CN114380800B - Pyridine-pyrimidine amine-benzimidazole derivative, and preparation method and application thereof - Google Patents

Abstract

The invention provides a pyridine-pyrimidine amine-benzimidazole derivative, a preparation method and application thereof, and belongs to the field of medicines. The derivative is a compound shown in a formula I, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof. The derivative has good inhibitory activity on CDK4 and CDK6 kinases, and can be used for preparing CDK4 and CDK6 kinase inhibitors; meanwhile, the derivative has remarkable inhibition activity on various tumor cells, can inhibit the growth of tumors in vivo, and has excellent effect; the derivative has small administration dosage and high safety; in addition, the derivative can pass through the blood brain barrier, and solves one of the biggest problems in brain tumor treatment. The derivative can be used for preparing medicines for preventing and/or treating tumors and autoimmune diseases, and has good application prospects.

Description

Pyridine-pyrimidine amine-benzimidazole derivative, and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a pyridine-pyrimidine amine-benzimidazole derivative, and a preparation method and application thereof.

Background

Malignant tumors have high mortality and the incidence rate of the malignant tumors increases year by year, and the malignant tumors exceed cardiovascular and cerebrovascular diseases and become the first killer for human health. CDK4/6, cyclin dependent kinases 4 and 6, are key conditional proteins of the human cell division proliferation cycle, and are common downstream targets of a plurality of growth promoting signal pathways such as ER, PI3K/mTOR and the like.

CDK4/6 regulates the cell cycle through the Cyclin D-CDK4/6-RB pathway. During cell proliferation, CDK4/6 forms a complex with Cyclin D (Cyclin D) and phosphorylates retinoblastoma protein (Rb). Rb, once phosphorylated, releases the tightly bound transcription factor E2F. E2F activation further pushes the cell cycle from G1 phase to S phase, into the cell proliferation cycle.

It was found that there is a deregulation of the pathway Cyclin D-CDK4/6-RB in almost all malignant tumors. This pathway is continuously activated, which manifests as CDK4/6 hyperactivity, ultimately leading to uncontrolled cell growth and proliferation and spread of cancer cells. And inhibit CDK4/6, make tumor cell unable to form Cyclin D-CK4/6 complex, block cell cycle in growing phase, thus achieve the goal of inhibiting tumor proliferation. Thus, targeted inhibition of CDK4/6 with novel small molecule inhibitors is a potential therapeutic approach.

Disclosure of Invention

The invention aims to provide pyridine-pyrimidine amine-benzimidazole derivatives, and a preparation method and application thereof.

The present invention provides a compound of formula I, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof:

wherein, the liquid crystal display device comprises a liquid crystal display device,

n ₁ an integer selected from 1 to 3;

n ₂ an integer selected from 0 to 5;

the dotted line is a bond or none;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

X ₂ 、Y ₂ Independently selected from N or CR ₄ ；

R ₁ 、R ₂ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

each R ₃ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

each R ₄ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxy, amino, nitro.

Further, the method comprises the steps of,

n ₁ an integer selected from 1 to 3;

n ₂ selected from 0 or 1;

the dotted line is a bond or none;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

X ₂ 、Y ₂ Independently selected from N or CR ₄ ；

R ₁ 、R ₂ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen;

each R ₃ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen;

each R ₄ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen.

Further, the compound is represented by formula II:

wherein, the liquid crystal display device comprises a liquid crystal display device,

n ₁ an integer selected from 1 to 3;

n ₂ an integer selected from 0 to 5;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

X ₂ 、Y ₂ Independently selected from N or CR ₄ ；

R ₁ 、R ₂ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

each R ₃ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

each R ₄ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

preferably, the method comprises the steps of,

n ₁ an integer selected from 1 to 3;

n ₂ selected from 0 or 1;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

X ₂ 、Y ₂ Independently selected from N or CR ₄ ；

R ₁ 、R ₂ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen;

each R ₃ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen;

each R ₄ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen.

Further, the compound is represented by formula III:

wherein, the liquid crystal display device comprises a liquid crystal display device,

n ₁ an integer selected from 1 to 3;

n ₂ an integer selected from 0 to 5;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

X ₂ 、Y ₂ Independently selected from N or CR ₄ ；

R ₁ 、R ₂ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

each R ₃ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

each R ₄ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

preferably, the method comprises the steps of,

n ₁ an integer selected from 1 to 3;

n ₂ selected from 0 or 1;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

X ₂ 、Y ₂ Independently selected from N or CR ₄ ；

R ₁ 、R ₂ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen;

each R ₃ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen;

each R ₄ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen.

Further, the compound is represented by formula IV:

wherein, the liquid crystal display device comprises a liquid crystal display device,

n ₁ an integer selected from 1 to 3;

n ₂ an integer selected from 0 to 5;

the dotted line is a bond or none;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

R ₁ 、R ₂ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

each R ₃ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

preferably, the method comprises the steps of,

n ₁ an integer selected from 1 to 3;

n ₂ selected from 0 or 1;

the dotted line is a bond or none;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

R ₁ 、R ₂ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen;

each R ₃ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen.

Further, the compound is represented by formula V:

wherein, the liquid crystal display device comprises a liquid crystal display device,

n ₁ an integer selected from 1 to 3;

n ₂ an integer selected from 0 to 5;

the dotted line is a bond or none;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

Each R ₃ Are independently selected from hydrogen, C ₁ ～C ₈ Alkyl, C ₁ ～C ₈ Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;

preferably, the method comprises the steps of,

n ₁ an integer selected from 1 to 3;

n ₂ selected from 0 or 1;

the dotted line is a bond or none;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

Each R ₃ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen.

