N-pyrimidinyl aniline CDK12 / 13 inhibitor as well as preparation and application thereof

Abstract

The invention belongs to the field of medical chemistry, and particularly relates to an N-pyrimidinylaniline CDK12 / 13 inhibitor as shown in a formula I or an isomer and pharmaceutically acceptable salt thereof, a preparation method of the N-pyrimidinylaniline CDK12 / 13 inhibitor and a pharmaceutical composition containing the N-pyrimidinylaniline CDK12 / 13 inhibitor. According to the present invention, the meta-position of pyridyl of the compound represented by the formula I is linked with the aromatic group phenyl group through the flexible amino group, such that the affinity of the compound and the CDK12 / 13 protein is increased, the CDK12 / 13 kinase selectivity is improved, the water solubility of the compound is significantly improved, the synthesis difficulty is reduced, and the druggability of the compound is substantially improved,

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to an N-pyrimidinylaniline CDK12 / 13 inhibitor or its isomers, pharmaceutically acceptable salts, their preparation methods, pharmaceutical compositions containing these compounds, and the use of these compounds or compositions for preparing drugs for treating CDK12 / 13-mediated diseases. Background Art

[0002] Cyclin-dependent kinases (CDKs) are a family of protein kinases that catalyze serine / threonine phosphorylation and are closely related to the cell cycle and transcription. They have been confirmed to be oncogenic drivers. 21 subtypes of CDKs have been found. Different CDK subtypes have different functions, but they have highly similar ATP binding pockets. Therefore, although many reported pan-CDK inhibitors have significant anti-tumor effects, they also have problems such as large toxic side effects, which seriously limit their clinical application. Therefore, it is of great significance to develop selective CDK inhibitors.

[0003] Studies have shown that CDK12 and CDK13 play a series of important biological functions in gene transcription regulation, participation in pre-mRNA splicing, promotion of cell proliferation and differentiation, and DNA damage response. With the continuous deepening of the pathogenic mechanism and genomic research of breast cancer and other tumors, studies have found that CDK12 and CDK13 are abnormally expressed or mutated in a variety of cancers, which are closely related to their occurrence and development. CDK12 / 13 is expected to become a new biomarker and a new target for personalized treatment of breast cancer and prostate cancer patients. By inhibiting CDK12 / 13 kinases, the sensitivity of tumor cells to DNA damaging drugs can be enhanced, and genomic instability can be increased, thereby effectively inhibiting tumor growth. Therefore, the development of CDK12 / 13 inhibitors has become a potential new strategy for tumor treatment. Summary of the invention

[0004] One object of the present invention is to provide an N-pyrimidinylaniline CDK12 / 13 inhibitor or its isomers and pharmaceutically acceptable salts shown in general formula I. The compound of general formula I is linked to an aromatic group phenyl at the meta position of the pyridyl group through a flexible amino group, which increases the affinity of the compound of the present invention to the CDK12 / 13 protein and improves its CDK12 / 13 kinase selectivity. At the same time, the water solubility of the compound of the present invention is significantly improved, the difficulty of synthesis is reduced, and its drugability is greatly improved.

[0005] Another object of the present invention is to provide a method for preparing the compound of formula I or a pharmaceutically acceptable salt thereof.

[0006] Another object of the present invention is to provide a composition comprising a compound of the general formula I of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, as well as a composition comprising a compound of the general formula I of the present invention or a pharmaceutically acceptable salt thereof and another one or more drugs.

[0007] Another object of the present invention is to provide use of the compound of general formula I of the present invention or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing CDK12 / 13 related diseases.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a CDK12 / 13 inhibitor or an isomer or a pharmaceutically acceptable salt thereof as shown in Formula I:

[0010]

[0011] in

[0012] R 1 is a halogen;

[0013] R 2 Selected from alkyl or cycloalkyl;

[0014] R 3 Selected from alkyl or cycloalkyl;

[0015] n is selected from 0 or 1. When n is 0, It is a tetrahydropyrrole ring.

[0016] In some preferred embodiments, R 1 is selected from chlorine or bromine; preferably, R 1 For bromine.

[0017] In some preferred embodiments, R 2 Selected from C 1-3 Alkyl, C 3-6 Cycloalkyl; preferably, R 2 Selected from methane, ethyl, propyl or cyclopropyl.

[0018] In some preferred embodiments, R 3 Selected from C 1-3 Alkyl, C 3-6 Cycloalkyl; preferably, R 3 Selected from methane, ethyl, propyl or cyclopropyl.

[0019] In some preferred embodiments, is selected from a tetrahydropyrrole ring or a piperidine ring; preferably, It is a tetrahydropyrrole ring.

[0020] The present invention provides the following specific compounds or their isomers and pharmaceutically acceptable salts:

[0021]

[0022]

[0023] The inventors found that connecting a monoaromatic group with an amino flexible chain at the 4-position of the pyrimidine group can improve its affinity with the CDK12 / 13 protein, thereby improving its selectivity, and the introduction of the amino flexible chain increases the water solubility.

