CN112174945B - Indazole compound with anticancer effect and preparation method and application thereof - Google Patents

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. 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.

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Description

Indazole compound with anticancer effect and preparation method and use thereof

technical field

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

Background technique

Cancer is recognized as one of the major public health problems in the world. Traditional methods of cancer treatment include surgical resection, radiotherapy, and chemotherapy. Epigenetic targeted therapy has been effective in the treatment of certain types of cancer since the late 1990s. It can treat cancer as effectively as chemotherapy, but the side effects are much reduced compared with chemotherapy. Therefore, it is currently being studied and is A very promising field. Epigenetics 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 the external environment, the normal epigenetic state of cells may be disrupted, resulting in abnormal activation of tumor-promoting genes or inactivation of tumor-suppressor genes, promoting tumor formation. Post-translational modification of histones is an important part of epigenetic regulation. Among them, lysine acetylation on histones is an important mechanism for regulating chromatin structure and activating transcription, and plays an important role in tumor cell survival and growth. one of the mechanisms. The acetylation level of histones is jointly regulated by a variety of enzyme families and proteins, and the entire regulatory process is completed through the "write (write), read (read), and erase (erase)" mechanism. The key to this process is the recognition of specific histone post-translational modifications by the reader bromodomain proteins. Abnormal levels of acetylation are closely related to the occurrence of various diseases, such as inflammation, immune and metabolic diseases, and various malignant tumors. Therefore, the bromodomain and terminal domain (BET) subfamily, as proteins that play an important role in the epigenetic regulation of gene transcription, have become a hotly studied antitumor target. Bromodomain-containing protein 4 (BRD4), one of the important members of the BET family, has been regarded as the most attractive candidate target for targeted gene transcription therapy in several cancers.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide indazole compounds with anti-cancer effect and preparation methods and uses thereof.

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

其余三个独立地选自-H、卤素、烷基、烷氧基、-NH₂、-NO₂或-CN，其中，R₁₀、R₁₁、R₁₂、R₁₃、R₁₄独立地选自-H或烷基；One and only one of R ₁ , R ₂ , R ₃ , and R ₄ is selected from

The remaining three are independently selected from -H, halogen, alkyl, alkoxy, -NH ₂ , -NO ₂ or -CN, wherein R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ are independently selected from -H or alkyl;

R ₅ is selected from -H or -XR ₇ , wherein X is -CH ₂ - or absent, and R ₇ is selected from aryl, cycloalkyl, acyl or alkyl;

R ₆ is selected from -H or -NH-R ₈ , wherein R ₈ is aryl.

其余三个独立地选自-H、卤素、未取代的C1～C6烷基、卤素取代的C1～C6烷基、未取代的C1～C6烷氧基、卤素取代的C1～C6烷基、-NH₂、-NO₂或-CN，其中，R₁₀、R₁₁、R₁₂、R₁₃、R₁₄独立地选自-H或未取代的C1～C6烷基。Further, among R ₁ , R ₂ , R ₃ and R ₄ , there is one and only one selected from

The remaining 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, - NH ₂ , -NO ₂ or -CN, wherein R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ and R ₁₄ are independently selected from -H or unsubstituted C1-C6 alkyl.

其余三个独立地选自-H、卤素、-OCH₃或-CH₃，其中，R₁₀、R₁₁、R₁₂、R₁₃、R₁₄独立地选自-H、-CH₃或-CH₂CH₃。Preferably, there is and only one of R ₁ , R ₂ , R ₃ , R ₄ is selected from

The remaining three are independently selected from -H, halogen, -OCH ₃ or -CH ₃ , wherein R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ are independently selected from -H, -CH ₃ or -CH _{2 CH3} .

或者R₃为

其余三个独立地选自-H或-OCH₃，其中，R₁₀、R₁₁、R₁₄均为-CH₃，R₁₂为-H，R₁₃为-CH₃或-CH₂CH₃。Preferably, among R ₁ , R ₂ , R ₃ and R ₄ , R ₂ or R ₃ is

or _R3 for

The remaining three are independently selected from -H or -OCH ₃ , wherein R ₁₀ , R ₁₁ and R ₁₄ are all -CH ₃ , R ₁₂ is -H, and R ₁₃ is -CH ₃ or -CH ₂ CH ₃ .

R₁、R₂、R₄独立地选自-H或-OCH₃。Most preferably, _R is

R ₁ , R ₂ , R ₄ are independently selected from -H or -OCH ₃ .

或C1～C6烷基，其中，R₉为C1～C6烷氧基。Further, R ₅ is selected from -H or -XR ₇ , wherein X is -CH ₂ - or does not exist, and R ₇ is selected from 5-14-membered aryl, 3-6 membered cycloalkyl,

or a C1-C6 alkyl group, wherein R ₉ is a C1-C6 alkoxy group.

或C1～C6烷基，其中，R₉为C1～C6烷氧基。Preferably, R ₇ is selected from 6-membered aryl, 3-6 membered cycloalkyl,

or a C1-C6 alkyl group, wherein R ₉ is a C1-C6 alkoxy group.

其中，R₉为-OCH₂CH₃。Preferably, R ₇ is selected from substituted or unsubstituted phenyl, 3-6 membered cycloalkyl or

wherein, R ₉ is -OCH ₂ CH ₃ .

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, -NO ₂ .

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 is selected from

Most preferably, R ₅ is -XR ₇ , wherein X is -CH ₂ -, and R ₇ is selected from

Further, R ₆ is selected from -H or -NH-R ₈ , wherein R ₈ is a 5- to 14-membered aryl group.

Preferably, _R8 is a 6-membered aryl group.

Preferably, _R8 is 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.

或-CN。Preferably, the substituted phenyl group contains at least one substituent selected from the group consisting of -F, -Cl, -OCH ₃ , -CF ₃ ,

or -CN.

Most preferably, _R6 is -H.

