CN114380800A - Pyridine-pyrimidine amine-benzimidazole derivative and preparation method and application thereof - Google Patents

Pyridine-pyrimidine amine-benzimidazole derivative and preparation method and application thereof Download PDF

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CN114380800A
CN114380800A CN202210096582.2A CN202210096582A CN114380800A CN 114380800 A CN114380800 A CN 114380800A CN 202210096582 A CN202210096582 A CN 202210096582A CN 114380800 A CN114380800 A CN 114380800A
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赵立峰
张力丹
余洛汀
李潇
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Sichuan University
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Abstract

The invention provides a pyridine-pyrimidine amine-benzimidazole derivative, a preparation method and application thereof, belonging to the field of medicines. The derivative is a compound shown in formula I, or a salt, a stereoisomer, a solvate, a hydrate or a prodrug thereof. The derivative has good inhibitory activity on CDK4 and CDK6 kinase, and can be used for preparing CDK4 and CDK6 kinase inhibitors; meanwhile, the derivative has obvious inhibitory activity on various tumor cells, can inhibit the growth of in vivo tumors and has excellent effect; the derivative has small administration dosage and high safety; in addition, the present inventionThe ramification of the Ming can pass through the blood brain barrier, and solves one of the biggest problems of the treatment of brain tumor. The derivative can be used for preparing medicines for preventing and/or treating tumors and autoimmune diseases, and has good application prospect.
Figure DDA0003491030780000011

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-pyrimidinamine-benzimidazole derivative, and a preparation method and application thereof.
Background
Malignant tumors have high lethality, and the incidence rate thereof is increasing year by year, which exceeds cardiovascular and cerebrovascular diseases and becomes the first killer of human health. CDK4/6, cyclin dependent kinases 4 and 6, are key condition proteins of human cell division and proliferation cycle, and are common downstream targets of multiple 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) which can phosphorylate retinoblastoma protein (Rb). Once Rb is phosphorylated, it releases the transcription factor E2F that is tightly bound to it. Activation of E2F further pushed the cell cycle from G1 into S phase into the cell proliferation cycle.
The deregulation of the Cyclin D-CDK4/6-RB pathway has been found in almost all malignancies. The activation of CDK4/6 is continued, which can induce uncontrolled cell growth and proliferation of cancer cells. And the CDK4/6 is inhibited, so that tumor cells cannot form Cyclin D-CK4/6 complex, and the cell cycle is retarded in the growth phase, thereby achieving the purpose of inhibiting tumor proliferation. Therefore, targeted inhibition of CDK4/6 with novel small molecule inhibitors is a potential therapeutic approach.
Disclosure of Invention
The invention aims to provide a pyridine-pyrimidinamine-benzimidazole derivative, and a preparation method and application thereof.
The invention provides a compound shown as a formula I, or a salt, a stereoisomer, a solvate, a hydrate or a prodrug thereof:
Figure BDA0003491030760000011
wherein,
n1an integer selected from 1 to 3;
n2an integer selected from 0 to 5;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R3Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R4Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxyl, carboxyl, amino and nitro.
Further, the air conditioner is provided with a fan,
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R4Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
Further, the compound is represented by formula II:
Figure BDA0003491030760000021
wherein,
n1an integer selected from 1 to 3;
n2an integer selected from 0 to 5;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R3Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R4Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
preferably, the first and second electrodes are formed of a metal,
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R4Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
Further, the compound is represented by formula III:
Figure BDA0003491030760000031
wherein,
n1an integer selected from 1 to 3;
n2an integer selected from 0 to 5;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R3Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R4Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
preferably, the first and second electrodes are formed of a metal,
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R4Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
Further, the compound is represented by formula IV:
Figure BDA0003491030760000041
wherein,
n1an integer selected from 1 to 3;
n2an integer selected from 0 to 5;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
R1、R2Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R3Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
preferably, the first and second electrodes are formed of a metal,
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
R1、R2Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
Further, the compound is represented by formula V:
Figure BDA0003491030760000042
wherein,
n1an integer selected from 1 to 3;
n2an integer selected from 0 to 5;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
Each R3Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
preferably, the first and second electrodes are formed of a metal,
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
Each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
Further, the compound is represented by formula Va:
Figure BDA0003491030760000051
wherein,
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
X1、Y1each independently selected from N or CR3；
Each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
alternatively, the compound is represented by formula Vb:
Figure BDA0003491030760000052
wherein,
n1an integer selected from 1 to 3;
X1、Y1each independently selected from N or CR3；
Each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
Further, the compound is one of the following compounds:
Figure BDA0003491030760000053
Figure BDA0003491030760000061
Figure BDA0003491030760000071
the invention also provides application of the compound or the salt thereof, or the stereoisomer thereof, or the solvate thereof, or the hydrate thereof, or the prodrug thereof in preparation of a CDK4 and/or CDK6 kinase inhibitor.
Further, the kinase inhibitor is a drug for preventing and/or treating tumors;
preferably, the tumor is brain cancer, glioblastoma, leukemia, lymphoma, Bannayan-Zonana syndrome, cowden 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, giant cell tumor of bone, or thyroid cancer.
Further, the kinase inhibitor is a drug for preventing and/or treating autoimmune diseases;
preferably, the autoimmune disease is psoriasis or lupus erythematosus.
The invention also provides a medicament which is a medicinal preparation prepared by taking the compound, or the salt, the stereoisomer, the solvate, the hydrate or the 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 the IUPAC (international union of pure and applied chemistry) or CAS (chemical abstracts service, Columbus, OH) naming system.