[0024] In another aspect, the present invention provides a method for preparing the compound of the general formula (I) of the present invention, comprising the following synthetic steps:

[0025] Step 1: The compound of formula (1) undergoes a nucleophilic substitution reaction with aniline to obtain a compound of formula (2);

[0026] Step 2: reacting the compound of formula (2) with the compound of formula (3) to obtain the compound of formula (4);

[0027] Step 3: removing the protecting group from the compound of formula (4) to obtain the compound of formula (5);

[0028] Step 4: condensing the compound of formula (6) with the compound of formula (7) to obtain the compound of formula (8);

[0029] Step 5: The compound of formula (8) reacts with the compound of formula (5) to obtain a compound of general formula (I). The reaction scheme is as follows:

[0030]

[0031] Among them, R 1 , R 2 , R 3 and n have the meanings as described for the general formula (I).

[0032] In a third aspect, the present invention provides a pharmaceutical composition comprising the compound of the present invention or an isomer or a pharmaceutically acceptable salt thereof.

[0033] In some embodiments, the present invention provides a compound of the present invention or an isomer thereof, a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the compound of the present invention or an isomer thereof, a pharmaceutically acceptable salt thereof, wherein the compound or pharmaceutical composition is used to treat a CDK12 / 13-mediated disease.

[0034] In some embodiments, the present invention provides a pharmaceutical composition comprising a compound of the present invention or an isomer thereof, a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

[0035] The compound or isomer of the present invention, its pharmaceutically acceptable salt and pharmaceutically acceptable carrier, diluent or excipient can be mixed and prepared into a pharmaceutical preparation, suitable for oral or parenteral administration. The method of administration includes, but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal and oral routes. The preparation can be administered by any route, such as by infusion or push injection, by the route of absorption through the epithelium or skin mucosa (such as oral mucosa or rectum, etc.). Administration can be systemic or local. Examples of oral administration preparations include solid or liquid dosage forms, specifically, tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions, etc. The preparation can be prepared by methods known in the art, and includes carriers, diluents or excipients conventionally used in the field of pharmaceutical preparations.

[0036] In a fourth aspect, the present invention provides the use of the compound represented by Formula I of the present invention or its isomers, pharmaceutically acceptable salts, or pharmaceutical compositions comprising the same in the preparation of drugs for treating CDK12 / 13-mediated diseases.

[0037] In some preferred embodiments, the present invention provides use of a compound of formula I of the present invention or its isomers, pharmaceutically acceptable salts, or pharmaceutical compositions comprising the same in the preparation of drugs for treating CDK12 / 13-mediated diseases, wherein the CDK12 / 13-mediated diseases include but are not limited to breast cancer, ovarian cancer, and prostate cancer.

[0038] Explanation of terms

[0039] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0040] The "halogen" in the present invention refers to fluorine, chlorine, bromine or iodine.

[0041] The "pharmaceutically acceptable salt" of the present invention refers to the salt of the compound of the present invention, which is safe and effective when used in mammals and has the desired biological activity.

[0042] The "pharmaceutical composition" of the present invention refers to a mixture comprising any one of the compounds of the present invention, including corresponding isomers, prodrugs, solvates, pharmaceutically acceptable salts or chemically protected forms thereof, and one or more pharmaceutically acceptable carriers and / or one or more other drugs. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to an organism. The composition is generally used to prepare a drug for the treatment and / or prevention of a disease mediated by one or more kinases.

[0043] The "pharmaceutically acceptable carrier" of the present invention refers to a carrier that does not cause significant irritation to an organism and does not interfere with the biological activity and properties of the administered compound, including all solvents, diluents or other excipients, dispersants, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. Unless any conventional carrier medium is incompatible with the compound of the present invention. Some examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, as well as cellulose and cellulose acetate; malt, gelatin, etc.

[0044] The "excipient" of the present invention refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Excipients may include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a graph showing the effect of compound I-20 on the proliferation ability of breast cancer cells.

[0046] Figure 2 This is a graph showing the selective inhibition of CDK12 / CDK13 / CDK7 protein expression in breast cancer cells by compound I-20.

[0047] Figure 3 This is a graph showing the effect of compound I-20 on breast cancer cell apoptosis.

[0048] Figure 4 This is a graph showing the effect of compound I-20 on the cell cycle of breast cancer.

[0049] Figure 5 This is a graph showing the effect of compound I-20 on breast cancer cell migration. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with the examples, but the present invention is not limited to these examples. The materials used in the following examples are all commercially available unless otherwise specified.