Further, the compound is selected from:

The present invention provides a preparation method of the compound or a pharmaceutically acceptable salt thereof, comprising the following steps:

时，采用方法一：原料SM-1与SM-2经偶联反应得到目标产物：When there is one and only one of R ₁ , R ₂ , R ₃ , and R ₄ is

, adopt method 1: the target product is obtained by coupling reaction of raw materials SM-1 and SM-2:

R₁₅、R₁₆独立地选自-H或烷基，或者相连形成环烷基；Among them, R _1a , R _2a , R _3a , R _4a have one and only one halogen, and the other three are the same as R ₁ , R ₂ , R ₃ , R ₄ groups in the same position of the benzene ring in the product; A is

R ₁₅ and R ₁₆ are independently selected from -H or alkyl, or are connected to form a cycloalkyl;

时，采用方法二：原料SM-3与SM-4经偶联反应得到目标产物：Or, when there is one and only one of R ₁ , R ₂ , R ₃ , and R ₄ is

When , adopt method two: raw material SM-3 and SM-4 obtain the target product through coupling reaction:

其余三个与产物中处于苯环相同位置的R₁、R₂、R₃、R₄基团相同，R₁₇、R₁₈独立地选自-H或烷基，或者相连形成环烷基，Y为卤素。Among them, R _1b , R _2b , R _3b , R _4b have and only one is

The remaining three groups are the same as R ₁ , R ₂ , R ₃ and R ₄ in the same position as the benzene ring in the product, R ₁₇ and R ₁₈ are independently selected from -H or alkyl, or connected to form a cycloalkyl, Y is halogen.

Preferably, R ₁₅ and R ₁₆ are independently selected from -H or unsubstituted C1-C6 alkyl, or are connected to form a 5-membered cycloalkyl.

Most preferably, the halogen is -Br, and A is

Preferably, R ₁₇ and R ₁₈ are independently selected from -H or unsubstituted C1-C6 alkyl, or are connected to form a 5-membered cycloalkyl.

Y为-Br。Most preferably, one and only one of R _1b , R _2b , R _3b , R _4b is

Y is -Br.

Further, the described preparation method satisfies at least one of the following:

Method 1 and method 2 both add palladium catalyst and alkali to the reaction system;

Preferably, the palladium catalyst is Pd(dppf)Cl ₂ ;

Preferably, the alkali is sodium carbonate;

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

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

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

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

The reaction is carried out under a protective atmosphere;

The reaction temperature is 90°C;

The present invention provides the use of the compound or a pharmaceutically acceptable salt thereof in the preparation of BRD4 inhibitor drugs.

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

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

Preferably, the leukemia is acute myeloid leukemia.

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

Further, the preparation is a preparation 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 can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

The term "alkyl" is a straight or branched chain saturated hydrocarbon radical. Examples of C ₁ -C ₆ 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 (C ₅ ), 3-pentyl (C ₅ ), pentyl (C ₅ ), new Amyl (C ₅ ), 3-methyl-2-butyl (C ₅ ), tert-amyl (C ₅ ) and n-hexyl (C ₆ ). Unless otherwise specified, each instance of alkyl is independently optionally substituted, ie, unsubstituted or substituted with one or more substituents. "Substitution" means that a hydrogen atom in a molecule is replaced by a different atom or molecule. In some embodiments, the C ₁ -C ₆ alkyl group is a C ₁ -C ₆ alkyl group substituted with halogen (fluorine, chlorine, bromine, iodine). In the case where the C ₁ -C ₆ alkyl group is substituted with a substituent, the number of carbon atoms of the substituent is not counted.

The term "alkoxy" refers to the group -OR, wherein R is an alkyl group as defined above. Examples of C ₁ -C ₆ alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy group, n-hexyloxy and 1,2-dimethylbutoxy. When the C ₁ -C ₆ alkoxy group is substituted with a substituent, the number of carbon atoms of the substituent is not counted.

The term "aryl" refers to a group 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 specified, each instance of an aryl group is independently optionally substituted, ie, unsubstituted or substituted with one or more substituents.

The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group containing or not containing a heteroatom, which may be a single ring structure or two or more rings, wherein the heteroatom is selected from the group consisting of boron, phosphorus, sulfur, oxygen and/or nitrogen, etc. Unless otherwise specified, each instance of cycloalkyl is independently optionally substituted, ie, 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 a carrier, vehicle, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that make up a pharmaceutical dosage form, and is physiologically Compatible with receptors.

The term "pharmaceutically acceptable salts" refers to the acid and/or base salts of the compounds of the present invention with inorganic and/or organic acids and bases, also including zwitterionic salts (inner salts), and also including quaternary ammonium salts, For example alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the above-mentioned compound with a certain amount of acid or base as appropriate (eg, equivalent). These salts may be precipitated in solution and collected by filtration, recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium. The salts described in the present invention can be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate of the compound salt, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.

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.

The pharmaceutically acceptable adjuvants in the present invention refer to the substances contained in the dosage form other than the active ingredients.

The pharmaceutically acceptable auxiliary component of the present invention has certain physiological activity, but the addition of the component will not change the dominance of the above-mentioned pharmaceutical composition in the process of disease treatment, but only exert auxiliary effects, and these auxiliary effects are only It is the utilization of the known activity of the ingredient and is a commonly used adjuvant therapy in the field of medicine. If the above-mentioned auxiliary components are used in combination with the pharmaceutical composition of the present invention, it should still belong to the protection scope of the present invention.

The present invention provides a class of indazole derivatives with novel structures. Biological experiments show that these compounds have a significant inhibitory effect on BRD4 protein and are potential cancer therapeutic drugs targeting BRD4. Moreover, the IC ₅₀ value of some compounds for inhibiting the proliferation of acute myeloid leukemia cells can reach below 2 μM, which can block the cells in the G ₀ /G ₁ phase and effectively inhibit the transcription and expression of downstream genes. The application of the present invention can provide a new drug choice for clinical treatment of cancer.

Description of drawings

Fig. 1 is a graph showing the results of cytotoxicity detection in Test Example 1;

Figure 2 is a graph showing the results of cell cycle detection in Test Example 2;

FIG. 3 is a graph showing the results of Western blot analysis in Test Example 3. FIG.

Detailed ways

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Example 1 Compound 5a: Preparation of 6-(3,5-dimethylisoxazol-4-yl)-N-(4-fluorophenyl)-1H-indazol-3-amine

Compound 1 (1.0eq) and solid potassium hydroxide (2.0eq) were dissolved in DMF, stirred at room temperature, and after complete dissolution, elemental iodine I ₂ (1.0eq) was slowly added, and the reaction continued for 1 to 2 hours to obtain intermediate 2 . Yield 85%.