In the present invention, the minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, e.g. Ca～bAlkyl means any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, C1～C8The alkyl refers to a straight chain or branched chain alkyl containing 1-8 carbon atoms; c1～C8The alkoxy group means an alkoxy group having 1 to 8 carbon atoms.
In the present invention, halogen is fluorine, chlorine, bromine or iodine.
In the formula I, n1When is 1, the structure is
Figure BDA0003491030760000072
n1When is 2, the structure is
Figure BDA0003491030760000073
n1When is 3, the structure is
Figure BDA0003491030760000074
In formula I of the invention, when the dotted line is absent, the structure is
Figure BDA0003491030760000075
When the dotted line is a bond, the structure is
Figure BDA0003491030760000076
n2Is 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 obvious inhibitory activity on various tumor cells, can inhibit the growth of in vivo tumors and has excellent effect; the derivative has small administration dosage and high safety; in addition, the derivative can pass through a blood brain barrier, and solves one of the biggest problems of brain tumor treatment. The derivative can be used for preparing medicines for preventing and/or treating tumors and autoimmune diseases, and has good application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the tumor growth of groups of NOD-Bablc mice.
Fig. 2 shows the distribution of compound a2 in blood and brain tissue at different time points.
Detailed Description
Unless otherwise indicated, the starting materials and equipment used in the embodiments of the present invention are known products and obtained by purchasing commercially available products.
Example 1 Compound A15-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
Figure BDA0003491030760000081
The synthetic route for compound a1 is as follows:
Figure BDA0003491030760000082
the first step is as follows:
raw material 1(1g,4.61mmol) was dissolved in anhydrous DMF (15mL), anhydrous potassium carbonate (862mg,6.24mmol) was added, and the mixture was stirred at room temperature for 5 min. Then starting material 2(1.03g,5.532mmol) was added slowly and the reaction stirred at 55 ℃ for 2 h. 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 separated out in the reaction system, the filtration is carried out, the filter cake is washed by water for 3 times, and the drying is carried out to obtain an intermediate 3, wherein the white solid is 1.321g, and the yield is 89%.
The second step is that:
intermediate 3(1.2g,3.72mmol) was dissolved in methanol (25mL), 10% palladium on carbon (396mg,0.372mmol) was added, vacuum was applied, the mixture was replaced with hydrogen three times, and the reaction was stirred at room temperature in a hydrogen atmosphere for 2 hours. TLC monitors the reaction is complete, the reaction liquid is filtered by diatomite, washed 3 times by dichloromethane/methanol (10/1, v/v) mixed solvent, and the filtrate is decompressed and concentrated to dryness to obtain a crude product, namely an intermediate 4. The product was used in the next reaction without further purification.
The third step:
add intermediate 4(3.72mmol), starting material 5(1.282g,3.72mmol), Pd2(dba)3Toluene (20mL) was added to a mixture of (341mg,0.372mmol), X-phos (178mg,0.372mmol) and cesium carbonate (1.82g,5.58mmol), and the mixture was evacuated and replaced with nitrogen 3 times, and the reaction mixture was stirred at 100 ℃ for 5 hours under a nitrogen atmosphere. TLC to monitor the reaction completion, cool the reaction solution to room temperature, filter with diatomaceous earth, and useThe mixture of dichloromethane/methanol (10/1, v/v) was washed 3 times, and the filtrate was concentrated under reduced pressure and subjected to silica gel column chromatography to give intermediate 6 as an off-white solid (1.1 g, yield 51%).
The fourth step:
intermediate 6(1g,1.73mmol) was dissolved in dichloromethane (15mL), and trifluoroacetic acid (4mL) was slowly added dropwise with stirring and reacted at room temperature for 2 h. TLC monitors the reaction to be complete, the reaction solution is decompressed and concentrated to be dry, and is dissolved by a small amount of dichloromethane again, and the operation is repeated for 3 times to obtain a crude product, namely an intermediate 7. The product was used in the next reaction without further purification.
The fifth step:
intermediate 7(0.173mmol) was dissolved in anhydrous DMF (4mL), cesium carbonate (113mg,0.346mmol) was added, stirring was carried out at room temperature for 5min, then starting material 8 (25. mu.L, 0.2595mmol) was slowly added, and the temperature was raised to 60 ℃ for reaction for 3 h. TLC monitors the reaction to be complete, the reaction liquid is cooled to room temperature, a proper amount of ice water is added, extraction is carried out for three times by ethyl acetate, organic phases are combined, washed by water and saturated saline solution, dried by anhydrous sodium sulfate and filtered, filtrate is decompressed and concentrated to be dry, and further chromatographic separation is carried out by a silica gel column to obtain the target compound A1, white solid is 68mg, and the yield is 73%. HR-MS (ESI-TOF) m/z calcd for C28H34F2N8O[M+H]+537.2902,found 537.2900.
Example 2 Compound A25-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
Figure BDA0003491030760000091
Taking the intermediate 7 and 1-bromo-2- (2-methoxyethoxy) ethane as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A2 is obtained as a white solid with a yield of 78%. HR-MS (ESI-TOF) m/z calcd for C30H38F2N8O2[M+H]+581.3164,found 581.3163.