Further, the compound is represented by formula Va:

wherein, the liquid crystal display device comprises a liquid crystal display device,

n ₁ an integer selected from 1 to 3;

n ₂ selected from 0 or 1;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

Each R ₃ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen;

alternatively, the compound is of formula Vb:

wherein, the liquid crystal display device comprises a liquid crystal display device,

n ₁ an integer selected from 1 to 3;

X ₁ 、Y ₁ independently selected from N or CR ₃ ；

Each R ₃ Are independently selected from hydrogen, C ₁ ～C ₃ Alkyl, C ₁ ～C ₃ Alkoxy, halogen.

Further, the compound is one of the following compounds:

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the invention also provides the use of a compound as hereinbefore described, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof, in the preparation of a CDK4 and/or CDK6 kinase inhibitor.

Further, the kinase inhibitor is a medicament for preventing and/or treating tumors;

preferably, the tumor is brain cancer, glioblastoma, leukemia, lymphoma, bannayan-Zonana syndrome, coden disease, lhermitte-Duclos disease, breast cancer, inflammatory breast cancer, wilms 'tumor, ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, colon cancer, stomach cancer, bladder cancer, head and neck cancer, kidney cancer, lung cancer, liver cancer, melanoma, kidney cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, osteosarcoma, bone giant cell tumor or thyroid cancer.

Further, the kinase inhibitor is a medicament for preventing and/or treating autoimmune diseases;

preferably, the autoimmune disease is psoriasis or lupus erythematosus.

The invention also provides a medicine which is a pharmaceutical preparation prepared by taking the compound, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

The compounds and derivatives provided in the present invention are named according to IUPAC (international union of pure and applied chemistry) or CAS (chemical abstract service, columbus, OH) naming system.

In the present invention, the minimum and maximum values of the carbon atom content in the hydrocarbon group are represented by prefixes, for example, C _a～b Alkyl indicates any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C ₁ ～C ₈ Alkyl refers to straight or branched alkyl groups containing 1 to 8 carbon atoms; c (C) ₁ ～C ₈ Alkoxy refers to an alkoxy group containing 1 to 8 carbon atoms.

In the present invention, halogen is fluorine, chlorine, bromine or iodine.

In the formula I of the invention, n ₁ When 1, the structure is

n ₁ When 2, the structure is +.>

n ₁ In case of 3, the structure is +.>

In the formula I, the dotted line is zero, and the structure is

When the dotted line is a bond, the structure is +>

n ₂ 0, 1, 2, 3, 4 or 5.

The invention provides a pyridine-pyrimidine amine-benzimidazole derivative which has good inhibitory activity on CDK4 and CDK6 kinase and can be used for preparing CDK4 and CDK6 kinase inhibitors; meanwhile, the derivative has remarkable inhibition activity on various tumor cells, can inhibit the growth of tumors in vivo, and has excellent effect; the derivative has small administration dosage and high safety; in addition, the derivative can pass through the blood brain barrier, and solves one of the biggest problems in brain tumor treatment. The derivative can be used for preparing medicines for preventing and/or treating tumors and autoimmune diseases, and has good application prospects.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 shows the tumor growth of NOD-Bablc mice in each group.

Figure 2 shows the distribution of compound A2 in blood and brain tissue at various time points.

Detailed Description

Unless otherwise indicated, the materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

Example 1, compound A1 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (5- (4- (2-methoxyethyl) piperazin-1-yl) methyl) pyridin-2-yl) pyrimidin-2-amine

The synthetic route for compound A1 is as follows:

the first step:

raw material 1 (1 g,4.61 mmol) was dissolved in anhydrous DMF (15 mL), anhydrous potassium carbonate (862mg, 6.24 mmol) was added, and stirred at room temperature for 5min. Subsequently, raw material 2 (1.03 g,5.532 mmol) was slowly added and the reaction was stirred at 55℃for 2h. TLC monitors the reaction to be complete, the reaction liquid is cooled to room temperature, a proper amount of ice water is added under the stirring condition, a large amount of white solid is precipitated in the reaction system, the reaction system is filtered, a filter cake is washed with water for 3 times, and intermediate 3 is obtained after drying, the white solid is 1.321g, and the yield is 89%.

And a second step of:

intermediate 3 (1.2 g,3.72 mmol) was dissolved in methanol (25 mL), 10% palladium on carbon (390 mg,0.372 mmol) was added, the mixture was evacuated, replaced three times with hydrogen, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. TLC monitoring the reaction completion, the reaction solution was filtered with celite, washed 3 times with a mixed solvent of dichloromethane/methanol (10/1, v/v), and the filtrate was concentrated to dryness under reduced pressure to give crude product intermediate 4. Without further purification, it was used directly in the next reaction.

And a third step of:

toward intermediate 4 (3.72 mmol), starting material 5 (1.282 g,3.72 mmol), pd ₂ (dba) ₃ Toluene (20 mL) was added to a mixture of (3411 mg,0.372 mmol), X-phos (178 mg,0.372 mmol) and cesium carbonate (1.82 g,5.58 mmol), the mixture was evacuated and replaced 3 times with nitrogen, and the reaction mixture was stirred under nitrogen at 100℃for 5 hours. TLC monitoring reaction completion, cooling the reaction liquid to room temperature, filtering with diatomite, washing 3 times with dichloromethane/methanol (10/1, v/v) mixed solvent, concentrating the filtrate under reduced pressure, separating by silica gel column chromatography to obtain intermediate 6, 1.1g of off-white solid, and the yield is 51%.

Fourth step:

intermediate 6 (1 g,1.73 mmol) was dissolved in dichloromethane (15 mL) and trifluoroacetic acid (4 mL) was slowly added dropwise with stirring and reacted at room temperature for 2h. TLC monitoring reaction completion, the reaction solution was concentrated to dryness under reduced pressure, dissolved again with a small amount of dichloromethane, dried by spin, and operated repeatedly 3 times to obtain crude product intermediate 7. Without further purification, it was used directly in the next reaction.