Weigh intermediate 2 (1.0eq) and cesium carbonate (2.0eq) into a dry reaction flask, use dry DMF as a reaction solvent, and use a pipette to take p-fluoroaniline liquid (1.2eq) into the reaction flask. N ₂ replacement protection, and the water and O ₂ in the reaction flask were withdrawn to maintain an anhydrous and oxygen-free environment. Catalyst and ligands Pd(AcO) ₂ (0.1 eq), Xantphos (0.1 eq) were added. Nitrogen replacement was performed several times. Transfer to a 60° C. oil bath for reaction. After 6-7 hours, TLC detects the progress of the reaction. If the reaction is not complete, add Pd(AcO) ₂ and Xantphos to continue the reaction. After the reaction is completed, filter through celite, remove the solvent by rotary evaporation, extract with EA/H ₂ O/saturated brine, dry with anhydrous magnesium sulfate, and purify the crude product obtained after rotary evaporation by column chromatography. The purified product was obtained and put into the next reaction.

The product obtained above (1.0eq) and 3,5-dimethylisoxazoleboronic acid (1.5eq) were dissolved in a mixed solution of dioxane and water (dioxane:water=2:1), Stir at room temperature. Then solid sodium carbonate (3.0 eq) was added and stirred well. Finally, Pd(dppf)Cl ₂ (0.05eq) was weighed and added to the mixed solution, protected by nitrogen replacement, and transferred to a 90° C. oil bath for heating and reaction for 6 to 7 hours. After TLC detected that the reaction was complete, celite was filtered, extracted with water/ethyl acetate/saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained crude product was purified by column chromatography to obtain the target compound 5a. Yield 50%.

¹ HNMR(400MHz, Chloroform-d)δ9.55(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).

¹³ CNMR(101MHz,Chloroform-d)δ165.56,158.77,156.40,145.81,141.85,138.46,138.44,129.85,121.13,119.90,118.36,118.28,116.79,115.81,115.58,114.44,110.38,11.61,10.84.

ESI-HRMS: 323.1311, [M+1] ⁺ .

Example 2 Compound 5b: 6-(3,5-dimethylisoxazol-4-yl)-N-(4-fluoro-3-methoxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol- Preparation of 3-amine

1.0 eq of intermediate 2 was dissolved in dichloromethane solvent and stirred at room temperature. Then 3,4-dihydropyran (2.0 eq) and p-toluenesulfonic acid (0.03 eq) were added slowly and kept at room temperature overnight. After TLC detected that the reaction was complete, the mixture was extracted with water/ethyl acetate/saturated brine; dried over anhydrous magnesium sulfate. The crude product was obtained by distillation under reduced pressure and purified by column chromatography to obtain the target intermediate 3a. Yield 60%.

The intermediate 3a (1.0eq) and cesium carbonate (2.0eq) were placed in a dry reaction flask, and dry DMF was used as the reaction solvent, and m-methoxy-p-fluoroaniline (1.2eq) was added to the reaction flask with a pipette. N ₂ replacement protection, and the water and O ₂ in the reaction flask were withdrawn to maintain an anhydrous and oxygen-free environment. Catalyst and ligands Pd(AcO) ₂ (0.1 eq), Xantphos (0.1 eq) were added. Nitrogen replacement was performed several times. Transfer to a 60° C. oil bath for reaction. After 6-7 hours, TLC detects the progress of the reaction. If the reaction is not complete, add Pd(AcO) ₂ and Xantphos to continue the reaction. After the reaction is completed, filter through celite, remove the solvent by rotary evaporation, extract with EA/H ₂ O/saturated brine, dry with anhydrous magnesium sulfate, and purify the crude product obtained after rotary evaporation by column chromatography.

The obtained 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. Then solid sodium carbonate (3.0 eq) was added and stirred well. Finally, Pd(dppf)Cl ₂ (0.05eq) was weighed and added to the mixed solution, protected by nitrogen replacement, and transferred to a 90° C. oil bath for heating and reaction for 6 to 7 hours. After TLC detected that the reaction was complete, celite was filtered, extracted with water/ethyl acetate/saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained crude product was purified by column chromatography to obtain the target compound 5b. Yield 50%.

¹ HNMR (400MHz, Chloroform-d) δ8.15 (dd, J=8.7, 6.0 Hz, 1H), 7.66 (d, J=8.2 Hz, 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).

¹³ CNMR(101MHz,Chloroform-d)δ165.39,165.27,158.61,158.51,135.94,133.55,130.21,129.46,128.81,127.71,123.71,122.28,121.69,116.82,116.08,109.62,67.37,56.00,52.86,29.38,25.21 ,22.72,11.55,11.53,10.77.

ESI-HRMS: 437.1980, [M+1] ⁺ .

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

Compound 5c was prepared in a similar manner to Example 2.

¹ HNMR(400MHz, Chloroform-d)δ7.54(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).

¹³ CNMR(101MHz,Chloroform-d)δ165.50,158.76,144.51,140.91,138.34,133.76,131.02,129.69,121.41,119.90,118.14,118.06,116.89,115.72,115.50,110.52,67.52,29.43,25.18,22.74,11.64 , 10.86.

ESI-HRMS: 407.1883, [M+1] ⁺ .

Example 4 Compound 5d: 6-(3,5-dimethylisoxazol-4-yl)-N-(4-fluoro-2-methoxyphenyl)-1-(tetra-hydro-2H-pyran-2-yl)-1H- Preparation of indazol-3-amine

Compound 5d was prepared in a similar manner to Example 2.

¹ HNMR (400MHz, Chloroform-d) δ8.15 (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).

¹³ CNMR(101MHz,Chloroform-d)δ165.45,144.41,140.64,129.65,121.22,119.52,116.96,116.14,115.91,115.82,110.42,106.57,106.36,98.67,98.40,85.06,67.37,56.00,31.59,29.38,25.21 ,22.72,22.66,14.12,11.64.

ESI-HRMS: 437.1979, [M+1] ⁺ .

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

The compound 5e was prepared according to the method similar to Example 2, and the yield was 40%.

¹ HNMR (400MHz, Chloroform-d)δ7.57(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).