[0051] Example 1: Preparation of (E)-N-(3-(((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-1)

[0052] Step 1: Synthesis of 2,5-dichloro-N-phenylpyrimidin-4-amine

[0053] 2,4,5-trichloropyrimidine (1 g, 5.46 mmol), aniline (0.56 g, 6.02 mmol), and N,N-diisopropylethylamine (DIEA) (0.7 g, 5.43 mmol) were dissolved in 20 ml of isopropanol and stirred at N 2 The reaction was carried out under condensation reflux conditions with protection and an external temperature of 90°C. After 2 h of reaction, the reaction solution was poured into a beaker and water (H 2 O) and ethyl acetate (EA) (150 ml×3), washed with saturated sodium carbonate and saturated brine in sequence, the organic phases were combined, dried over anhydrous sodium sulfate powder, and the organic layer was spin-dried to obtain 1.3 g of a yellow solid with a yield of 98%.

[0054] Step 2: Synthesis of tert-butyl 4-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carboxylate

[0055] Weigh 2,5-dichloro-N-phenylpyrimidine-4-amine (1.3 g, 5.44 mmol), tert-butyl 4-aminopiperidine-1-carboxylate (1.08 g, 5.4 mmol), and N,N-diisopropylethylamine (DIEA) (0.70 g, 5.42 mmol) and dissolve them in N-methylpyrrolidone (NMP). 2 After protection and reflux reaction at 135°C for 3 h, the reaction solution was added to a beaker and water (H 2 O) and ethyl acetate (EA) (150 ml×3), washed with saturated sodium carbonate and saturated brine in sequence, the organic phases were combined, dried over anhydrous sodium sulfate, and the organic layer was spin-dried to obtain a dark yellow liquid. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to obtain 1.05 g of a white powdery solid compound in a yield of 48%.

[0056] Step 3: 5-Chloro-N 4 -phenyl-N 2 Synthesis of -(piperidin-4-yl)pyrimidine-2,4-diamine

[0057] 4-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carboxylic acid tert-butyl ester (1.05 g, 2.61 mmol) was added to 20 ml of 1,4-dioxane solution, and then 0.33 ml of concentrated hydrochloric acid was added. 2 The reaction was allowed to proceed at room temperature for 3 h under protection. TLC monitoring showed that the reaction was complete. The reaction was cooled to room temperature, 100 ml of ethyl acetate (EA) was added to quench the reaction, and saturated sodium carbonate (Na 2 CO 3 ) solution was adjusted to an alkaline pH and then transferred to a separatory funnel. After standing and stratification, the organic layer was rotary evaporated and the residue was purified by silica gel column chromatography to obtain 0.62 g of a light yellow solid compound with a yield of 77%.

[0058] Step 4: Synthesis of (E)-4-(3-(dimethylamino)-2-butenylamido)benzoic acid

[0059] Weigh N,N-dimethyl trans-crotonic acid (2g, 12.08mmol) and DMF (1ml) and dissolve them in acetonitrile (20ml). Under ice bath conditions, dissolve oxalyl chloride (1.6ml, 18.12mmol) in acetonitrile (8ml), add and stir to react. After the reaction is clarified, add to the reaction bottle of methyl 3-aminobenzoate (2g, 5.79mmol) and acetonitrile (15ml). After the addition is complete, move it into the reaction bottle at room temperature and stir to react for 1.5h. Pour the reaction solution into a beaker, add ethyl acetate (EA) and water to dilute, add saturated sodium carbonate solution to adjust the pH of the solution to alkaline, transfer it to a separatory funnel, separate the liquid, and obtain an oily product by column chromatography. Then add NaOH (0.22g, 1.5eq) dissolved in anhydrous ethanol (10ml) to the obtained product. The reaction mixture was condensed and refluxed at an external temperature of 50° C. After the reaction was complete, heating was stopped and HCl was added until the pH value was acidic. The resulting liquid was rotary evaporated to precipitate 1.24 g of a white solid with a yield of 62.3%.

[0060] Step 5: Synthesis of (E)-N-(3-(((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-1)

[0061] Take compound 5-chloro-N 4 -phenyl-N 2 -(Piperidin-4-yl)pyrimidine-2,4-diamine (0.62g, 2.04mmol) and compound (E)-4-(3-(dimethylamino)-2-butenylamino)benzoic acid (0.73g, 2.57mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.59g, 3.09mmol), (1-hydroxybenzotriazole) (0.40g, 3.05mmol), N,N-diisopropylethylamine (0.49g, 3.80mmol) were dissolved in N,N-dimethylformamide in a 50ml reaction bottle, and reacted at room temperature for 1.5h. TLC monitoring showed that the reaction was complete. Saturated saline solution, water, and ethyl acetate were added. After standing and stratification, the organic layer was rotary evaporated, and the residue was purified by silica gel column chromatography to obtain 150mg of a white solid compound with a yield of 13.8%. 1 HNMR (600 MHz, DMSO-d 6)δ11.11(s,1H),10.78(s,1H),8.62(s,1H),10.10(s,1H),8.31(s,1H),7.86(s,1H),7.69( t,J=14.1Hz,1H),7.61(d,J=7.7Hz,1H),7.42(q,J=8.4,7.9Hz,3H),7.25(t,J=7.4Hz,1H),7 .09(d,J=7.5Hz,1H),6.90-6.84(m,1H),6.55(d,J=15.3Hz,1H),4.29(s,1H),4.01-3.93(m, 2H),3.77(s,2H),2.99-2.91(m,2H),2.75(d,J=3.7Hz,6H),1.84-1.89(m,2H),1.47(s,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ175.41,169.16,163.85,159.90,156.04,155.62,142.11,139.69,139.52,137.18,129.41,128.69,126.26,12 3.59,122.37,121.87,120.42,117.76,106.46,101.84,60.12,55.33,46.70,45.58,41.14,34.78,31.68,29.90.