Fifth step:

intermediate 7 (0.173 mmol) was dissolved in anhydrous DMF (4 mL), cesium carbonate (113 mg, 0.348 mmol) was added, and after stirring at room temperature for 5min, starting material 8 (25. Mu.L, 0.2595 mmol) was slowly added and the temperature was raised to 60℃for 3h. TLC monitoring reaction completion, cooling the reaction solution to room temperature, adding appropriate amount of ice water, extracting with ethyl acetate three times, mixing organic phases, washing with water, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to dryness, further drying with siliconThe target compound A1 is separated by gel column chromatography, the white solid is 68mg, and the yield is 73%. HR-MS (ESI-TOF) m/z calcd for C ₂₈ H ₃₄ F ₂ N ₈ O[M+H] ⁺ 537.2902,found 537.2900.

EXAMPLE 2 Compound A2 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (5- (4- (2- (2-methoxyethoxy) ethyl) piperazin-1-yl) methyl) pyridin-2-yl) pyrimidin-2-amine

The intermediate 7 and 1-bromo-2- (2-methoxyethoxy) ethane are used as raw materials, and the compound A2 is obtained by referring to a fifth step of the synthesis method of the compound A1, and the yield is 78 percent. HR-MS (ESI-TOF) m/z calcd for C ₃₀ H ₃₈ F ₂ N ₈ O ₂ [M+H] ⁺ 581.3164,found 581.3163.

EXAMPLE 3 Compound A3 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (5- (4- (2- (2-methoxyethoxy) ethoxy) ethyl) piperazin-1-yl) methyl) pyridin-2-amine

The intermediate 7 and diethylene glycol-2-bromoethyl methyl ether are used as raw materials, and the compound A3 is obtained by referring to a fifth step of compound A1 synthesis method, and the yield is 69%. HR-MS (ESI-TOF) m/z calcd for C ₃₂ H ₄₂ F ₂ N ₈ O ₃ [M+H] ⁺ 625.3426,found 625.3425.

Example 4 Compound A4 (5- ((5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) pyrimidin-2-yl) amino) pyridin-2-yl) (4- (2-methoxyethyl) piperazin-1-yl) methanone

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The synthetic route for compound A4 is as follows:

the first step:

raw material 9 (2 g,11.9 mmol) was dissolved in anhydrous THF (30 mL), DIEA (2.95 mL,17.85 mmol) and HATU (4.98 g,13.09 mmol) were added sequentially, and after stirring at room temperature for 10min, raw material 2 (3.32 g,17.85 mmol) was slowly added and the reaction was continued under stirring at room temperature for 4h. TLC was used to monitor completion of the reaction, a proper amount of water was added, extraction was performed three times with ethyl acetate, the organic phases were combined, washed with water, then with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure, followed by separation by silica gel column chromatography to give intermediate 10 as a white solid 2.092g in 52% yield.

And a second step of:

the second step synthesis of reference compound A1 gives crude intermediate 11 starting from intermediate 10. Without further purification, it was used directly in the next reaction.

And a third step of:

the intermediate 12 is obtained as a yellow solid with a yield of 82% by referring to the third step of the synthesis method of the compound A1 by taking the intermediate 11 and the raw material 5 as raw materials.

Fourth step:

the fourth step of the synthesis method, which uses intermediate 12 as a raw material, refers to compound A1, gives a crude product intermediate 13. Without further purification, it was used directly in the next reaction.

Fifth step:

the intermediate 13 and the raw material 8 are used as raw materials, and the compound A4 is obtained by referring to a fifth step of synthesis method of the compound A1, and is yellow solid with the yield of 77%. HR-MS (ESI-TOF) m/z calcd for C ₂₈ H ₃₂ F ₂ N ₈ O ₂ [M+H] ⁺ 551.2694,found 551.2692.

Example 5 Compound A5 (5- ((5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) pyrimidin-2-yl) amino) pyridin-2-yl) (4- (2- (2-methoxyethoxy) ethyl) piperazin-1-yl) methanone

The intermediate 13 and 1-bromo-2- (2-methoxyethoxy) ethane are used as raw materials, and the compound A5 is obtained by referring to a fifth step of the synthesis method of the compound A1, and the yield is 66 percent. HR-MS (ESI-TOF) m/z calcd for C ₃₀ H ₃₆ F ₂ N ₈ O ₃ [M+H] ⁺ 595.2956,found 595.2952.

Example 6 Compound A6 (5- ((5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) pyrimidin-2-yl) amino) pyridin-2-yl) (4- (2- (2-methoxyethoxy) ethoxy) piperazin-1-yl) methanone

The intermediate 13 and diethylene glycol-2-bromoethyl methyl ether are used as raw materials, and the compound A6 is obtained by referring to a fifth step of compound A1 synthesis method, and the yield is 74 percent. HR-MS (ESI-TOF) m/z calcd for C ₃₂ H ₄₀ F ₂ N ₈ O ₄ [M+H] ⁺ 639.3219,found 639.3218.

EXAMPLE 7 Compound A7 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (6- ((4- (2-methoxyethyl) piperazin-1-yl) methyl) pyridin-3-yl) pyrimidin-2-amine

The synthetic route for compound A7 is as follows:

the first step:

intermediate 11 (918 mg,3.0 mmol) was dissolved in anhydrous THF (20 mL) and borane/dimethyl sulfide was slowly added dropwiseComplex BH ₃ /S(CH ₃ ) ₂ (2M in THF) (6 mL) and then the reaction was warmed to 60℃and stirred for 3h. TLC monitored completion of the reaction, the reaction mixture was cooled to room temperature, piperidine (2.5 mL) was added and stirring continued at room temperature for 30min. The reaction mixture was concentrated to dryness under reduced pressure, and intermediate 14 was isolated by silica gel column chromatography as a white solid (362 mg) in 41% yield.

And a second step of:

the intermediate 15, a white solid, was obtained in a yield of 76% by the third step of the synthesis method with reference to compound A1, starting from intermediate 14 and starting material 5.