Example 3 Compound A35-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-yl) pyrimidin-2-amine
Figure BDA0003491030760000101
Taking the intermediate 7 and diethylene glycol-2-bromoethyl methyl ether as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A3 is obtained as a white solid with the yield of 69%. HR-MS (ESI-TOF) m/z calcd for C32H42F2N8O3[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
Figure BDA0003491030760000102
The synthetic route for compound a4 is as follows:
Figure BDA0003491030760000103
the first step is as follows:
dissolve starting material 9(2g,11.9mmol) in anhydrous THF (30mL), add DIEA (2.95mL,17.85mmol) and HATU (4.98g,13.09mmol) in that order, stir at room temperature for 10min, then slowly add starting material 2(3.32g,17.85mmol), and continue the reaction with stirring at room temperature for 4 h. TLC, the reaction was monitored to completion, appropriate amount of water was added, extraction was carried out three times with ethyl acetate, the organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated to dryness under reduced pressure, and further separated by silica gel column chromatography to give intermediate 10 as a white solid 2.092g, yield 52%.
The second step is that:
taking the intermediate 10 as a raw material, and obtaining a crude product intermediate 11 by referring to a second step synthesis method of a compound A1. The product was used in the next reaction without further purification.
The third step:
taking the intermediate 11 and the raw material 5 as raw materials, and referring to the third synthesis method of the compound A1, obtaining an intermediate 12 which is yellow solid with the yield of 82%.
The fourth step:
taking the intermediate 12 as a raw material, and obtaining a crude product intermediate 13 by referring to the fourth synthesis method of the compound A1. The product was used in the next reaction without further purification.
The fifth step:
taking the intermediate 13 and the raw material 8 as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A4 is obtained as a yellow solid with the yield of 77%. HR-MS (ESI-TOF) m/z calcd for C28H32F2N8O2[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
Figure BDA0003491030760000111
Taking the intermediate 13 and 1-bromo-2- (2-methoxyethoxy) ethane as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A5 is obtained as a yellow solid with the yield of 66%. HR-MS (ESI-TOF) m/z calcd for C30H36F2N8O3[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
Figure BDA0003491030760000112
Taking intermediate 13 and diethylene glycol-2-bromoethyl methyl ether as raw materialsReferring to the fifth synthesis method of the compound A1, the compound A6 is obtained as a yellow solid with a yield of 74%. HR-MS (ESI-TOF) m/z calcd for C32H40F2N8O4[M+H]+639.3219,found 639.3218.
Example 7 Compound A75-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
Figure BDA0003491030760000121
The synthetic route for compound a7 is as follows:
Figure BDA0003491030760000122
the first step is as follows:
intermediate 11(918mg,3.0mmol) was dissolved in anhydrous THF (20mL) and borane/dimethyl sulfide complex BH was slowly added dropwise3/S(CH3)2(2M in THF) (6mL), then the reaction was warmed to 60 ℃ and stirred for 3 h. TLC monitored the reaction completion, the reaction was cooled to room temperature and piperidine (2.5mL) was added and stirring continued at room temperature for 30 min. The reaction mixture was concentrated to dryness under reduced pressure, and then separated by silica gel column chromatography to give intermediate 14 as a white solid (362 mg, yield 41%).
The second step is that:
taking the intermediate 14 and the raw material 5 as raw materials, and referring to the third synthesis method of the compound A1, the intermediate 15 is obtained and is white solid, and the yield is 76%.
The third step:
taking the intermediate 15 as a raw material, and obtaining a crude product intermediate 16 by referring to the fourth synthesis method of the compound A1. The product was used in the next reaction without further purification.
The fourth step:
taking the intermediate 16 and the raw material 8 as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A7 is obtained as a white solid with the yield of 63%. HR-MS (ESI-TOF)m/z:calcd for C28H34F2N8O[M+H]+537.2902,found 537.2902.
Example 8 Compound A85-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
Figure BDA0003491030760000123
Taking the intermediate 16 and 1-bromo-2- (2-methoxyethoxy) ethane as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A8 is obtained as a white solid with the yield of 70%. HR-MS (ESI-TOF) m/z calcd for C30H38F2N8O2[M+H]+581.3164,found 581.3161.
Example 9 Compound A95-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
Figure BDA0003491030760000131
Taking the intermediate 16 and diethylene glycol-2-bromoethyl methyl ether as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A9 is obtained as a light yellow solid with the yield of 59%. HR-MS (ESI-TOF) m/z calcd for C32H42F2N8O3[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
Figure BDA0003491030760000132
The synthetic route for compound a10 is as follows:
Figure BDA0003491030760000133
the first step is as follows:
by using the raw material 17 as a raw material and referring to the first synthesis method of the compound A4, the intermediate 18 is obtained as a white solid with the yield of 69%.
The second step is that:
taking the intermediate 18 as a raw material, and obtaining a crude product intermediate 19 by referring to the second synthesis method of the compound A1. The product was used in the next reaction without further purification.
The third step:
taking the intermediate 19 and the raw material 5 as raw materials, and referring to the third synthesis method of the compound A1, obtaining an intermediate 20 which is yellow solid with the yield of 77%.
The fourth step:
taking the intermediate 20 as a raw material, and obtaining a crude product intermediate 21 by referring to the fourth synthesis method of the compound A1. The product was used in the next reaction without further purification.