[0062] Example 2: Preparation of (E)-N-(3-((4-(5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)cyclohexyl)amino)phenyl)-4-(dimethylamino)but-2-enamide (I-2)

[0063] The operation was the same as in Example 1, and 47 mg of white powder was obtained by column chromatography. 1 H NMR (600 MHz, DMSO-d 6)δ11.22(s,1H),10.70(s,1H),10.05(s,1H),8.42(s,1H),8.36–8.17(m,1H),8.08(s,1H ),7.90-7.82(m,1H),7.66(d,J=8.0Hz,2H),7.51-7.55(m,1H),7.41(m,3H),7.27(d,J=7. 8Hz,1H),6.81-6.85(m,1H),6.55(d,J=15.3Hz,1H),3.93(t,J=5.8Hz,2H),3.74-3.76(m, 2H),2.75(d,J=4.3Hz,6H),2.00–1.91(m,2H),1.87(d,J=10.1Hz,2H),1.55–1.25(m,4H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.05,162.74,160.03,157.57,154.84,139.39,136.10,132.40,132.27,128.91,128.81,124.51,123. 63,122.73,122.47,119.42,116.17,99.99,96.37,59.26,56.96,50.72,48.04,47.22,42.06,31.04,30.96.

[0064] Example 3: Preparation of (E)-N-(3-(((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-3)

[0065] The operation was the same as in Example 1, and 76 mg of white powder was obtained by column chromatography. 1 H NMR (600 MHz, DMSO-d 6 )δ10.45(s,1H),8.88-8.52(m,1H),8.00(s,1H),7.86–7.66(m,3H),7.46(s,1H),7.41-7.34(m,3H),7.19–6.99(m,2H),6.88(s,1H),6.74( s,1H),6.34(s,1H),4.48(s,1H),3.51(s,2H),3.06(d,J=6.0Hz,2H), 2.94-2.86(m,2H),2.18(s,6H),1.93-1.82(m,2H),1.59-1.51(m,2H). 13 C NMR (151 MHz, DMSO-d 6)δ163.94,163.77,159.92,156.35,149.53,142.12,141.98,139.82,139.65,137.33,137.09,128.81,126.4 7,123.60,122.45,121.76,120.42,117.74,100.73,60.16,48.95,47.71,45.57,42.17,30.73,21.23,14.56.

[0066] Example 4: Preparation of (E)-N-(3-(((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)pyrrolidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-4)

[0067] The operation was the same as in Example 1 to obtain 53 mg of yellow-white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.17(d,J=11.6Hz,1H),8.54(s,1H),7.92-7.98(m,1H),7.89(s,1H),7.77(d,J=8. 1Hz,1H),7.62-7.65(m,2H),7.49–7.29(m,3H),7.26–7.11(m,2H),7.10–6.94(m,1H), 6.73-6.75(m,1H),6.35–6.17(m,1H),4.31(s,1H),3.80–3.60(m,2H),3.60–3.36(m,2 H),3.06(d,J=5.8Hz,2H),2.18(d,J=1.6Hz,6H),2.14–2.05(m,1H),2.03–1.87(m,1H). 13 C NMR (151 MHz, DMSO-d 6 )δ172.11,168.61,163.88,160.21,156.07,142.13,139.53,137.82,129.11,129.05,128.73,128.67,128.63,12 6.25,123.68,122.52,122.39,122.36,120.85,118.46,60.19,54.80,51.99,47.72,45.61,44.81,32.00,29.65.