¹³ CNMR(101MHz,Chloroform-d)δ165.52,158.75,143.89,140.87,129.75,129.01,125.21,121.56,119.81,117.69,116.86,116.00,110.57,85.08,67.50,29.41,25.17,22.69,11.64,10.86.

ESI-HRMS: 423.1591, [M+1] ⁺ .

Example 6 Compound 5f: Preparation of 6-(3,5-dimethylisoxazol-4-yl)-N-(4-fluorophenyl)-1-(4-methylbenzyl)-1H-indazol-3-amine

Weigh the solid sodium hydride (1.5eq) into a parallel reaction tube, replace with nitrogen, and extract water vapor and oxygen. 1.0 eq of Intermediate 2 was dissolved in DMSO solvent, added to the reaction tube, and stirred for 15 minutes. Then p-methylbenzyl bromide (1.2 eq) was added, and repeated nitrogen replacements were performed to maintain an anhydrous and oxygen-free environment. React overnight at room temperature. The degree of reaction was detected by TLC. After the reaction was completed, water was added to quench, and a solid was precipitated. Filter with a baffle funnel and wash with water several times. Drying yields intermediate 4a-2. Yield 85%.

Take intermediate 4a-2 (1.0eq) and cesium carbonate (2.0eq) in a dry reaction flask, dry DMF as a solvent, and add p-fluoroaniline liquid (1.2eq) to the reaction flask with a pipette. _N2 replacement protection, maintain an anhydrous and oxygen-free environment. Catalyst Pd(AcO) ₂ (0.1 eq) and ligand Xantphos (0.1 eq) were added. The reaction was carried out in an oil bath at 60°C, and the progress of the reaction was detected by TLC after 6 to 7 hours. If the reaction was not complete, Pd(AcO) ₂ and Xantphos were added to continue the reaction. After the reaction is completed, filter through celite, remove the solvent by rotary evaporation, extract with EA/H ₂ O/saturated brine, dry with anhydrous magnesium sulfate, and purify the crude product obtained after rotary evaporation by column chromatography. The purified product was obtained and put into the next reaction.

The product obtained above (1.0eq) and 3,5-dimethylisoxazoleboronic acid (1.5eq) were dissolved in a mixed solution of dioxane and water (dioxane:water=2:1), Stir at room temperature. Then add sodium carbonate solid (3.0eq) and Pd(dppf)Cl ₂ (0.05eq), under nitrogen protection, transfer to 90°C oil bath for heating reaction for 6-7 hours. After TLC detected that the reaction was complete, celite was filtered, extracted with water/ethyl acetate/saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained crude product was purified by column chromatography to obtain the target compound 5f. Yield 50%.

Example 7 Compound 5g: Preparation of 6-(3,5-dimethylisoxazol-4-yl)-1-(4-methylbenzyl)-N-(4-(trifluoromethyl)phenyl)-1H-indazol-3-amine

The compound 5g was prepared according to the method similar to Example 6, and the yield was 50%.

¹ HNMR(400MHz, Chloroform-d)δ7.53(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).

¹³ CNMR(101MHz,Chloroform-d)δ165.50,158.64,145.65,142.44,140.59,137.73,133.59,129.48,129.46,127.43,126.56,126.52,121.02,119.94,116.70,115.71,115.29,110.03,53.03,21.09,11.55 , 10.76.

ESI-HRMS: 477.1895, [M+1] ⁺ .

Example 8 Compound 5h: Preparation of 4-((6-(3,5-dimethylisoxazol-4-yl)-1-(4-methylbenzyl)-1H-indazol-3-yl)amino)benzonitrile

The compound 5h was prepared according to the method similar to Example 6, and the yield was 50%.

¹ HNMR (400MHz, Chloroform-d)δ7.55(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).

¹³ CNMR(101MHz,Chloroform-d)δ165.53,158.60,146.53,141.72,140.51,137.82,133.61,133.43,129.64,129.49,127.41,121.29,119.86,119.74,116.63,115.71,115.59,110.10,102.26,53.10,21.10 ,11.56,10.76.

ESI-HRMS: 434.1972, [M+1] ⁺ .

Example 9 Compound 5i: Preparation of 6-(3,5-dimethylisoxazol-4-yl)-N-(4-methoxyphenyl)-1-(4-methylbenzyl)-1H-indazol-3-amine

Compound 5i was prepared in a similar manner to Example 6 in 50% yield.

¹ HNMR(400MHz, Chloroform-d)δ7.50(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).

¹³ CNMR(101MHz,Chloroform-d)δ165.38,158.69,154.33,140.82,137.47,136.06,133.98,129.37,129.11,127.38,120.33,120.21,118.78,116.83,115.21,114.56,109.77,55.67,52.79,21.09,11.54 , 10.76.

ESI-HRMS: 439.2123, [M+1] ⁺ .

Example 10 Compound 5j: Preparation of 6-(3,5-dimethylisoxazol-4-yl)-N-(4-fluorophenyl)-1-(4-methoxybenzyl)-1H-indazol-3-amine

The sodium hydride solid (1.5eq) was weighed into a parallel reaction tube, replaced with nitrogen, and water and oxygen were removed. The intermediate 2 (1.0 eq) was taken and dissolved in DMSO solvent, p-methoxybenzyl bromide (1.2 eq) was added, and repeated nitrogen replacement was performed to maintain an anhydrous and oxygen-free environment. React overnight at room temperature. The degree of reaction was detected by TLC. After the reaction was completed, water was added to quench, and a solid was precipitated. Filter through a baffle funnel and wash with water several times. Dry to obtain intermediate 4a-3. Yield 85%.

Take intermediate 4a-3 (1.0eq) and cesium carbonate (2.0eq), p-fluoroaniline liquid (1.2eq) and dissolve in dry DMF solvent. _N2 replacement protection, maintain an anhydrous and oxygen-free environment. Catalyst Pd(AcO) ₂ (0.1 eq) and ligand Xantphos (0.1 eq) were added. The reaction was carried out in an oil bath at 60°C, and the progress of the reaction was detected by TLC after 6 to 7 hours. After the reaction is complete, filter through celite, remove the solvent by rotary evaporation, extract with EA/H ₂ O/saturated brine, dry with anhydrous magnesium sulfate, and purify the crude product obtained after rotary evaporation by column chromatography. The purified product was obtained and put into the next reaction.