The fifth step:
taking the intermediate 21 and the raw material 8 as raw materials, and referring to the fifth synthesis method of the compound A1, obtaining the compound A10 as a yellow solid with the yield of 65%. HR-MS (ESI-TOF) m/z calcd for C32H39F2N7O4[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
Figure BDA0003491030760000141
Taking the intermediate 21 and 1-bromo-2- (2-methoxyethoxy) ethane as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A11 is obtained as a yellow solid with a yield of 71%. HR-MS (ESI-TOF) m/z calcd for C34H43F2N7O5[M+Na]+690.3192,found 690.3185.
Example 12 Compound A125-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
Figure BDA0003491030760000142
The synthetic route for compound a12 is as follows:
Figure BDA0003491030760000143
the first step is as follows:
5-bromo-2-nitropyridine (1g,4.93mmol) was dissolved in anhydrous DMF (15mL), and anhydrous potassium carbonate (921mg,6.67mmol) was added, followed by stirring at room temperature for 5 min. Then starting material 2(1.03g,5.532mmol) was added slowly and the reaction stirred at 55 ℃ for 2 h. 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 separated out in the reaction system, the filtration is carried out, the filter cake is washed by water for 3 times, and the drying is carried out to obtain an intermediate 22, wherein the white solid is 1.211g, and the yield is 89%.
The second step is that:
intermediate 22(1.2g,3.89mmol) was dissolved in methanol (25mL), 10% palladium on carbon (396mg,0.372mmol) was added, vacuum was applied, the mixture was replaced with hydrogen three times, and the reaction was stirred at room temperature in a hydrogen atmosphere for 2 hours. TLC monitors the reaction is complete, the reaction liquid is filtered by suction by using kieselguhr, is washed by using a mixed solvent of dichloromethane/methanol (10/1, v/v) for 3 times, and the filtrate is decompressed and concentrated to be dry to obtain a crude product, namely an intermediate 23. The product was used in the next reaction without further purification.
The third step:
add intermediate 23(3.72mmol), starting material 5(1.282g,3.72mmol), Pd2(dba)3Toluene (20mL) was added to a mixture of (341mg,0.372mmol), X-phos (178mg,0.372mmol) and cesium carbonate (1.82g,5.58mmol), the mixture was evacuated, nitrogen gas was substituted 3 times, and the reaction mixture was purged with nitrogenThe reaction is stirred for 5 hours at 100 ℃ in a gas environment. TLC monitors the reaction to be complete, the reaction solution is cooled to room temperature, filtered by suction through kieselguhr, washed 3 times by using a mixed solvent of dichloromethane/methanol (10/1, v/v), and the filtrate is concentrated under reduced pressure and then separated by silica gel column chromatography to obtain an intermediate 24 which is 1.05g of an off-white solid with the yield of 52%.
The fourth step:
intermediate 24(1g,1.73mmol) was dissolved in dichloromethane (15mL) and trifluoroacetic acid (4mL) was added dropwise slowly with stirring and reacted at room temperature for 2 h. TLC monitors the reaction to be complete, the reaction solution is decompressed and concentrated to be dry, and is dissolved by a small amount of dichloromethane again, and the operation is repeated for 3 times to obtain a crude product, namely an intermediate 25. The product was used in the next reaction without further purification.
The fifth step:
intermediate 25(0.172mmol) was dissolved in anhydrous DMF (4mL), cesium carbonate (112mg,0.345mmol) was added, stirring was carried out at room temperature for 5min, then starting material 8 (25. mu.L, 0.2595mmol) was slowly added, and the temperature was raised to 60 ℃ for reaction for 3 h. TLC monitors the reaction to be complete, the reaction liquid is cooled to room temperature, a proper amount of ice water is added, extraction is carried out for three times by ethyl acetate, organic phases are combined, washed by water and saturated saline solution, dried by anhydrous sodium sulfate and filtered, filtrate is decompressed and concentrated to be dry, and further chromatographic separation is carried out by a silica gel column to obtain the target compound A12, white solid is 70mg, and the yield is 74%. HR-MS (ESI-TOF) m/z calcd for C27H32F2N8O[M+H]+522.2667,found 522.2667.
Example 13 Compound A135-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
Figure BDA0003491030760000161
Taking the intermediate 25 and 1-bromo-2- (2-methoxyethoxy) ethane as raw materials, and referring to the fifth synthesis method of the compound A12, the compound A13 is obtained as a white solid with a yield of 79%. HR-MS (ESI-TOF) m/z calcd for C29H36F2N8O2[M+H]+566.2929,found 566.2929.
Example 14 Compound A145-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
Figure BDA0003491030760000162
Taking the intermediate 25 and diethylene glycol-2-bromoethyl methyl ether as raw materials, and referring to the fifth synthesis method of the compound A12, the compound A14 is obtained as a white solid with the yield of 70%. HR-MS (ESI-TOF) m/z calcd for C31H40F2N8O3[M+H]+610.3191,found 610.3191.
Example 15 Compound A155-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
Figure BDA0003491030760000163
The synthetic route for compound a15 is as follows:
Figure BDA0003491030760000171
the first step is as follows:
2-bromo-5-nitropyridine (1g,4.93mmol) was dissolved in anhydrous DMF (15mL), and anhydrous potassium carbonate (922mg,6.70mmol) was added, followed by stirring at room temperature for 5 min. Then starting material 2(1.02g,5.530mmol) was added slowly and the reaction stirred at 55 ℃ for 2 h. 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 separated out in the reaction system, the filtration is carried out, the filter cake is washed by water for 3 times, and the drying is carried out to obtain an intermediate 26, wherein the white solid is 1.210g, and the yield is 89%.