And a third step of:

the fourth step of the synthesis, with reference to compound A1, gives the crude intermediate 16 starting from intermediate 15. Without further purification, it was used directly in the next reaction.

Fourth step:

the intermediate 16 and the raw material 8 are used as raw materials, and the compound A7 is obtained by referring to a fifth step of the synthesis method of the compound A1, and is white solid with the yield of 63%. HR-MS (ESI-TOF) m/z calcd for C ₂₈ H ₃₄ F ₂ N ₈ O[M+H] ⁺ 537.2902,found 537.2902.

Example 8, compound A8 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (6- ((4- (2- (2-methoxyethoxy) ethyl) piperazin-1-yl) methyl) pyridin-3-yl) pyrimidin-2-amine

The intermediate 16 and 1-bromo-2- (2-methoxyethoxy) ethane are used as raw materials, and the compound A8 is obtained by referring to a fifth step of the synthesis method of the compound A1, and the yield is 70 percent. HR-MS (ESI-TOF) m/z calcd for C ₃₀ H ₃₈ F ₂ N ₈ O ₂ [M+H] ⁺ 581.3164,found 581.3161.

Example 9, compound A9 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (6- ((4- (2- (2-methoxyethoxy) ethoxy) ethyl) piperazin-1-yl) methyl) pyridin-3-yl) pyrimidin-2-amine

The intermediate 16 and diethylene glycol-2-bromoethyl methyl ether are used as raw materials, and the compound A9 is obtained by referring to a fifth step of compound A1 synthesis method, so that the yield is 59%. HR-MS (ESI-TOF) m/z calcd for C ₃₂ H ₄₂ F ₂ N ₈ O ₃ [M+H] ⁺ 625.3426,found 625.3431.

Example 10 Compound A10 (4- ((5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) pyrimidin-2-yl) amino) -3-methoxyphenyl) (4- (2- (2-methoxyethoxy) ethyl) piperazin-1-yl) methanone

The synthetic route for compound a10 is as follows:

the first step:

the intermediate 18, a white solid, was obtained in 69% yield by the first step synthesis of reference compound A4 starting from starting material 17.

And a second step of:

the second step synthesis of reference compound A1 gives crude intermediate 19 starting from intermediate 18. Without further purification, it was used directly in the next reaction.

And a third step of:

the intermediate 20 is obtained as a yellow solid in 77% yield by referring to the third step of the synthesis method of the compound A1 by taking the intermediate 19 and the raw material 5 as raw materials.

Fourth step:

the fourth step of the synthesis, starting from intermediate 20, is to provide crude intermediate 21, with reference to compound A1. Without further purification, it was used directly in the next reaction.

Fifth step:

the intermediate 21 and the raw material 8 are used as raw materials, and the compound A10 is obtained by referring to a fifth step of synthesis method of the compound A1, and is yellow solid with the yield of 65%. HR-MS (ESI-TOF) m/z calcd for C ₃₂ H ₃₉ F ₂ N ₇ O ₄ [M+Na] ⁺ 646.2930,found 646.2950.

Example 11 Compound A11 (4- ((5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) pyrimidin-2-yl) amino) -3-methoxyphenyl) (4- (2- (2-methoxyethoxy) ethoxy) piperazin-1-yl) methanone

The intermediate 21 and 1-bromo-2- (2-methoxyethoxy) ethane are used as raw materials, and the compound A11 is obtained by referring to a fifth step of the synthesis method of the compound A1, and is yellow solid with the yield of 71%. HR-MS (ESI-TOF) m/z calcd for C ₃₄ H ₄₃ F ₂ N ₇ O ₅ [M+Na] ⁺ 690.3192,found 690.3185.

EXAMPLE 12 Compound A12 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (5- (4- (2-methoxyethyl) piperazin-1-yl) pyridin-2-yl) pyrimidin-2-amine

The synthetic route for compound a12 is as follows:

the first step:

5-bromo-2-nitropyridine (1 g,4.93 mmol) was dissolved in anhydrous DMF (15 mL), anhydrous potassium carbonate (921 mg,6.67 mmol) was added, and stirred at room temperature for 5min. Subsequently, raw material 2 (1.03 g,5.532 mmol) was slowly added and the reaction was stirred at 55℃for 2h. TLC monitors the reaction completely, cool the reaction liquid to room temperature, add a proper amount of ice water under stirring, precipitate a large amount of white solid in the reaction system, filter, wash the filter cake with water for 3 times, dry to get intermediate 22, 1.211g of white solid, yield 89%.

And a second step of:

intermediate 22 (1.2 g,3.89 mmol) was dissolved in methanol (25 mL), 10% palladium on carbon (390 mg,0.372 mmol) was added, the mixture was evacuated, replaced three times with hydrogen, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. TLC showed that the reaction was complete, the reaction solution was filtered through celite, washed 3 times with a mixed solvent of dichloromethane/methanol (10/1, v/v), and the filtrate was concentrated to dryness under reduced pressure to give crude product intermediate 23. Without further purification, it was used directly in the next reaction.

And a third step of:

toward intermediate 23 (3.72 mmol), starting material 5 (1.282 g,3.72 mmol), pd ₂ (dba) ₃ Toluene (20 mL) was added to a mixture of (3411 mg,0.372 mmol), X-phos (178 mg,0.372 mmol) and cesium carbonate (1.82 g,5.58 mmol), the mixture was evacuated and replaced 3 times with nitrogen, and the reaction mixture was stirred under nitrogen at 100℃for 5 hours. TLC monitoring reaction completion, cooling the reaction liquid to room temperature, filtering with diatomite, washing 3 times with dichloromethane/methanol (10/1, v/v) mixed solvent, concentrating the filtrate under reduced pressure, separating by silica gel column chromatography to obtain intermediate 24, 1.05g of off-white solid, yield 52%.