The product obtained above (1.0eq) and 3,5-dimethylisoxazoleboronic acid (1.5eq) were dissolved in a mixed solution of dioxane and water (dioxane:water=2:1), Stir at room temperature. Then add sodium carbonate solid (3.0eq) and Pd(dppf)Cl ₂ (0.05eq), under nitrogen protection, transfer to 90°C oil bath for heating reaction for 6-7 hours. After TLC detected that the reaction was complete, celite was filtered, extracted with water/ethyl acetate/saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained crude product was purified by column chromatography to obtain the target compound 5j. Yield 50%.

¹ HNMR (400MHz, Chloroform-d)δ7.55(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).

¹³ CNMR(101MHz,Chloroform-d)δ165.43,159.26,158.66,143.94,140.64,138.73,129.30,128.95,128.75,120.51,120.05,117.81,116.79,115.78,115.56,115.32,114.12,109.84,55.30,52.57,11.58 ,10.80.

ESI-HRMS: 443.1875, [M+1] ⁺ .

Example 11 Compound 5k: Preparation of 6-(3,5-dimethylisoxazol-4-yl)-N-(3-isopropylphenyl)-1-(4-methoxybenzyl)-1H-indazol-3-amine

Compound 5k was prepared in a similar manner to Example 10 in 50% yield.

¹ HNMR (400MHz, Chloroform-d) δ7.59 (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).

¹³ CNMR(101MHz,Chloroform-d)δ165.40,159.24,158.69,150.07,143.84,142.60,140.62,129.13,129.07,129.02,128.85,120.39,120.32,118.78,116.84,115.67,114.57,114.09,113.89,109.76,55.29 ,52.54,34.21,23.97,11.59,10.81.

ESI-HRMS: 467.2435, [M+1] ⁺ .

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

The compound 51 was prepared according to the method similar to Example 2, and the yield was 50%.

¹ HNMR (400MHz, Chloroform-d)δ7.55(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, 3.3 Hz, 1H), 1.84–1.73 (m, 2H), 1.70–1.55 (m, 2H).

¹³ CNMR(101MHz,Chloroform-d)δ165.21,158.95,144.68,140.02,128.78,122.06,120.27,118.36,116.90,116.76,115.73,115.43,111.18,110.42,102.46,85.12,67.66,29.46,25.16,22.83,11.47 , 10.74.

ESI-HRMS: 407.1871, [M+1] ⁺ .

Example 13 Compound 5m: 5-(3,5-dimethylisoxazol-4-yl)-N-(4-fluoro-2-methoxyphenyl)-1-(tetrahy-dro-2H-pyran-2-yl)-1H- Preparation of indazol-3-amine

Compound 5m was prepared in a similar manner to Example 2 in 50% yield.

¹ HNMR (400MHz, Chloroform-d) δ 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).

¹³ CNMR(101MHz,Chloroform-d)δ165.18,158.98,144.36,139.74,128.82,121.94,119.80,117.19,116.82,115.94,115.86,110.35,106.52,106.31,98.71,98.43,85.09,67.53,56.00,53.42,29.40 ,25.20,22.81,11.48,10.75.

ESI-HRMS: 437.1977, [M+1] ⁺ .

Example 14 Compound 5n: Preparation of 5-(3,5-dimethylisoxazol-4-yl)-N-(4-fluoro-2-methoxyphenyl)-1-(4-methoxybenzyl)-1H-indazol-3-amine

Compound 5n was prepared in a similar manner to Example 10 in 50% yield.

¹ HNMR (400MHz, Chloroform-d) δ8.02 (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).

¹³ CNMR(101MHz,Chloroform-d)δ165.14,159.20,143.92,129.14,128.73,128.67,127.93,121.15,120.06,116.76,116.35,115.82,115.73,114.08,109.65,106.55,106.34,98.85,98.58,56.00,55.27 ,52.33,11.52,10.78.

ESI-HRMS: 473.1974, [M+1] ⁺ .

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

Purchase the raw material of 6-bromo-5methoxy-1H-indazole, dissolve it (1.0eq) and sodium hydride (1.5eq) in DMSO solvent, add it to the reaction tube, replace it with nitrogen, heat it properly with a hair dryer, and extract it The water and _O2 in the reaction tube were stirred for 15 minutes. Subsequently, p-fluorobenzyl bromide (1.2 eq) was added and repeated nitrogen replacements were performed to maintain an anhydrous and oxygen-free environment. overnight at room temperature. The degree of reaction was detected by TLC. After the reaction was completed, water was added to quench, and the solid was precipitated and filtered with a baffle funnel, and washed with water for several times. The obtained crude product was purified by column chromatography and put into the next reaction.

The above product (1.0eq) and 3,5-dimethylisoxazoleboronic acid (1.5eq) were dissolved in a mixture of dioxane and water (dioxane:water=2:1), stirred at room temperature . Then add sodium carbonate solid (3.0eq) and Pd(dppf)Cl ₂ (0.05eq), under nitrogen protection, transfer to 90°C oil bath for heating reaction for 6-7 hours. After TLC detected that the reaction was complete, celite was filtered, extracted with water/ethyl acetate/saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained crude product was purified by column chromatography to obtain the target compound 5o. Yield 60%.

¹ HNMR(400MHz, Chloroform-d)δ7.96(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).

¹³ CNMR(101MHz,Chloroform-d)δ165.90,160.06,157.50,140.43,133.60,132.49,132.45,128.98,128.90,123.79,118.69,115.85,115.64,115.35,113.42,89.63,55.48,52.20,11.52,10.68.

ESI-HRMS: 352.1446, [M+1] ⁺ .