The second step is that:
intermediate 26(1.19g,3.88mmol) was dissolved in methanol (25mL), 10% palladium on carbon (395mg,0.371mmol) was added, vacuum was applied, hydrogen gas was substituted three times, and the reaction was stirred at room temperature under hydrogen atmosphere for 2 h. TLC to monitor the reaction is complete, the reaction liquid is filtered by suction through kieselguhr, washed 3 times by a mixed solvent of dichloromethane/methanol (10/1, v/v), and the filtrate is decompressed and concentrated to be dry to obtain a crude product, namely an intermediate 27. The product was used in the next reaction without further purification.
The third step:
taking the intermediate 27 and the raw material 5 as raw materials, and referring to the third synthesis method of the compound A12, the intermediate 28 is obtained as a yellow solid with the yield of 78%.
The fourth step:
taking the intermediate 28 as a raw material, and obtaining a crude product intermediate 29 by referring to the fourth synthesis method of the compound A12. The product was used in the next reaction without further purification.
The fifth step:
taking the intermediate 29 and the raw material 8 as raw materials, and referring to the compound A12 in the fifth synthesis step, the compound A15 is obtained as a white solid with the yield of 63%. HR-MS (ESI-TOF) m/z calcd for C27H32F2N8O[M+H]+522.2667,found 522.2667.
Example 16, Compound A165-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
Figure BDA0003491030760000181
Taking the intermediate 29 and 1-bromo-2- (2-methoxyethoxy) ethane as raw materials, and referring to the fifth synthesis method of the compound A12, the compound A16 is obtained as a white solid with a yield of 70%. HR-MS (ESI-TOF) m/z calcd for C29H36F2N8O2[M+H]+566.2929,found 566.2929.
Example 17 Compound A175-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
Figure BDA0003491030760000182
Taking the intermediate 29 and diethylene glycol-2-bromoethyl methyl ether as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A17 is obtained as a light yellow solid with the yield of 59%. HR-MS (ESI-TOF) m/z calcd for C31H40F2N8O3[M+H]+610.3191,found 610.3191.
Example 18 Compound A185-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
Figure BDA0003491030760000183
The synthetic route for compound a18 is as follows:
Figure BDA0003491030760000184
the first step is as follows:
1-bromo-4-nitrobenzene (1g,4.61mmol) was dissolved in anhydrous DMF (15mL), anhydrous potassium carbonate (860mg,6.22mmol) was added, and stirring was carried out at room temperature for 5 min. Then starting material 2(1.01g,5.530mmol) was added slowly and the reaction stirred at 55 ℃ for 2 h. 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 separated out in the reaction system, the filtration is carried out, the filter cake is washed by water for 3 times, and the drying is carried out to obtain an intermediate 30, wherein the white solid is 1.221g, and the yield is 86%.
The second step is that:
intermediate 30(1.2g,3.71mmol) was dissolved in methanol (25mL), 10% palladium on carbon (395mg,0.371mmol) was added, vacuum was applied, hydrogen gas was substituted three times, and the reaction was stirred at room temperature under hydrogen atmosphere for 2 h. TLC monitors the reaction is complete, the reaction liquid is filtered by suction by using kieselguhr, is washed by using a mixed solvent of dichloromethane/methanol (10/1, v/v) for 3 times, and the filtrate is decompressed and concentrated to be dry to obtain a crude product, namely an intermediate 31. It was used in the next reaction without further purification.
The third step:
add intermediate 31(3.72mmol), starting material 5(1.281g,3.71mmol), Pd2(dba)3Toluene (20mL) was added to a mixture of (340mg,0.3712mmol), X-phos (177mg,0.371mmol) and cesium carbonate (1.81g,5.57mmol), the mixture was evacuated, nitrogen was substituted for 3 times, and the reaction mixture was stirred at 100 ℃ for 5 hours under a nitrogen atmosphere. TLC (thin layer chromatography) is used for monitoring the reaction completion, the reaction solution is cooled to room temperature, the diatomite is filtered by suction, dichloromethane/methanol (10/1, v/v) mixed solvent is used for washing for 3 times, the filtrate is concentrated under reduced pressure, and then silica gel column chromatography is carried out to separate the intermediate 32, white solid 1.05g and yield 50%.
The fourth step:
intermediate 32(1.0g,1.73mmol) was dissolved in dichloromethane (15mL), and trifluoroacetic acid (4mL) was slowly added dropwise with stirring and reacted at room temperature for 2 h. TLC monitors the reaction to be complete, the reaction solution is decompressed and concentrated to be dry, and is dissolved by a small amount of dichloromethane again, and the operation is repeated for 3 times to obtain a crude product, namely an intermediate 33. The product was used in the next reaction without further purification.
The fifth step:
intermediate 33(0.173mmol) was dissolved in anhydrous DMF (4mL), cesium carbonate (113mg,0.346mmol) was added, stirring was carried out at room temperature for 5min, then starting material 8 (25. mu.L, 0.2595mmol) was slowly added, and the temperature was raised to 60 ℃ for reaction for 3 h. TLC monitors the reaction to be complete, the reaction liquid is cooled to room temperature, a proper amount of ice water is added, extraction is carried out for three times by ethyl acetate, organic phases are combined, washed by water and saturated saline solution, dried by anhydrous sodium sulfate and filtered, filtrate is decompressed and concentrated to be dry, and further chromatographic separation is carried out by a silica gel column to obtain the target compound A18, white solid is 68mg, and the yield is 73%. HR-MS (ESI-TOF) m/z calcd for C28H33F2N7O[M+H]+522.2715,found 522.2715.