Fourth step:

intermediate 24 (1 g,1.73 mmol) was dissolved in dichloromethane (15 mL) and trifluoroacetic acid (4 mL) was slowly added dropwise with stirring and reacted at room temperature for 2h. TLC monitored the reaction was complete, the reaction was concentrated to dryness under reduced pressure, dissolved again with a small amount of dichloromethane, dried by spinning, and operated repeatedly 3 times to afford crude intermediate 25. Without further purification, it was used directly in the next reaction.

Fifth step:

intermediate 25 (0.172 mmol) was dissolved in anhydrous DMF (4 mL), cesium carbonate (112 mg,0.345 mmol) was added, and after stirring at room temperature for 5min, starting material 8 (25. Mu.L, 0.2595 mmol) was slowly added and the temperature was raised to 60℃for 3h. TLC monitoring reaction is complete, the reaction solution is cooled to room temperature, a proper amount of ice water is added, extraction is carried out three times by ethyl acetate, the organic phases are combined,washing with water, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to dryness, and further separating by silica gel column chromatography to obtain the target compound A12, 70mg of white solid, and yield 74%. HR-MS (ESI-TOF) m/z calcd for C ₂₇ H ₃₂ F ₂ N ₈ O[M+H] ⁺ 522.2667,found 522.2667.

EXAMPLE 13 Compound A13 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (5- (4- (2- (2-methoxyethoxy) ethyl) piperazin-1-yl) pyridin-2-yl) pyrimidin-2-amine

The intermediate 25 and 1-bromo-2- (2-methoxyethoxy) ethane are used as raw materials, and the compound A13 is obtained by referring to a fifth step of the synthesis method of the compound A12, and the yield is 79 percent. HR-MS (ESI-TOF) m/z calcd for C ₂₉ H ₃₆ F ₂ N ₈ O ₂ [M+H] ⁺ 566.2929,found 566.2929.

EXAMPLE 14 Compound A14 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (5- (4- (2- (2-methoxyethoxy) ethoxy) ethyl) piperazin-1-yl) pyridin-2-yl) pyrimidin-2-amine

The intermediate 25 and diethylene glycol-2-bromoethyl methyl ether are used as raw materials, and the compound A14 is obtained by referring to a fifth step of compound A12 synthesis method, and the yield is 70%. HR-MS (ESI-TOF) m/z calcd for C ₃₁ H ₄₀ F ₂ N ₈ O ₃ [M+H] ⁺ 610.3191,found 610.3191.

Example 15 Compound A15 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (6- ((4- (2-methoxyethyl) piperazin-1-yl) pyridin-3-yl) pyrimidin-2-amine

The synthetic route for compound a15 is as follows:

the first step:

2-bromo-5-nitropyridine (1 g,4.93 mmol) was dissolved in anhydrous DMF (15 mL), anhydrous potassium carbonate (922 mg,6.70 mmol) was added, and stirred at room temperature for 5min. Subsequently, raw material 2 (1.02 g,5.530 mmol) was slowly added and the reaction stirred at 55℃for 2h. The TLC monitoring reaction is complete, the reaction liquid is cooled to room temperature, a proper amount of ice water is added under the stirring condition, a large amount of white solid is precipitated in the reaction system, the reaction system is filtered, the filter cake is washed with water for 3 times, and intermediate 26 is obtained after drying, the white solid is 1.210g, and the yield is 89%.

And a second step of:

intermediate 26 (1.19 g,3.88 mmol) was dissolved in methanol (25 mL), 10% palladium on carbon (399mg, 0.371 mmol) was added, the mixture was evacuated, replaced with hydrogen gas three times, and the mixture was stirred under a hydrogen atmosphere at room temperature for 2 hours. TLC showed that the reaction was complete, the reaction solution was filtered through celite, washed 3 times with a mixed solvent of dichloromethane/methanol (10/1, v/v), and the filtrate was concentrated to dryness under reduced pressure to give crude intermediate 27. Without further purification, it was used directly in the next reaction.

And a third step of:

the intermediate 28, a yellow solid, was obtained in 78% yield from intermediate 27 and starting material 5 by the third step synthesis of compound a 12.

Fourth step:

starting from intermediate 28, the fourth step synthesis of reference compound a12 affords crude intermediate 29. Without further purification, it was used directly in the next reaction.

Fifth step:

the intermediate 29 and the raw material 8 are used as raw materials, and the compound A15 is obtained by referring to a fifth step of the synthesis method of the compound A12, and is white solid with the yield of 63%. HR-MS (ESI-TOF) m/z calcd for C ₂₇ H ₃₂ F ₂ N ₈ O[M+H] ⁺ 522.2667,found 522.2667.

EXAMPLE 16 Compound A16 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (6- ((4- (2- (2-methoxyethoxy) ethyl) piperazin-1-yl) pyridin-3-yl) pyrimidin-2-amine

The intermediate 29 and 1-bromo-2- (2-methoxyethoxy) ethane are used as raw materials, and the compound A16 is obtained by referring to a fifth synthesis method of the compound A12, and the yield is 70 percent. HR-MS (ESI-TOF) m/z calcd for C ₂₉ H ₃₆ F ₂ N ₈ O ₂ [M+H] ⁺ 566.2929,found 566.2929.

EXAMPLE 17 Compound A17 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (6- ((4- (2- (2-methoxyethoxy) ethoxy) ethyl) piperazin-1-yl) pyridin-3-yl) pyrimidin-2-amine

The intermediate 29 and diethylene glycol-2-bromoethyl methyl ether are used as raw materials, and the compound A17 is obtained as a pale yellow solid by referring to a fifth step of the compound A1, and the yield is 59%. HR-MS (ESI-TOF) m/z calcd for C ₃₁ H ₄₀ F ₂ N ₈ O ₃ [M+H] ⁺ 610.3191,found 610.3191.