Example 16 Preparation of Compound 11a: 5-(1-(4-fluorobenzyl)-1H-indazol-6-yl)-1-methyl-3-(methylamino)pyridin-2(1H)-one

Dissolve compound 1 (1.0eq) and solid sodium hydride (1.5eq) in DMSO solvent, add them into a reaction tube, place in a parallel reaction tube, replace with nitrogen, extract the water vapor and O ₂ in the reaction tube, and then add p-fluorobenzyl bromide (1.2eq), and repeated nitrogen replacement for several times to maintain an anhydrous and oxygen-free environment. overnight at room temperature. The degree of reaction was detected by TLC. After the reaction was completed, water was added to quench, and a solid was precipitated. Filter with a baffle funnel, wash with water several times, and dry. The obtained product was 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) were dissolved in a mixed solvent of dioxane and water (two Oxane:water=2:1), nitrogen protection, transfer to a 90°C oil bath for heating and stirring, and the reaction lasts for 6-7 hours. After TLC detected that the reaction was complete, celite was filtered, extracted with water/ethyl acetate/saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained crude product was purified by column chromatography to obtain the target compound 11a. Yield 70%.

¹ HNMR (400MHz, Chloroform-d) δ8.04 (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).

¹³ CNMR (101MHz, Chloroform-d) δ140.09, 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, 106.75.

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 a similar manner to Example 16 in 70% yield.

¹ HNMR (400MHz, Chloroform-d) δ8.03 (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).

¹³ CNMR(101MHz,Chloroform-d)δ159.25,157.40,140.05,139.63,136.93,133.26,128.87,128.66,123.49,121.86,121.49,121.35,120.10,114.16,106.37,105.76,55.27,52.55,37.62,29.87.

ESI-HRMS: 375.1808, [M+1] ⁺ .

Example 18 Preparation of Compound 11c: 1-methyl-3-(methylamino)-5-(1-(4-methylbenzyl)-1H-indazol-6-yl)pyridine-2(1H)-one

Compound 11c was prepared in a similar manner to Example 16 in 70% yield.

¹ HNMR (400MHz, Chloroform-d) δ 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).

¹³ CNMR(101MHz,Chloroform-d)δ157.40,140.14,139.62,137.54,136.94,133.78,133.28,129.43,127.24,123.48,121.85,121.49,121.36,120.10,106.36,105.77,52.81,37.61,29.87,21.09.

ESI-HRMS: 359.1861, [M+1] ⁺ .

Example 19 Compound 11d: Preparation of 5-(1-(3,5-dimethoxybenzyl)-1H-indazol-6-yl)-1-methyl-3-(methylamino)pyridine-2(1H)-one

Compound 11d was prepared in a similar manner to Example 16 in 70% yield.

¹ HNMR (400MHz, Chloroform-d) δ8.04 (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).

¹³ CNMR(101MHz,Chloroform-d)δ161.13,157.40,140.26,139.63,139.19,137.05,133.41,123.47,121.82,121.50,121.39,120.21,106.33,105.75,105.35,99.36,55.31,53.01,37.61,29.85.

ESI-HRMS: 405.1919, [M+1] ⁺ .

Example 20 Compound 11e: Preparation of 5-(1-(3,4-difluorobenzyl)-1H-indazol-6-yl)-1-methyl-3-(methylamino)pyridine-2(1H)-one

Compound 11e was prepared in a similar manner to Example 16 in 70% yield.

¹ HNMR (400MHz, Chloroform-d) δ8.06 (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).

¹³ CNMR(101MHz,Chloroform-d)δ157.41,140.14,139.68,137.49,133.90,123.51,123.20,123.16,123.13,123.10,121.69,121.40,120.53,117.68,117.50,116.36,116.18,105.90,105.57,51.74,37.62 , 29.84.

ESI-HRMS: 381.1528, [M+1] ⁺ .

Example 21 Compound 11f: 5-(1-(4-fluoro-3-nitrobenzyl)-1H-indazol-6-yl)-1-methyl-3-(methyl-amino)pyridin-2(1H)-one preparation

Compound 11f was prepared in a similar manner to Example 16 in 70% yield.

¹ HNMR (400MHz, Chloroform-d) δ 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).

¹³ CNMR(101MHz,CDCl ₃ )δ157.39,140.14,139.72,137.85,134.41,134.27,134.18,134.01,124.80,123.56,121.85,121.61,121.44,120.80,119.11,118.90,105.60,105.46,51.17,37.65,29.85.

ESI-HRMS: 408.1459, [M+1] ⁺ .

Example 22 Preparation of compound 11g: 1-methyl-3-(methylamino)-5-(1-(4-(trifluoromethyl)benzyl)-1H-indazol-6-yl)pyridin-2(1H)-one

Compound 11g was prepared in a similar manner to Example 16 in 70% yield.

¹ HNMR(400MHz, Chloroform-d)δ8.07(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).

¹³ CNMR(101MHz,Chloroform-d)δ157.35,140.82,140.23,139.65,137.43,133.90,127.44,127.37,126.80,125.79,125.75,123.51,121.86,121.71,121.36,120.52,105.96,105.74,52.28,37.65,29.81 .

ESI-HRMS: 413.1579, [M+1] ⁺ .

Example 23 Compound 11h: 5-(1-(3-fluoro-4-methoxybenzyl)-1H-indazol-6-yl)-1-methyl-3-(methyl-amino)pyridin-2(1H)-one preparation

Compound 11h was prepared in a similar manner to Example 16. Yield 70%.

¹ HNMR (400MHz, Chloroform-d) δ8.04(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) ).

¹³ CNMR(101MHz,Chloroform-d)δ158.94,141.64,140.04,136.53,133.57,127.02,124.25,124.22,123.50,123.10,123.06,121.73,120.27,120.12,119.57,113.57,113.11,106.02,56.31,56.29,38.56 , 38.25.

ESI-HRMS: 393.1727, [M+1] ⁺ .

Example 24 Preparation of Compound 11i: 3-(ethylamino)-5-(1-(4-fluorobenzyl)-1H-indazol-6-yl)-1-methylpyridin-2(1H)-one

Dissolve compound 1 (1.0eq) and solid sodium hydride (1.5eq) in DMSO solvent, add them into a reaction tube, place in a parallel reaction tube, replace with nitrogen, extract the water vapor and O ₂ in the reaction tube, and then add p-fluorobenzyl bromide (1.2eq), and repeated nitrogen replacement for several times to maintain an anhydrous and oxygen-free environment. overnight at room temperature. The degree of reaction was detected by TLC. After the reaction was completed, water was added to quench, and a solid was precipitated. Filter with a baffle funnel, wash with water several times, and dry. The obtained product was put into the next reaction.