Example 19 Compound A195-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
Figure BDA0003491030760000201
Taking the intermediate 33 and 1-bromo-2- (2-methoxyethoxy) ethane as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A2 is obtained as a white solid with a yield of 78%. HR-MS (ESI-TOF) m/z calcd for C30H37F2N7O2[M+H]+565.2977,found 565.2977.
EXAMPLE 20 Compound A205-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-yl) pyrimidin-2-amine
Figure BDA0003491030760000202
Taking the intermediate 33 and diethylene glycol-2-bromoethyl methyl ether as raw materials, and referring to the fifth synthesis method of the compound A1, the compound A20 is obtained as a white solid with the yield of 68%. HR-MS (ESI-TOF) m/z calcd for C32H41F2N7O3[M+H]+609.3239,found 609.3239.
The beneficial effects of the present invention are demonstrated by the following in vitro tests.
Test example 1, CDK4, CDK6 kinase inhibitory Activity test
The purpose of this experiment was to detect the inhibitory activity of the compounds of the invention on kinases in vitro, using an isotopic labeling approach. In vitro activity inhibition assays were performed on CDK4 and CDK6 kinases, respectively, in this experiment. Abemaciciclib was a positive control. IC for kinase inhibitory Activity of test Compounds50(median inhibitory concentration). IC (integrated circuit)50Values can be obtained by calculation of the rate of inhibition of kinase activity by the test compound at a range of different concentrations.
1. Experimental Material
20mM 3- (N-morpholinyl) propanesulfonic acid (MOPS); 1mM ethylenediaminetetraacetic acid (EDTA); 0.01% Brij 35 (Brij-35); 5% Glycerol (glycol); 0.1% mercaptoethanol (mercptoeethanol); 1mg/ml cattleSerum Albumin (BSA); l0 solution of 0mM manganese dichloride (MnC 1)2) (ii) a Stop buffer (3% phosphate buffer); wash buffer (75mM phosphate solution); methanol (methanol); a Filtermat A film; CDK4/6 kinase, test compound.
2. Experimental methods
Adding the kinase to be detected and corresponding substrate together with different concentrations of compound to be detected or DMSO into reaction buffer (8mM propanesulfonate, 0.2mM EDTA, 10mM magnesium acetate and Km concentration gamma-33P-ATP solution), and incubating at room temperature for 40 min; 3% phosphate solution was added to stop the reaction; pipette 10. mu.L of the reaction mixture onto P30 filter paper; the filter paper was washed 3 times with 75mM phosphate solution; then the filter paper is washed by methanol for 1 time; after the filter paper is air dried, scintillation fluid is added and the phosphorylated substrate is measured by scintillation counting. Inhibition [% ], [% ] - (1-compound treatment group/blank group ] × 100%; median Inhibitory Concentration (IC)50) And fitting the inhibition ratios corresponding to the concentrations.
3. Results of the experiment
By the above experimental methods, the compounds of the present invention were tested for their inhibitory activity against CDK4/6 kinase, respectively. Table 1 shows the IC of the test compounds for CDK4/6 kinase inhibitory activity50The value is obtained.
The experimental result shows 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 Activity50Value of
Figure BDA0003491030760000211
*A:IC50<50nM；B:50nM≤IC50<100nM；C:100nM≤IC50<150nM；D:IC50＞150nM
Test example 2 in vitro tumor cell proliferation inhibition test of Compound
The purpose of the experiment is to detect the in vitro tumor cell proliferation inhibition activity of the compound, and the adopted method is an MTT (tetramethyl azozolium) colorimetric method.
1. Experimental Material
1.1 Primary reagents
Breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-436 and MDA-MB-468, colorectal cancer cell lines SW620 and lung cancer cell lines A549 were purchased from American Type Culture Collection (ATCC), RPMI 1640 medium and Fetal Bovine Serum (FBS) were purchased from GIBICO corporation, USA; penicillin and streptomycin were purchased from Dalibao bio; cultured cells were purchased from Corning, such as plates and 96-well plates; centrifuge tubes of various specifications were purchased from BD corporation; MTT reagent was purchased from Donjind, Japan institute of Homond chemistry. The test compound is synthesized by the inventor, 100% DMSO is used for preparing 10mM stock solution during in vitro experiments, the stock solution is placed in a refrigerator at the temperature of minus 20 ℃ and is kept in the dark for standby, and the culture solution is used for diluting to the required concentration during the tests.
1.2 cell lines and culture
The breast cancer cell strains MCF-7, MDA-MB-231, MDA-MB-436 and MDA-MB-468 used in the experiment, the colorectal cancer cell strain SW620 and the lung cancer cell A549 are cultured in a conventional RPMI 1640 complete medium containing 10 percent Fetal Bovine Serum (FBS), 100IU/mL penicillin and 100 mu g/mL streptomycin at 37 ℃ and 5 percent CO2Cultured in an incubator.