[0068] Example 5: Preparation of N-((1R,4R)-4-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)cyclohexyl)-3-((E)-4-(dimethylamino)but-2-enamide)benzamide (I-5)

[0069] The operation was the same as in Example 1 to obtain 65 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.20(s,1H),8.57(s,1H),8.24(s,1H),8.02(t,J=1.9Hz,1H),7.96(s,1H),7.90–7.83(m ,1H),7.79(d,J=8.0Hz,2H),7.52(d,J=7.7Hz,1H),7.39(t,J=7.9Hz,1H),7.33(t,J=7.7Hz, 2H),7.01-7.06(m,1H),6.96(s,1H),6.76-6.78(m,1H),6.24-6.28(m,1H),3.76(s,1H),3.5 1(s,1H),3.05-3.08(m,2H),2.19(s,6H),1.97(s,2H),1.91-1.83(m,2H),1.48-1.30(m,4H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.05,163.81,160.10,155.96,155.54,155.26,153.53,145.14,141.92,139.69,139.62,136.18,128.93,128. 64,126.38,123.41,122.36,122.20,122.08,119.19,69.99,60.16,50.30,48.51,45.56,31.63,22.73,22.27,9.71.

[0070] Example 6: Preparation of 3-acrylamido-N-(4-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)cyclohexyl)benzamide (I-6)

[0071] The same operation as in Example 1 was performed to obtain 77 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6)δ10.41(s,1H),10.10(s,1H),8.44–8.35(m,1H),8.31–8.23(m,1H),8.06(s,1H ),7.93–7.86(m,1H),7.65(d,J=7.9Hz,2H),7.54(d,J=7.7Hz,1H),7.42-7.40(m, ,3H),7.27(t,J=7.7Hz,1H),6.53-6.49(m,1H),6.31-6.28(m,1H),5.82–5.71(m ,2H),3.77(s,2H),1.96(d,J=11.4Hz,2H),1.90–1.76(m,2H),1.61–1.14(m,4H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.02,163.76,157.67,152.39,142.37,139.49,137.13,136.03,132.29,128.98,128.79,127.46,126 .41,124.88,124.72,122.54,122.29,119.21,105.49,103.38,66.82,50.79,47.96,31.05,30.88,27.86.

[0072] Example 7: Preparation of (E)-N-(3-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)cyclohexyl)-3-(4-(dimethylamino)but-2-enamide)benzamide (I-7)

[0073] The same operation as in Example 1 was performed to obtain 47 mg of a white solid powder. 1H NMR (600 MHz, Methanol-d 4)δ8.08(t,J=2.0Hz,1H),7.86(s,1H),7.82-7.78(m,1H),7.72–7.61(m,2H),7.58-7.55(m,1H),7.42 (t,J=7.9Hz,1H),7.36(t,J=7.8Hz,2H),7.14-7.11(m,1H),6.91-6.88(m,1H),6.61-6.57(m,1H),4. 02-3.99(m,1H),3.97–3.90(m,2H),3.80-3.76(m,1H),2.88(s,6H),2.32(d,J=11.8Hz,1H),2.01(t, J=11.8Hz,2H),1.87(m,J=13.7,3.3Hz,1H),1.53–1.40(m,1H),1.41–1.28(m,2H),1.27-1.23(m,1H). 13 C NMR (151MHz, Methanol-d 4 )δ177.23,167.79,163.12,159.02,156.36,155.55,152.69,138.46,138.34,135.65,132.21,131.83,128.71,12 8.21,123.77,122.90,122.75,122.06,121.85,119.01,57.55,56.47,49.67,48.29,42.07,38.32,31.57,23.01.

[0074] Example 8: Preparation of N-(3-(3-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)acrylamide (I-8)

[0075] The operation was the same as in Example 1 to obtain 46 mg of yellow-white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.01(s,1H),8.59(s,1H),8.28(s,1H),7.63(m,3H),7.47–7.26(m,3H),7.27–6.82(m,4H),6.72(s,1H),3.62( t,J=6.3Hz,2H),3.45-3.43(m,2H)3.36(s,2H),2.58(q,J=5.1,4.0Hz,2H),1.85(s,2H),1.60(m,2H),1.53(m,1H). 13 C NMR (151 MHz, DMSO-d 6)δ169.92,164.63,157.42,153.90,152.68,147.82,146.63,142.72,139.85,137.02,128.88,128.77 ,126.60,126.41,125.20,124.56,119.27,116.96,103.42,99.99,68.57,66.82,58.39,47.73,37.40.

[0076] Example 9: Preparation of (S,E)-N-(3-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)pyrrolidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-9)

[0077] The operation was the same as in Example 1 to obtain 98 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.20(d,J=11.0Hz,1H),8.59(s,1H),7.99(m,1H),7.90(s,1H),7.77(d,J=7.9Hz,1H), 7.65(dd,J=20.7,8.1Hz,1H),7.50(s,1H),7.41–7.24(m,3H),7.19(dd,J=23.5,7.6Hz,2H ),7.07(t,J=7.3Hz,1H),6.79–6.71(m,1H),6.27(dd,J=16.5,2.7Hz,1H),4.26(s,1H),3. 86–3.60(m,2H),3.61–3.41(m,2H),3.06(d,J=5.7Hz,2H),2.18(s,6H),2.10–1.86(m,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ168.60,162.78,157.60,153.37,143.39,139.26,137.50,132.40,129.27,129.21,128.96,128.76,126.49 ,124.82,124.78,122.81,121.24,118.61,103.95,101.71,66.80,57.02,54.11,51.60,47.53,42.22,31.59.