The product obtained above (1.0eq) and intermediate 9b (1.5eq), solid sodium carbonate (3.0eq) and Pd(dppf)Cl2 ₍ 0.05eq) were dissolved in a mixed solvent of dioxane and water (two Oxane:water=2:1), nitrogen protection, transfer to a 90°C oil bath for heating and stirring, and the reaction lasts for 6-7 hours. After TLC detected that the reaction was complete, celite was filtered, extracted with water/ethyl acetate/saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained crude product was purified by column chromatography to obtain the target compound 11i. Yield 70%.

¹ HNMR (400MHz, Chloroform-d) δ8.04 (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).

¹³ CNMR(101MHz,Chloroform-d)δ157.37,140.09,138.64,137.24,133.56,129.00,128.91,123.49,121.77,121.59,121.24,120.32,115.81,115.59,106.14,105.93,52.25,37.82,37.70,14.17.

ESI-HRMS: 377.1778, [M+1] ⁺ .

Example 25 Preparation of compound 11j: 5-(1-(3,5-dimethoxybenzyl)-1H-indazol-6-yl)-3-(ethylamino)-1-methyl-pyridin-2(1H)-one

Compound 11j was prepared in a similar manner to Example 24 in 70% yield.

¹ HNMR (400MHz, Chloroform-d) δ8.04 (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).

¹³ CNMR(101MHz,Chloroform-d)δ161.13,140.25,139.18,138.60,137.08,133.41,123.46,121.81,121.49,121.27,120.21,106.32,106.04,105.39,105.34,99.37,55.31,53.01,37.81,37.68,14.19 .

ESI-HRMS: 419.2071, [M+1] ⁺ .

Example 26 Preparation of Compound 11k: 5-(1-(cyclohexylmethyl)-1H-indazol-6-yl)-1-methyl-3-(methylamino)pyri-din-2(1H)-one

Compound 11k was prepared in a similar manner to Example 16 in 70% yield.

¹ HNMR (400MHz, Chloroform-d) δ 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).

¹³ CNMR(101MHz,Chloroform-d)δ157.38,140.44,139.64,136.71,132.63,122.94,122.21,121.42,121.33,119.94,106.25,106.02,55.15,38.81,30.98,30.93,29.90,29.69,26.28,25.71,25.66 .

ESI-HRMS: 351.2177, [M+1] ⁺ .

Example 27 Compound 111: Preparation of ethyl2-(6-(1-methyl-5-(methylamino)-6-oxo-1,6-dihydropyridin-3-yl)-1H-indazol-1-yl)acetate

Compound 111 was prepared in a similar manner to Example 16 in 70% yield.

¹ HNMR (400MHz, Chloroform-d) δ 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).

¹³ CNMR(101MHz,Chloroform-d)δ157.38,140.44,139.64,136.71,132.63,122.94,122.21,121.42,121.33,119.94,106.25,106.02,55.15,38.81,30.98,30.93,29.90,29.69,26.28,25.71,25.66 .

ESI-HRMS: 341.1601, [M+1] ⁺ .

Example 28 Preparation of compound 11m: 5-(1-(cyclopropylmethyl)-1H-indazol-6-yl)-1-methyl-3-(methylamino)pyridin-2(1H)-one

Compound 11m was prepared in a similar manner to Example 16 in 70% yield.

¹ HNMR(400MHz, Chloroform-d)δ8.00(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 ).

¹³ CNMR(101MHz, CDCl ₃ )δ157.41,139.92,139.67,136.78,132.76,123.23,122.01,121.46,121.32,119.95,106.15,105.77,53.47,37.62,29.91,11.30,4

ESI-HRMS: 309.1719, [M+1] ⁺ .

The beneficial effects of the present invention are proved by the following biological experiments.

Test Example 1 BRD4(1,2) protein activity test and cell proliferation inhibition test

1.1BRD4(1,2) protein activity test

The compounds of the present invention were all tested for BRD4 protein by Shanghai Ruizhi Chemical Company. Compounds were tested for inhibition of BRD4(1,2) protein activity using the HTRF method. The test method is as follows:

The compound was diluted 5 times starting from 25 μM, and 5 concentration gradients were set up, and 5 μl of prepared BRD4(1,2) protein solution, GST-tagged substrate, 5 μl [Lys(5, 8,12,16)AC]H4(1-21)-biotin, SA acceptor, anti-GST donor solution, 5μl blank buffer was added to the control group. Incubate for 3 hours at room temperature, blocked from light. The fluorescence emission at 665nm and 620nm wavelengths was detected by an HTRF-compatible tester, and the inhibition rate of BRD4(1,2) protein by the compounds at different concentrations was calculated, and the inhibition value was obtained by fitting with Excel software, and then calculated by GraphPad fitting. _IC50 value. The experimental results are shown in Table 1 and Table 2.

1.2 Cell proliferation inhibition experiment

1.2.1 Experimental materials

Human acute myeloid leukemia cell MV4-11 and normal human stem cell LO2, thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma company; DMEM medium and RPMI1640 medium, fetal bovine serum and trypsin were purchased from GibcoBRL company; MV4-11 cells were grown in complete medium consisting of 10% fetal bovine serum in DMEM (FBS; Gibco, Auckland, NZ) and 1% antibiotics (100 U/mL penicillin and 100 U/mL streptomycin) at 37°C , in an incubator under 5% CO ₂ conditions.

1.1.2 Experimental principle and method

MTT method, also known as MTT colorimetric method, is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan (Formazan) and deposit in cells, but dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value can be measured at 540 or 720 nm with an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number. Therefore, the higher the number of viable cells, the higher the amount of precipitated formazan, and the higher the absorbance measured by the microplate reader.