2. Experimental methods
Adjusting the cell concentration to 1-2 × 10 by using a whole cell culture solution4Cell suspension per ml (HCC827 cell concentration 6X 10)4Cell density of H1975 cell 4X 103Pieces/ml) were inoculated into 96-well plates, 200. mu.L of cell suspension per well, and cultured overnight. The following day, the supernatants were aspirated and the cells were treated with a gradient concentration of test compound, respectively. Setting a negative control group without drug and a solvent control group with the same volume, wherein the concentration of DMSO is 0.1%, each dose group is provided with 3 multiple wells, and the concentration of DMSO is 5% CO at 37 DEG C2Culturing under the condition. After 72 hours, 20 μ L of MTT reagent with a concentration of 5mg/ml was added to each well, and after further culturing for 2 to 4 hours, the supernatant was discarded, 150 μ L of DMSO was added to each well, and mixed by shaking for 15 minutes, and the value of absorbance (a) (a value is proportional to the number of living cells) was measured with a microplate reader (λ. lamda. ═ 570nm), and the average value was taken. Relative inhibition of cell proliferationThe yield was (control a 570-experimental a 570)/control a570 × 100%. The experiment was repeated at least 3 times. The experimental data are expressed by mean, and the statistical data are tested by t, P<A difference of 0.05 is statistically significant. IC was used for inhibition of cell proliferation by each of the following compounds50And (4) showing.
3. Results of the experiment
By adopting the method, the proliferation inhibition activity test is carried out on breast cancer cell strains MCF-7, MDA-MB-231, MDA-MB-436 and MDA-MB-468, colorectal cancer cell strains SW620 and lung cancer cells A549. Table 2 shows the proliferation inhibitory activity (IC) of the test compounds50). The results show that most of the compounds in the examples show significant proliferation inhibition activity on 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 treatment advantages compared with positive compounds Abemaciclib.
TABLE 2 proliferation of tumor cell lines by Compounds (MTT method)
Figure BDA0003491030760000231
Note: ND is not determined.
A:IC50<0.1μM；B:0.1μM≤IC50<1μM；C:IC50≥1μM.
Test example 3 and in vivo antitumor test of Compound A2
The purpose of this experiment was to examine the in vivo anti-tumor effect of the compounds of the invention. Experimental the in vivo anti-tumor activity of Compound A2 was tested in a BALB/c mouse subcutaneous tumor model. The cell strain used was human colon cancer cell strain COLO 205.
1. Experimental Material
Fetal bovine serum, medium, pancreatin, etc. were purchased from Gibco BRL (Invitrogen Corporation, USA), medium was purchased from ATCC (American Type Culture Collection), human colon cancer cell line COLO205 was purchased from American ATCC, and NOD-Bablc mice were purchased from Beijing Huafukang animal Experimental center.
2. Experimental methods
6-8 weeks of NOD-Bablc mice were used at approximately 1X 107One/0.1 ml/COLO 205 cell concentration was inoculated into the subcutaneous posterior costal region of mice, and after tumor growth reached a certain volume, mice were randomly grouped and gavage oral and tail vein administration was started.
Observation indexes are as follows: the length and the length of the tumor were measured once every 3 days, the tumor volume (length x length 2 x 0.52) was calculated, and the presence or absence of diarrhea, cramping, rash, significant weight loss, and other reactions were observed.
3. Results of the experiment
The experimental measurements of tumor growth for various groups are shown in FIG. 1. The experimental results show that the tested compound A2 has obvious in vivo growth inhibition effect on COLO 205. No adverse reactions such as weight loss, rash, diarrhea, etc. were observed in the mice during the administration, indicating that the test compound A2 was very toxic in the administered dose range at the tested dose.
Test example 4 blood brain tissue distribution test of Compound A2
The purpose of this experiment was to examine the in vivo anti-tumor effect of the compounds of the invention. In vivo anti-tumor activity of compound A2 was tested using a BALB/c mouse brain tumor in situ model. The cell line used was human brain astrocyte U87-LUC.
1. Experimental Material
Fetal bovine serum, medium, pancreatin, etc. were purchased from Gibco BRL Corporation (Invitrogen Corporation, USA), medium was purchased from ATCC (American Type Culture Collection), and NOD-Bablc mice were purchased from the Beijing Wakkuk animal Experimental center.
2. Experimental methods
SPF grade ICR mice were used and weighed before administration, and the dose was calculated from the body weight. Administration is by intravenous injection.
Plasma sample treatment: blood was collected by cardiac puncture, about 0.2mL of each sample was collected, heparin sodium was anticoagulated, the samples were placed on ice after collection, and plasma was centrifuged within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8 ℃). Plasma samples were stored in a-80 ℃ freezer prior to analysis.
Tissue sample treatment: after the animals were euthanized, brain tissue was collected, washed with normal saline to avoid cross contamination, filter paper was blotted dry, and then placed into labeled tubes/self-sealing bags (one tube/self-sealing bag for each tissue), and the samples were temporarily placed on ice before being stored in a super low temperature refrigerator.
Grouping experiments: drug solvent control group (12.5% EL + 12.5% EtOH + 75% water)
50mg/kg q.d of Compound A2-M;
compound A2-H100 mg/kg q.d;
50mg/kg q.d of compound Abemaciclib;
(Each group of drugs dissolved in 12.5% EL + 12.5% EtOH + 75% water)
And (4) analyzing results: pharmacokinetic parameters are calculated by using Phoenix WinNonlin7.0 through blood concentration data at different time points, and parameters such as AUC0-T, AUC0- ∞, MRT0- ∞, Cmax, Tmax, T1/2 and the like, and mean values and standard deviations thereof are provided.
