of 10% fetal bovine serum DMEM (FBS; Gibco, Auckland, N.Z.) and 1% antibiotics (100U/mL penicillin and 100U/mL streptomycin) at 37 ℃ in 5% CO₂Culturing in an incubator under the conditions.

1.1.2 Experimental principles and methods

The MTT method is also called MTT colorimetric method, and is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit the Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and an enzyme linked immunosorbent assay detector is used for measuring the light absorption value of formazan at the wavelength of 540 or 720nm, so that the quantity of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number. Therefore, the larger the number of living cells, the larger the amount of formazan precipitated, and the larger the absorbance measured by the microplate reader.

The stock solutions of the test compounds were diluted to different concentrations (3 duplicate wells) with a solution volume of 100. mu.L per well and added to 96-well plates. Collecting tumor cells in logarithmic growth phase, and culturing at 2.5-10 × 10³The density of individual cells/well was seeded into 96-well plates at a cell suspension volume of 100 μ L per well and cells were treated with 1% DMSO medium and pure medium as negative controls. The well plate was then placed at 37 ℃ and 5% CO₂Culturing in a cell culture incubator for 24 h. After formazan formation, the culture was stopped, 150. mu.L of LDMSO (adherent cells) was added to each well, and 50. mu.L of 20% SDS solution was directly added to suspension cells MV4-11, followed by shaking for 15-20 min. Detecting the absorbance (OD) of each well cell under the wavelength of 570nm by using a microplate reader_570nm) And taking the average value to record the result. Cell proliferation inhibition rate (control group OD)_570nmExperimental group OD_570nm) Control group OD_570nm100%. Half maximal Inhibitory Concentration (IC) was calculated for each compound by Graphpadprism5₅₀). Proliferation experiments were repeated three times for each tumor cell. The experimental results are shown in tables 1 and 2. The normal human stem cell LO2 was also subjected to a cytotoxicity test using the MTT method, and the results are shown in fig. 1.

TABLE 1 inhibitory Rate of the partial Compounds of the invention against BRD4(1, 2) and IC for inhibition of MV4-11₅₀

TABLE 2 IC inhibition of BRD4(1, 2) and MV4-11 by a portion of the compounds of the invention₅₀

Note: for IC₅₀The value: a represents IC₅₀Less than 2 μ M; b represents IC₅₀2-10 μ M; c represents IC₅₀Greater than 10. mu.M.

Inhibition for 1 μ M dose: a represents more than 40%; b represents between 20% and 40%; c represents less than 20%.

JQ1 is a positive control drug, a known BRD4 inhibitor, and has the following structural formula:

test example 2 cell cycle arrest test

MV4-11 log phase cells were collected and appropriate cell suspension concentrations were made to be about 10X 10 per well⁵Individual cells (2 mL in volume) were seeded into 6-well plates; then placing the mixture in an incubator for 24 hours, and incubating the mixture at 37 ℃ under 5% CO₂(ii) a The supernatant was added to 2mL of the media at different concentrations, and DMSO was used as a control. Then continuously placing the mixture at 37 ℃ and 5% CO₂Incubate for 48 hours. Final cell treatment: (1) collecting cells in each hole, numbering the cells according to concentration in sequence, loading the cells into a flow tube, centrifuging the cells at the temperature of 4 ℃ at 2000r/min for 3 minutes, and removing supernatant; (2) resuspending the cells with 3ml PBS, centrifuging in the same manner, and discarding PBS; (3) fixing the cells with 75% ethanol for 30min, centrifuging in the same manner, and removing the supernatant; (4) then 3ml PBS is used for resuspending the cells, the centrifugation is carried out by the same method, and PBS is discarded; (5) adding 1mL of PI dye solution containing TritonX-100, uniformly mixing, and keeping out of the sun for 20 minutes at room temperature; and (4) detecting by using a flow cytometer. Cell cycle was examined using Flow Cytometry (FCM), PI staining. MV4-11 cells were treated with compound 11e for 48h before harvesting, fixed with 75% ethanol and stained with sodium Propyliodide (PI). Data were analyzed using FlowJo software. The results of the experiment are shown in FIG. 2.

As can be seen from fig. 2: in comparison with DMSO control, MV4-11 cell line was treated with a low concentration (0.1. mu.M) of compound 11eThe phases showed significant changes. When the concentration is increased to 0.5. mu.M and 2.5. mu.M, the number of cells in the G0/G1 phase is increased. The above experimental results show that the compound 11e of the present invention can block cells at G₀/G₁And exhibits concentration dependence.

Test example 3 immunoblotting analysis experiment

MV4-11 cells were treated with compound 11e at various concentrations for 72 hours, and the cells were harvested and lysed with a RicA lysate (China, Beijing, Byunnan) supplemented with cocktail and a phosphatase inhibitor, after 30 minutes on ice, 13000 rpm was applied, centrifuged at 4 ℃ for 15 minutes, and the supernatant was collected. Protein concentration determination was performed using the BCA method. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to ensure consistent total protein loading for each sample, and then proteins were transferred to nitrocellulose (NC membrane) at 100V for 2 hours. NC membranes were blocked with 5% skim milk powder in TBS/T for 1.5 hours at room temperature, followed by 4 degrees overnight incubation of primary antibody. After incubation of horseradish peroxidase (HRP) conjugated secondary antibodies, the membrane was reacted with a chemiluminescent substrate and then exposed. The quality of western blot experiments was assessed using ImageJ to combine the results of three independent experiments. The results of the experiment are shown in FIG. 3.