[0078] Example 10: Preparation of (R,E)-N-(3-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-10)

[0079] The operation was the same as in Example 1 to obtain 101 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ11.10(s,1H),10.70(m,1H),10.04(s,1H),8.55(s,1H),8.29(s,1H),7.88(s,1 H),7.77–7.52(m,2H),7.33(s,2H),7.27–6.89(m,3H),6.84(m,1H),6.56(d,J=15 .3Hz,1H),4.03(q,J=7.1Hz,2H),3.93(t,J=6.1Hz,2H),3.57(s,2H),3.18–2.92( m,2H),2.76(d,J=4.3Hz,6H),1.99(s,1H),1.85(s,1H),1.70(s,1H),1.54(s,1H). 13 CNMR (151MHz, DMSO-d 6 )δ169.60, 162.59, 156.80, 152.53, 139.43, 137.50, 137.12, 132.38, 128.84, 125.53, 124.31, 122.33, 120.70, 11 7.96, 114.11, 111.32, 104.98, 104.52, 103.14, 103.07, 66.81, 62.46, 60.11, 57.03, 47.93, 42.22, 34.15, 28.70.

[0080] Example 11: Preparation of (R,E)-N-(3-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)pyrrolidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-11)

[0081] The same operation as in Example 1 was performed to obtain 74 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6)δ10.21(d,J=11.7Hz,1H),8.56(s,1H),7.98(m,1H),7.93–7.83(m,1H),7.77(d,J=8.2H z,1H),7.71–7.61(m,1H),7.56(d,J=8.3Hz,1H),7.41–7.25(m,3H),7.18(dd,J=24.3,7.7 Hz,2H),7.10–6.96(m,1H),6.80-6.75(m,1H),6.34–6.23(m,1H),3.79–3.61(m,2H),3.6 1–3.45(m,2H),3.40-3.31(m,2H),3.12(d,J=5.9Hz,2H),2.22(s,6H),2.17–2.04(m,1H). 13 C NMR (151 MHz, DMSO-d 6 )δ168.58,163.75,156.03,143.37,141.38,139.49,137.77,129.14,128.73,126.72,125.29,123.79,123.6 5, 122.51, 122.41, 120.84, 119.01, 118.43, 110.87, 99.99, 59.92, 54.83, 11.87, 47.73, 45.35, 44.84, 32.00.

[0082] Example 12: Preparation of (S,E)-N-(3-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-12)

[0083] The operation was the same as in Example 1 to obtain 112 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.19(s,1H),8.68-8.52(m,1H),8.00(s,1H),7.88–7.60(m,3H),7.5 4(d,J=8.3Hz,1H),7.47–7.23(m,3H),7.13-7.05(m,3H),6.82–6.59(m, 1H),6.29(d,J=15.7Hz,1H),3.73-3.69(m,2H),3.51(s,2H),3.17(d,J= 6.1Hz,2H),2.98(s,2H),2.26(s,6H),1.78(d,,1H),1.57-1.51(m,2H). 13 C NMR (151 MHz, DMSO-d6 )δ170.39,163.00,162.73,158.15,157.38,139.35,139.11,137.35,137.01,132.39,132.29,129.37,128.72,12 5.03,122.64,120.71,119.64,118.31,117.06,114.81,66.79,60.08,57.13,49.08,47.89,36.34,31.29,30.62.

[0084] Example 13: Preparation of (S,E)-N-(4-(3-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-13)

[0085] The operation was the same as in Example 1 to obtain 107 mg of white solid powder. 1 H NMR (600 MHz, Methanol-d 4 )δ8.00(s,1H),7.80(s,1H),7.60(s,2H),7.43(t,J=7.8Hz,3H),7.37(s ,1H),7.31(s,1H),7.08(s,1H),7.05-6.91(m,1H),6.64(d,J=15.2Hz,1 H),4.15(s,1H),3.93-4.03(m,2H),3.88(s,1H),3.21-3.19(m,2H)2.94 (s,6H),2.21–1.92(m,2H),1.85(s,2H),1.65(s,1H).1.32-1.29(m,1H) 13 C NMR (151MHz, Methanol-d 4 )δ174.96,174.26,168.58,164.52,160.77,160.11,159.61,156.56,155.36,150.34,147.02,145.38,136.25,13 5.91,133.53,132.95,130.98,128.37,127.16,123.62,64.92,53.70,52.84,40.35,35.14,34.22,27.14,22.44.