3. Results of the experiment
The distribution of compound a2 in blood and brain tissue at different time points is shown in figure 2. The experimental results show that the concentration of the drug in the brain tissue is higher than that in the plasma of the test compound A2 at different time points. The compound A2 can penetrate through the blood brain barrier and directly enter the brain to play a role, and can be used for developing medicines aiming at brain tumors.
In conclusion, the pyridine-pyrimidinamine-benzimidazole derivative provided by the invention has good inhibitory activity on CDK4 and CDK6 kinase, and can be used for preparing CDK4 and CDK6 kinase inhibitors; meanwhile, the derivative has obvious inhibitory activity on various tumor cells, can inhibit the growth of in vivo tumors and has excellent effect; the derivative has small administration dosage and high safety; in addition, the derivative can pass through a blood brain barrier, and solves one of the biggest problems of brain tumor treatment. The derivative can be used for preparing medicines for preventing and/or treating tumors and autoimmune diseases, and has good application prospect.

Claims (12)

1. A compound represented by formula I, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof:
Figure FDA0003491030750000011
wherein,
n1an integer selected from 1 to 3;
n2an integer selected from 0 to 5;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R3Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R4Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxyl, carboxyl, amino and nitro.
2. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof, characterized in that:
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R4Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
3. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof, characterized in that: the compound is represented by formula II:
Figure FDA0003491030750000021
wherein,
n1an integer selected from 1 to 3;
n2an integer selected from 0 to 5;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R3Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R4Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
preferably, the first and second electrodes are formed of a metal,
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R4Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
4. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof, characterized in that: the compound is represented by formula III:
Figure FDA0003491030750000022
wherein,
n1an integer selected from 1 to 3;
n2an integer selected from 0 to 5;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R3Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
each R4Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
preferably, the first and second electrodes are formed of a metal,
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
X1、Y1each independently selected from N or CR3；
X2、Y2Each independently selected from N or CR4；
R1、R2Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R4Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
5. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof, characterized in that: the compound is represented by formula IV:
Figure FDA0003491030750000031
wherein,
n1an integer selected from 1 to 3;
n2an integer selected from 0 to 5;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
R1、R2Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy radicalHalogen, hydroxyl, carboxyl, amino, nitro;
each R3Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
preferably, the first and second electrodes are formed of a metal,
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
R1、R2Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
6. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof, characterized in that: the compound is represented by formula V:
Figure FDA0003491030750000041
wherein,
n1an integer selected from 1 to 3;
n2an integer selected from 0 to 5;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
Each R3Are respectively and independently selected from hydrogen and C1～C8Alkyl radical, C1～C8Alkoxy, halogen, hydroxy, carboxyl, amino, nitro;
preferably, the first and second electrodes are formed of a metal,
n1selected from 1 to 3 integersCounting;
n2is selected from 0 or 1;
the dotted line is a bond or nothing;
X1、Y1each independently selected from N or CR3；
Each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
7. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof, characterized in that: the compound is shown as formula Va:
Figure FDA0003491030750000042
wherein,
n1an integer selected from 1 to 3;
n2is selected from 0 or 1;
X1、Y1each independently selected from N or CR3；
Each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen;
alternatively, the compound is represented by formula Vb:
Figure FDA0003491030750000051
wherein,
n1an integer selected from 1 to 3;
X1、Y1each independently selected from N or CR3；
Each R3Are respectively and independently selected from hydrogen and C1～C3Alkyl radical, C1～C3Alkoxy, halogen.
8. The compound according to claim 1, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a hydrate thereof, or a prodrug thereof, characterized in that: the compound is one of the following compounds:
Figure FDA0003491030750000052
Figure FDA0003491030750000061
9. use of a compound according to any one of claims 1 to 8, 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.
10. Use according to claim 9, characterized in that: the kinase inhibitor is a medicament for preventing and/or treating tumors;
preferably, the tumor is brain cancer, glioblastoma, leukemia, lymphoma, Bannayan-Zonana syndrome, cowden 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, giant cell tumor of bone, or thyroid cancer.
11. Use according to claim 9, characterized in that: the kinase inhibitor is a medicament for preventing and/or treating autoimmune diseases;
preferably, the autoimmune disease is psoriasis or lupus erythematosus.
12. A medicament, characterized by: the pharmaceutical preparation is prepared by taking the compound or the salt thereof, the stereoisomer thereof, the solvate thereof, the hydrate thereof or the prodrug thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105294683A (en) * 2014-07-26 2016-02-03 广东东阳光药业有限公司 CDK small-molecule inhibitor compounds and application thereof
CN113880816A (en) * 2020-07-01 2022-01-04 杭州百诚医药科技股份有限公司 Piperazine-containing aminopyrimidine derivative and application thereof
CN113880809A (en) * 2020-07-03 2022-01-04 盛世泰科生物医药技术(苏州)有限公司 Pyrimidine derivative and preparation method and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105294683A (en) * 2014-07-26 2016-02-03 广东东阳光药业有限公司 CDK small-molecule inhibitor compounds and application thereof
CN105294655A (en) * 2014-07-26 2016-02-03 广东东阳光药业有限公司 CDK small-molecule inhibitor compounds and application therefore
CN113880816A (en) * 2020-07-01 2022-01-04 杭州百诚医药科技股份有限公司 Piperazine-containing aminopyrimidine derivative and application thereof
CN113880809A (en) * 2020-07-03 2022-01-04 盛世泰科生物医药技术(苏州)有限公司 Pyrimidine derivative and preparation method and application thereof

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