As can be seen in FIG. 3, the expression of c-Myc decreased in a dose-dependent manner with Compound 11e, indicating that Compound 11e of the present invention is able to affect transcription and expression of downstream genes.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof:

R₁、R₂、R₃、R₄among which only one is selected from

R₆is selected from-H or-NH-R₈Wherein R is₈Is an aryl group.

2. The compound of claim 1, wherein: r₁、R₂、R₃、R₄Among which only one is selected from

The other three are independently selected from-H, halogen, unsubstituted C1-C6 alkyl, halogen substituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, halogen substituted C1-C6 alkyl, -NH₂、-NO₂or-CN, wherein R₁₀、R₁₁、R₁₂、R₁₃、R₁₄Independently selected from-H or unsubstituted C1-C6 alkyl;

preferably, R₁、R₂、R₃、R₄Among which only one is selected from

The other three are independently selected from-H, halogen, -OCH₃or-CH₃Wherein R is₁₀、R₁₁、R₁₂、R₁₃、R₁₄Independently selected from-H, -CH₃or-CH₂CH₃；

Preferably, R₁、R₂、R₃、R₄Middle R₂Or R₃Is composed of

Or R₃Is composed of

The other three are independently selected from-H or-OCH₃Wherein R is₁₀、R₁₁、R₁₄Are all-CH₃，R₁₂is-H, R₁₃is-CH₃or-CH₂CH₃；

Most preferably, R₃Is composed of

R₁、R₂、R₄Independently selected from-H or-OCH₃。

3. The compound of claim 1, wherein: r₅Is selected from-H or-X-R₇Wherein X is-CH₂-or is absent, R₇Selected from 5-to 14-membered aryl, 3-to 6-membered cycloalkyl,

Or C1-C6 alkyl, wherein R₉Is C1-C6 alkoxy;

preferably, R₇Selected from 6-membered aryl, 3-to 6-membered cycloalkyl,

Or C1-C6 alkyl, wherein R₉Is C1-C6 alkoxy;

Wherein R is₉is-OCH₂CH₃；

Preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of: halogen, alkyl, alkoxy, -NO₂；

Preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of: halogen, unsubstituted C1-C6 alkyl, halogen substituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, halogen-substituted C1-C6 alkyl, -NO₂；

Preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of: -F, -CH₃、-CF₃、-OCH₃、-NO₂；

Preferably, the 3-6 membered cycloalkyl group is selected from

Most preferably, R₅is-X-R₇Wherein X is-CH₂-，R₇Is selected from

4. The compound of claim 1, wherein: r₆Is selected from-H or-NH-R₈Wherein R is₈Is a 5-to 14-membered aryl group;

preferably, R₈Is a 6-membered aryl group;

preferably, R₈Is substituted or unsubstituted phenyl;

preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of: halogen, alkyl, alkoxy or-CN;

preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of: halogen, unsubstituted C1-C6 alkyl, halogen substituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, halogen substituted C1-C6 alkoxy or-CN;

or-CN;

most preferably, R₆is-H.

5. The compound according to any one of claims 1 to 4, wherein: the compound is selected from:

6. a process for the preparation of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, characterized in that: the method comprises the following steps:

when R is₁、R₂、R₃、R₄Among them and only one is

preferably, R₁₅、R₁₆Independently selected from-H or unsubstituted C1-C6 alkyl, or joined to form a 5-membered cycloalkyl;

most preferably, the halogen is-Br and A is

Or, when R is₁、R₂、R₃、R₄Among them and only one is

wherein R is_1b、R_2b、R_3b、R_4bOne and only one is

The other three R are at the same position of benzene ring in the product₁、R₂、R₃、R₄The radicals R are the same₁₇、R₁₈Independently selected from-H or alkyl, or joined to form cycloalkyl, Y is halogen;

preferably, R₁₇、R₁₈Independently selected from-H or unsubstituted C1-C6 alkyl, or joined to form a 5-membered cycloalkyl;

most preferably, R_1b、R_2b、R_3b、R_4bOne and only one is

Y is-Br.

7. The method of claim 6, wherein: at least one of the following is satisfied:

preferablyThe palladium catalyst is Pd (dppf) Cl₂；

Preferably, the base is sodium carbonate;

preferably, SM-2: SM-1: pd (dppf) Cl₂: the molar ratio of sodium carbonate is 1: 1.5: 0.05: 3;

the reaction solvent is a mixed solvent of dioxane and water;

preferably, the ratio of dioxane: the volume ratio of water is 2: 1;

carrying out reaction under a protective atmosphere;

the reaction temperature was 90 ℃.

8. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of BRD 4.

9. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment and/or prophylaxis of cancer.

10. Use according to claim 9, characterized in that: the medicament is a medicament for treating and/or preventing leukemia; preferably, the leukemia is acute myeloid leukemia.

11. The pharmaceutical composition for preventing and treating the cancer is characterized in that: the compound or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 is used as an active ingredient, and an acceptable auxiliary material or an auxiliary ingredient is added to prepare the preparation.

12. The pharmaceutical composition of claim 11, wherein: the preparation is an agent for treating and/or preventing leukemia; preferably, the leukemia is acute myeloid leukemia.