The stock solutions of the compounds to be tested were diluted into different concentrations (3 duplicate wells) corresponding to the culture medium, and the solution volume of each well was 100 μL, and added to a 96-well plate. Tumor cells in logarithmic growth phase were collected and seeded into 96-well plates at a density of 2.5-10×10 ³ cells/well, with a cell suspension volume of 100 μL per well, and cells were treated with 1‰ DMSO medium and pure medium as a negative control. The plate was then placed in a 37°C, 5% _CO2 cell culture incubator for 24h. After the formation of formazan, the culture was stopped, and 150 μL of DMSO (adherent cells) was added to each well. For suspension cells MV4-11, 50 μL of 20% SDS solution was directly added and shaken for 15-20 min. The absorbance (OD _570nm ) of each well of cells at 570nm wavelength was detected with a microplate reader, and the average value was taken to record the results. Cell proliferation inhibition rate=(OD _570nm of control group-OD _570nm of experimental group)/OD _570nm of control group*100%. The median inhibitory concentration ( _IC50 ) of each compound was calculated by GraphpadPrism5. The proliferation experiments for each tumor cell were repeated three times. The experimental results are shown in Table 1 and Table 2. The MTT method was also used to test the cytotoxicity of normal human stem cells LO2, and the results are shown in Figure 1.

Table 1 Inhibitory rate of some compounds of the present invention to BRD4(1, 2) and IC ₅₀ of inhibiting MV4-11

Table 2 IC ₅₀ of some compounds of the present invention inhibiting BRD4(1, 2) and MV4-11

Note: For _IC50 values: A means _IC50 is less than 2 μM; B means _IC50 is between 2 and 10 μM; C means _IC50 is greater than 10 μM.

For the inhibition rate at 1 μM dose: A means greater than 40%; B means between 20% and 40%; C means less than 20%.

JQ1 is a positive control drug, a known BRD4 inhibitor, its structural formula is as follows:

Test Example 2 Cell cycle arrest experiment

Collect MV4-11 log phase cells, prepare an appropriate cell suspension concentration, and inoculate about 10×10 ⁵ cells per well (volume 2 mL) into a 6-well plate; 37°C, 5% CO ₂ ; take the supernatant and add 2 mL of medicated culture medium with different concentrations, and DMSO is used as the control group. The cells were further incubated in a 37°C, 5% CO ₂ incubator for 48 hours. Final treatment of cells: (1) Collect the cells in each well, load them into a flow tube according to their concentration, centrifuge at 4°C, 2000 r/min for 3 minutes, and discard the supernatant; (2) Resuspend the cells with 3 mL of PBS, Centrifuge in the same way and discard the PBS; (3) Fix the cells with 75% ethanol for 30 min, centrifuge in the same way, and discard the supernatant; (4) Resuspend the cells with 3 mL of PBS, centrifuge in the same way, and discard the PBS; (5) ) Add 1 mL of PI staining solution containing TritonX-100, mix well, and protect from light at room temperature for 20 minutes; flow cytometer detection. Cell cycle was detected by flow cytometry (FCM) and PI staining. MV4-11 cells were harvested after being treated with compound 11e for 48 h, fixed with 75% ethanol, and stained with sodium propidium iodide (PI). Data were analyzed using FlowJo software. The experimental results are shown in Figure 2.

It can be seen from Figure 2 that compared with the DMSO control group, when the MV4-11 cell line was treated with a low concentration (0.1 μM) of compound 11e, the cell cycle showed significant changes. When the concentration increased to 0.5μM and 2.5μM successively, the number of cells in G0/G1 phase increased. The above experimental results show that the compound 11e of the present invention can arrest cells in the G ₀ /G ₁ phase in a concentration-dependent manner.

Test Example 3 Western blot analysis experiment

After MV4-11 cells were treated with different concentrations of compound 11e for 72 hours, cells were harvested and lysed with RIPA lysis buffer (Beijing, China, Beijing, China) supplemented with cocktails and phosphatase inhibitors. After lysis on ice for 30 minutes, 13,000 Turn, centrifuge at 4 degrees for 15 minutes, and take the supernatant. Protein concentration determination was performed using the BCA method. Perform sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to ensure that the total amount of protein loaded in each sample is the same, and then transfer the protein to a nitrocellulose membrane (NC) at 100V for 2 hours. membrane). NC membranes were blocked with 5% nonfat dry milk in TBS/T for 1.5 hours at room temperature, and then incubated with primary antibody overnight at 4 degrees. After incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody, the membrane was reacted with a chemiluminescent substrate and then exposed to light. The quality of the western blot experiments was assessed using ImageJ to synthesize the results of three independent experiments. The experimental results are shown in Figure 3.

It can be seen from Figure 3 that the expression of c-Myc decreased in a dose-dependent manner with compound 11e, indicating that compound 11e of the present invention can affect the transcription and expression of downstream genes.

Claims (12)

1. The compound represented by formula I or a pharmaceutically acceptable salt thereof:

Among R ₁ , R ₂ , R ₃ and R ₄ , R ₃ is

The remaining three are independently selected from -H, wherein R ₁₄ is -CH ₃ , R ₁₂ is -H, and R ₁₃ is -CH ₃ or -CH ₂ CH ₃ ; R ₅ is selected from -XR ₇ , wherein X is -CH ₂ -, and R ₇ is selected from substituted or unsubstituted phenyl, unsubstituted 3-6 membered cycloalkyl or

Wherein, R ₉ is -OCH ₂ CH ₃ ; in R ₇ , the substituted phenyl group contains at least one substituent selected from the following group: -F, -CH ₃ , -CF ₃ , -OCH ₃ , -NO ₂ ; R ₆ is selected from -H. 2. compound as claimed in claim 1 is characterized in that: _R3 is

R ₁ , R ₂ , R ₄ are independently selected from -H. 3. The compound of claim 1, wherein: In R ₅ , the unsubstituted 3-6 membered cycloalkyl is selected from

4. The compound of claim 1, wherein: R ₅ is -XR ₇ , wherein X is -CH ₂ -, and R ₇ is selected from

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

6. Use of the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of BRD4 inhibitor drugs. 7. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating and/or preventing cancer. 8. The use according to claim 7, wherein the medicine is a medicine for treating and/or preventing leukemia. 9. The use according to claim 8, wherein the leukemia is acute myeloid leukemia. 10. A pharmaceutical composition for preventing and treating cancer, characterized in that: it is prepared by using the compound described in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof as an active ingredient, and adding acceptable auxiliary ingredients. preparation. 11. The pharmaceutical composition of claim 10, wherein the preparation is a preparation for treating and/or preventing leukemia. 12. The pharmaceutical composition of claim 11, wherein the leukemia is acute myeloid leukemia.

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