[0086] Example 14: Preparation of (R,E)-N-(4-(3-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)pyrrolidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-14)

[0087] The operation was the same as in Example 1 to obtain 94 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.26(d,J=12.0Hz,1H),8.58(s,1H),8.04-7.99(m,1H),7.86–7.64(m,3H),7.64–7.42(m,3H),7.41–7.16(m,3H),7.06(m,1H),6.76 6.72(m,1H),6.34-6.30(m,1H),4.29(s,1H),3.79–3.58(m,2H),3.43-3.38(m,2H),3.11(d,J=6.2Hz,2H),2.21(s,6H),2.05–1.71(m,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ168.51,163.83,162.90,160.19,156.04,141.55,140.98,139.40,131.73,128.78,128.64,126.70,124.65,123.8 1,123.72,122.53,122.36,118.89,118.78,111.26,99.99,59.87,54.86,51.99,47.79,45.28,44.95,36.29,29.50.

[0088] Example 15: Preparation of (R,E)-N-(3-((5-bromo-4-(phenylamino)pyrimidin-2-yl)amino)pyrrolidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-15)

[0089] The operation was the same as in Example 1 to obtain 78 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6)δ10.20(d,J=10.4Hz,1H),8.06(m,1H),7.89(s,1H),7.73(d,J=7.6Hz,1H),7.69– 7.62(m,1H),7.62(s,1H),7.51-7.45(m,1H)7.46–7.27(m,3H),7.18(m,2H),7.11–6 .95(m,1H),6.84–6.65(m,1H),6.37–6.17(m,1H),3.72-3.65(m,2H),3.60(d,J=7. 2Hz,2H),3.59–3.38(m,1H),3.09(t,J=8.0Hz,2H),2.19(s,6H),2.10-1.94(m,2H). 13 C NMR (151 MHz, DMSO-d 6 )δ168.69,163.91,160.55,156.70,142.03,139.39,137.66,132.00,129.21,129.15,128.77,126.35,123.87,1 23.84,122.72,122.56,122.46,120.92,118.41,65.53,60.06,57.16,54.84,51.86,47.75,46.11,45.46,44.88.

[0090] Example 16: Preparation of (S,E)-N-(4-(3-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)pyrrolidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-16)

[0091] The same operation as in Example 1 was performed to obtain 64 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.26(d,J=13.5Hz,1H),8.57(s,1H),7.95(s,1H),7.77(d,J=8.0Hz,1H),7.71(m, J=17.8,8.3Hz,2H),7.50(m,,3H),7.40–7.11(m,3H),7.06(m,1H),6.75(m,1H),6.29( m,1H),4.19(m,1H),3.74(m,1H),3.67–3.59(m,1H),3.55–3.41(m,2H),3.06(t,J=5. 1Hz,2H),2.18(d,J=3.2Hz,6H),2.10(m,1H),1.98(m,1H),1.89(q,J=6.4,5.2Hz,1H).13 C NMR (151 MHz, DMSO-d 6 )δ174.23,168.41,163.90,160.28,156.03,154.78,142.24,141.15,139.49,131.68,128.74,128.67,128.62 ,126.27,123.74,123.64,122.55,122.37,118.88,118.80,60.18,54.82,51.96,48.95,47.77,45.62,44.93.

[0092] Example 17: Preparation of N-(3-(4-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)cyclopropanecarboxamide (I-17)

[0093] The same operation as in Example 1 was performed to obtain 116 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.35(s,1H),8.58(m,1H),7.98(s,1H),7.72(s,3H),7.56(d,J=8.1Hz,1H),7.36(t,J=7.9Hz,1H),7.32(t,J=7.9Hz,2H),7.23–6.88(m,3H ),4.40(s,1H),3.76(s,2H),3.62(s,1H),3.08(s,1H),1.88(d,J=66.0Hz,2H),1.80–1.74(m,1H),1.39(d,J=46.6Hz,2H),0.92–0.71(m,4H). 13 CNMR (151MHz, DMSO-d 6 )δ 13 C NMR (151MHz, DMSO) δ172.32,169.21,168.41,162.76,159.91,156.03,139.80,139.53,137.14,129.36,128. 68,123.59,122.37,121.47,120.10,117.53,99.99,55.37,46.71,38.71,36.25,32.44,21.76,15.07,7.75.

[0094] Example 18: Preparation of N-(4-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)cyclohexyl)-3-(cyclopropanecarboxamido)benzamide (I-18)

[0095] The operation was the same as in Example 1 to obtain 119 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.33(s,1H),8.66(s,1H),8.31–8.15(m,1H),7.97(s,2H),7.80(t,J=7.8Hz,3H) ,7.50(d,J=7.8Hz,1H),7.45-7.35(m,3H),7.07(t,J=7.4Hz,1H),7.02(s,1H),3.77( s,1H),1.98(d,J=12.2Hz,2H),1.88(d,J=12.1Hz,2H),1.80(t,J=7.1Hz,1H),1.44(q ,J=12.2Hz,2H),1.36(q,J=12.8,12.2Hz,2H),1.28-1.25(m,1H),0.92–0.69(m,4H). 13 C NMR (151 MHz, DMSO-d 6 )δ172.24,166.08,159.67,156.01,154.92,139.75,139.56,136.08,128.92,128.63,123.54,122.30,1 22.16,121.98,121.93,118.81,101.55,54.07,50.38,49.57,48.43,36.24,31.60,23.23,14.98,7.68.