EXAMPLE 18 Compound A18 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (5- (4- (2-methoxyethyl) piperazin-1-yl) phenyl-2-yl) pyrimidin-2-amine

The synthetic route for compound a18 is as follows:

the first step:

1-bromo-4-nitrobenzene (1 g,4.61 mmol) was dissolved in anhydrous DMF (15 mL), anhydrous potassium carbonate (860 mg,6.22 mmol) was added, and stirred at ambient temperature for 5min. Subsequently, raw material 2 (1.01 g,5.530 mmol) was slowly added and the reaction was stirred at 55℃for 2h. The TLC monitoring reaction is complete, the reaction liquid is cooled to room temperature, a proper amount of ice water is added under the stirring condition, a large amount of white solid is precipitated in the reaction system, the reaction system is filtered, the filter cake is washed with water for 3 times, and intermediate 30, white solid 1.221g and yield 86% are obtained by drying.

And a second step of:

intermediate 30 (1.2 g,3.71 mmol) was dissolved in methanol (25 mL), 10% palladium on carbon (399mg, 0.371 mmol) was added, the mixture was evacuated, replaced with hydrogen three times, and the mixture was stirred under a hydrogen atmosphere at room temperature for 2 hours. TLC showed that the reaction was complete, the reaction solution was filtered through celite, washed 3 times with a mixed solvent of dichloromethane/methanol (10/1, v/v), and the filtrate was concentrated to dryness under reduced pressure to give crude intermediate 31. Without further purification, the reaction mixture was used in the next reaction.

And a third step of:

intermediate 31 (3.72 mmol), starting material 5 (1.281g, 3.71 mmol), pd ₂ (dba) ₃ Toluene (20 mL) was added to a mixture of (340 mg,0.3712 mmol), X-phos (177 mg,0.371 mmol) and cesium carbonate (1.81 g,5.57 mmol), the mixture was evacuated and replaced 3 times with nitrogen, and the reaction mixture was stirred under nitrogen at 100℃for 5 hours. TLC monitoring reaction completion, cooling the reaction liquid to room temperature, filtering with diatomite, washing 3 times with dichloromethane/methanol (10/1, v/v) mixed solvent, concentrating the filtrate under reduced pressure, separating by silica gel column chromatography to obtain intermediate 32, white solid 1.05g, yield 50%.

Fourth step:

intermediate 32 (1.0 g,1.73 mmol) was dissolved in dichloromethane (15 mL) and trifluoroacetic acid (4 mL) was slowly added dropwise with stirring and reacted at room temperature for 2h. TLC monitored the reaction was complete, the reaction was concentrated to dryness under reduced pressure, dissolved again with a small amount of dichloromethane, dried by spinning, and operated repeatedly 3 times to afford crude intermediate 33. Without further purification, it was used directly in the next reaction.

Fifth step:

intermediate 33 (0.173 mmol) was dissolved in anhydrous DMF (4 mL), cesium carbonate (113 mg, 0.348 mmol) was added, and after stirring at room temperature for 5min, starting material 8 (25. Mu.L, 0.2595 mmol) was slowly added and the temperature was raised to 60℃for 3h. TLC monitoring reaction completion, cooling the reaction liquid to room temperature, adding a proper amount of ice water, extracting with ethyl acetate three times, combining organic phases, washing with water, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to dryness, further separating the target compound A18 by silica gel column chromatography, and obtaining a white solid with a yield of 73%. HR-MS (ESI-TOF) m/z calcd for C ₂₈ H ₃₃ F ₂ N ₇ O[M+H] ⁺ 522.2715,found 522.2715.

EXAMPLE 19 Compound A19 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (5- (4- (2- (2-methoxyethoxy) ethyl) piperazin-1-yl) phenyl-2-yl) pyrimidin-2-amine

The intermediate 33 and 1-bromo-2- (2-methoxyethoxy) ethane are used as raw materials, and the compound A2 is obtained by referring to a fifth synthesis method of the compound A1, and is white solid with the yield of 78%. HR-MS (ESI-TOF) m/z calcd for C ₃₀ H ₃₇ F ₂ N ₇ O ₂ [M+H] ⁺ 565.2977,found 565.2977.

EXAMPLE 20 Compound A20 5-fluoro-4- (4-fluoro-1-isopropyl-2-methyl-1H-benzo [ d ] imidazol-6-yl) -N- (5- (4- (2- (2-methoxyethoxy) ethoxy) ethyl) piperazin-1-yl) phenyl-2-amine

Intermediate 33 and diethylene glycol-2-bromoethyl methyl ether are used as raw materials, and the compound A20 is obtained by referring to a fifth step of compound A1 synthesis method, and the yield is 68%. HR-MS (ESI-TO)F)m/z:calcd for C ₃₂ H ₄₁ F ₂ N ₇ O ₃ [M+H] ⁺ 609.3239,found 609.3239.

The beneficial effects of the invention are demonstrated by the following in vitro tests.

Test example 1, CDK4, CDK6 kinase inhibitory Activity assay

The aim of this experiment was to detect the inhibitory activity of the compounds of the invention against in vitro kinases by means of isotope labelling. In this experiment, CDK4 and CDK6 kinases were tested for inhibition of in vitro activity, respectively. Abemaciclib is a positive control. IC for kinase inhibitory Activity of test Compounds ₅₀ (half inhibition concentration). IC (integrated circuit) ₅₀ Values can be obtained by calculation of the inhibition rate of the kinase activity by the test compound at a range of different concentrations.

1. Experimental materials

20mM 3- (N-morpholino) propanesulfonic acid (MOPS); 1mM ethylenediamine tetraacetic acid (EDTA); 0.01% Brij 35 (Brij-35); 5% Glycerol (Glycerol); 0.1% mercaptoethanol (mercptoethanol); 1mg/ml Bovine Serum Albumin (BSA); l0mM manganese dichloride solution (MnC 1 ₂ ) The method comprises the steps of carrying out a first treatment on the surface of the Stop buffer (3% phosphate buffer); washing buffer (75 mM phosphate solution); methanol (methanol); filtermat a film; CDK4/6 kinase, test compounds.

2. Experimental method

Adding the kinase to be detected and the corresponding substrates and the compounds to be detected or DMSO with different concentrations into a reaction buffer solution (8 mM propanesulfonate, 0.2mM EDTA,10mM magnesium acetate and Km concentration gamma-33P-ATP solution), and incubating for 40min at room temperature; adding 3% phosphate solution to terminate the reaction; pipette 10. Mu.L of the reaction mixture drop onto P30 filter paper; the filter paper was washed 3 times with 75mM phosphate solution; cleaning the filter paper with methanol for 1 time; after the filter paper was air-dried, scintillation fluid was added and the phosphorylated substrate was measured by scintillation counting. Inhibition% = (1-compound treatment group count/blank group count) ×100%; half maximal Inhibitory Concentration (IC) ₅₀ ) Fitting the inhibition ratios corresponding to the concentrations.

3. Experimental results

The test method tests theCompounds of the invention are directed to the inhibitory activity of CDK4/6 kinase, respectively. Table 1 shows the IC of the test compounds for CDK4/6 kinase inhibitory activity ₅₀ Values.

Experimental results show that the tested compound has strong inhibitory activity on CDK4/6 kinase. Can be used for preparing CDK4 kinase and CDK6 kinase inhibitors.

TABLE 1 IC of test compounds for CDK4/6 kinase inhibitory Activity ₅₀ Value of

*A:IC ₅₀ <50nM；B:50nM≤IC ₅₀ <100nM；C:100nM≤IC ₅₀ <150nM；D:IC ₅₀ ＞150nM

Test example 2 in vitro tumor cell proliferation inhibition experiments of Compounds

The aim of the experiment is to detect the proliferation inhibition activity of the compound on in vitro tumor cells, and the adopted method is MTT (tetramethyl azoazole salt) colorimetric method.

1. Experimental materials

1.1 major reagents

Breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-436 and MDA-MB-468, colorectal cancer cell line SW620, lung cancer cell A549 were all purchased from American standard biological Collection (American Type Culture Collection, ATCC), RPMI 1640 medium, fetal Bovine Serum (FBS) were all purchased from GIBICO company, USA; penicillin and streptomycin were purchased from Dalianbao biosome; cultured cells were purchased from Corning company using plates, 96-well plates, etc.; centrifuge tubes of various sizes were purchased from BD company; MTT reagent was purchased from the Japan same-kernel chemical institute (Donjindo). The tested compounds are synthesized by the inventor, 10mM stock solution is prepared by using 100% DMSO in vitro experiments, the stock solution is stored in a refrigerator at the temperature of minus 20 ℃ for standby, and the stock solution is diluted to the required concentration in the test.

1.2 cell lines and cultures

The breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-436 and MDA-MB-468, colorectal cancer cell line SW620 and lung cancer cell line A549 used in the experimentConventional RPMI 1640 complete medium containing 10% Fetal Bovine Serum (FBS), 100IU/mL penicillin, 100. Mu.g/mL streptomycin was cultured at 37℃in 5% CO ₂ Is cultured in an incubator of (a).

2. Experimental method

The cell concentration is regulated to be 1 to 2 multiplied by 10 by using the complete cell culture solution ⁴ Each/ml of cell suspension (HCC 827 cell concentration 6X 10) ⁴ H1975 cells 4X 10 per ml ³ At/ml), 200 μl of cell suspension per well was inoculated in 96-well plates and incubated overnight. The next day, the supernatant was aspirated and the cells were then individually treated with gradient concentrations of the test compound. Simultaneously setting a negative control group without medicine and an equal volume solvent control group, wherein the concentration of DMSO is 0.1%, 3 compound holes are arranged in each dosage group, and the concentration of CO is 5% at 37 DEG C ₂ Culturing under the condition. After 72 hours, 20. Mu.L of MTT reagent at a concentration of 5mg/ml was added to each well, and after further incubation for 2 to 4 hours, the supernatant was discarded, 150. Mu.L of DMSO was added to each well, and the mixture was mixed by shaking for 15 minutes, and the absorbance (A) value (A value proportional to the number of living cells) was measured by an enzyme-labeled instrument (lambda=570 nm), and the average value was obtained. Relative cell proliferation inhibition ratio= (control group a 570-experimental group a 570)/control group a570×100%. Experiments were repeated at least 3 times. Experimental data are expressed by mean, data statistics are tested by t-test, P<A difference of 0.05 is statistically significant. IC is used for inhibiting cell proliferation by the following compounds ₅₀ And (3) representing.

3. Experimental results

By the method, proliferation inhibition activity tests are carried out on breast cancer cell strains MCF-7, MDA-MB-231, MDA-MB-436 and MDA-MB-468, colorectal cancer cell strain SW620 and lung cancer cell A549. Table 2 shows the proliferation inhibitory activity (IC) ₅₀ ). The results show that most of the compounds of the examples show remarkable proliferation inhibition activity on the tested tumor cell lines, wherein the proliferation inhibition activity of the compounds A8 and A9 on various tumor cell lines is in a low micromolar level, and the compounds have certain therapeutic advantages compared with the positive compound Abemaciclib.

TABLE 2 proliferation of Compounds against tumor cell lines (MTT method)

Note that: ND: non-determined.

A:IC ₅₀ <0.1μM；B:0.1μM≤IC ₅₀ <1μM；C:IC ₅₀ ≥1μM.

Test example 3 in vivo antitumor test of Compound A2

The aim of this experiment was to detect the in vivo antitumor effect of the compounds of the invention. Experimental BALB/c mice were tested for anti-tumor activity in vivo in a subcutaneous tumor model. The cell line used was human colon cancer cell line COLO205.

1. Experimental materials

Fetal bovine serum, culture medium, pancreatin, etc. were purchased from Gibco BRL company (Invitrogen Corporation, USA), culture medium was purchased from ATCC (American Type Culture Collection), human colon cancer cell line COLO205 was purchased from ATCC company, USA, and NOD-Bablc mice were purchased from the beijing verruca animal experiment center.

2. Experimental method

NOD-Bablc mice were used for 6-8 weeks at about 1X 10 ⁷ The concentration of each cell per 0.1 ml/COLO 205 was inoculated into the subcutaneous posterior rib of the mice, and after the tumor was grown to a certain volume, the mice were randomly grouped and started to be orally administrated by gastric lavage and tail vein administration.

And (3) observing the indexes: the tumor length and diameter of the mice body weight meter and the neck were measured every 3 days and the tumor volume was calculated (length diameter x short diameter 2 x 0.52), and the presence or absence of diarrhea, cramps, rash, significant weight loss and other reactions were observed.

3. Experimental results

The experimental results of the tumor growth of the various groups are shown in FIG. 1. The experimental results show that the tested compound A2 has obvious in-vivo growth inhibition effect on COLO205. No adverse reactions such as weight loss, rash, diarrhea and the like were found in the mice during the administration, indicating that the toxicity of the test compound A2 was very low in the administration dosage range.

Test example 4 blood brain tissue distribution experiment of Compound A2

The aim of this experiment was to detect the in vivo antitumor effect of the compounds of the invention. In-situ model of BALB/c mice brain tumor is used for experiment, and the in-vivo anti-tumor activity of the compound A2 is tested. The cell strain used was human brain astrocyte U87-LUC.

1. Experimental materials

Fetal bovine serum, culture medium, pancreatin, etc. were purchased from Gibco BRL company (Invitrogen Corporation, USA), culture medium was purchased from ATCC (American Type Culture Collection), NOD-Bablc mice were purchased from the Beijing Warcang animal laboratory center.

2. Experimental method

SPF grade ICR mice were used, weighed prior to dosing, and dosing amounts were calculated from body weight. Administration is by intravenous injection.

Plasma sample treatment: blood was collected by cardiac puncture, each sample was collected at about 0.2mL, heparin sodium was anticoagulated, placed on ice after collection, and plasma was centrifuged within 1 hour (centrifugation conditions: 6800g,6 minutes, 2-8deg.C). The plasma samples were stored in a-80 ℃ freezer prior to analysis.

Tissue sample treatment: after animals were euthanized, brain tissue was collected, rinsed with normal saline to avoid cross contamination, blotted dry with filter paper, and then placed in labeled tubes/self-sealing bags (one tissue per tube/self-sealing bag), and samples were temporarily placed on ice before storage in an ultra-low temperature refrigerator.

Experimental grouping: drug solvent control (12.5% EL+12.5% EtOH+75% Water)

50mg/kg q.d of compound A2-M;

100mg/kg q.d of compound A2-H;

compound Abemaciclib 50mg/kg q.d;

(each group of drugs was dissolved in 12.5% EL+12.5% EtOH+75% water)

Analysis of results: pharmacokinetic parameters were calculated using Phoenix WinNonlin7.0 from blood concentration data at different time points, providing parameters such as AUC0-T, AUC 0-infinity, MRT 0-infinity, cmax, tmax, and T1/2, as well as their mean and standard deviation.

3. Experimental results

The distribution of compound A2 in blood and brain tissue at various time points is shown in figure 2. The experimental results show that the concentration of the drug in brain tissue is higher than that in plasma of the tested compound A2 at different time points. The compound A2 can penetrate through the blood brain barrier, directly enter the brain to play a role, and can be used for developing medicaments for treating brain tumors.

In summary, the present invention provides a pyridine-pyrimidine amine-benzimidazole derivative, which has good inhibitory activity on CDK4 and CDK6 kinases, and can be used to prepare CDK4 and CDK6 kinase inhibitors; meanwhile, the derivative has remarkable inhibition activity on various tumor cells, can inhibit the growth of tumors in vivo, and has excellent effect; the derivative has small administration dosage and high safety; in addition, the derivative can pass through the blood brain barrier, and solves one of the biggest problems in brain tumor treatment. The derivative can be used for preparing medicines for preventing and/or treating tumors and autoimmune diseases, and has good application prospects.

Claims (8)

1. A compound, or salt thereof, characterized by: the compound is represented by formula Va:

wherein, the liquid crystal display device comprises a liquid crystal display device,

n ₁ an integer selected from 1 to 3;

n ₂ selected from 1;

X ₁ selected from CR ₃ ；

Y ₁ Selected from N;

R ₃ selected from hydrogen.

2. The compound, or salt thereof, according to claim 1, wherein: the compound is one of the following compounds:

3. use of a compound according to claim 1 or 2, or a salt thereof, in the preparation of a CDK4 and/or CDK6 kinase inhibitor.

4. Use according to claim 3, characterized in that: the kinase inhibitor is a medicament for preventing and/or treating tumors.

5. Use according to claim 4, characterized in that: the tumor is brain cancer, glioblastoma, leukemia, lymphoma, bannayan-Zonana syndrome, coden disease, lhermitte-Duclos disease, breast cancer, inflammatory breast cancer, wilms 'tumor, ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma, colon cancer, gastric cancer, bladder cancer, head and neck cancer, renal cancer, lung cancer, liver cancer, melanoma, renal cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, osteosarcoma, bone giant cell tumor or thyroid cancer.

6. Use according to claim 3, characterized in that: the kinase inhibitor is a medicament for preventing and/or treating autoimmune diseases.

7. Use according to claim 6, characterized in that: the autoimmune disease is psoriasis or lupus erythematosus.

8. A medicament, characterized in that: a pharmaceutical preparation prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components into the compound or the salt thereof as an active ingredient in the invention as claimed in claim 1 or 2.

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