[0096] Example 19: Preparation of (S,E)-N-(4-(3-((5-bromo-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-19)

[0097] The same operation as in Example 1 was performed to obtain 89 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.21(s,1H),8.29(s,1H),8.03(s,1H),7.70(s,3H),7.51(s,1H),7.43-7.33(m,3H),7.05(s,2H),6.96–6.44(m,2H),6.28(d,J= 15.4Hz,1H),3.82-3.74(m,2H),3.33(s,2H),3.06(d,J=5.8Hz,2H),2.97(s,1H),2.18(s,6H),1.98-1.88(m,2H),1.65-1.50(m,2H). 13C NMR (151 MHz, DMSO-d 6 )δ166.27,163.83,156.63,144.09,142.98,142.26,156.63,144.09,142.26,130.74,128.73,126.21,125.9 3,124.42,123.75,122.76,119.09,106.10,104.67,99.99,70.58,68.46,60.18,52.35,45.64,30.43,29.46.

[0098] Example 20: Preparation of (R,E)-N-(4-(3-((5-bromo-4-(phenylamino)pyrimidin-2-yl)amino)pyrrolidine-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-20)

[0099] The same operation as in Example 1 was performed to obtain 95 mg of white solid powder. 1 H NMR (600 MHz, DMSO-d 6 )δ10.24(d,J=11.2Hz,1H),8.32(s,1H),8.14-8.06(m,1H),7.78–7.63(m,3H),7.58 -7.49(m,3H),7.33(t,J=7.7Hz,3H),7.12–6.98(m,1H),6.82-6.75(m,1H),6.35-6. 29(m,1H),3.73(s,1H),3.67–3.58(m,1H),3.58–3.39(m,2H),3.06(t,J=5.1Hz,2H) ,2.18(d,J=3.3Hz,6H),2.14–2.05(m,1H),2.03–1.83(m,1H),1.24(d,J=5.2Hz,1H). 13 C NMR (151 MHz, DMSO-d 6 )δ180.36,168.39,163.87,160.61,160.52,156.70,142.35,141.09,139.47,131.72,128.73,128.66,12 6.18,123.86,123.76,122.75,122.58,118.87,118.80,60.17,54.78,51.90,47.76,45.63,44.92,32.04.

[0100] Experimental Example 1 Cell proliferation inhibition activity test

[0101] MTT assay to detect the 48h IC of drugs on breast cancer cells 50 Value: The experiment set up solvent control group and drug experimental group. Each drug experimental group set up 8 concentrations, and each concentration set up 3 parallel wells. Each experiment was repeated three times. In the 96-well plate, the cell concentration added to each well was 1×10 5 100 μL of cell suspension containing 3×10 cells / mL, i.e., each well contains 3×10 3 When inoculating, make sure that the cells are evenly distributed in each well. To prevent liquid evaporation, cells are not inoculated in the wells around the 96-well plate, and PBS is added to keep the cells moist. After the cells adhere to the wall, 100 μL of target compound of different concentrations is added to each well in the drug experimental group. The 96-well plate is placed at 37°C and 5% CO 2 Continue to culture in the incubator and terminate the culture after 72 hours. After 48 hours of drug treatment, take out the 96-well plate, add MTT solution to each well, continue to incubate in the cell culture incubator for 4 hours, and measure the absorbance value (A) of each well at 570nm on an automatic microplate reader. Calculate the inhibition rate by the formula. Perform linear regression analysis on the inhibition rate and drug concentration, and use the linear equation to calculate IC 50 The test results were expressed as mean ± standard deviation, and the experimental results were statistically analyzed using SPSS15.0 software, with P < 0.05 indicating a significant difference. The results are shown in Table 1.

[0102] Table 1 Effects of the tested compounds at different concentrations on the proliferation of two breast cancer tumor cells

[0103]

[0104]

[0105] The experimental results show that compounds I-1, I-2, I-5, I-13, I-14, I-19 and I-20 showed strong proliferation inhibition activity against two breast cancer cells. In particular, compound I-20 had an inhibitory IC of 1.34 for MDA-MB-231 cells. 50 It is 10 times that of the positive drug THZ-531.

[0106] Experimental Example 2 Cell cloning experiment

[0107] Cell cloning experiment to detect the effect of drugs on the proliferation of breast cancer cells: cells in the logarithmic growth phase were taken and 1×10 3Breast cancer cells were inoculated in a 12-well cell culture plate. After the cells adhered overnight, different final concentrations of I-20 (0 / 25 / 50 / 100 nM) were added to treat the cells for 14 days, and fresh complete culture medium containing the corresponding concentration of drugs was replaced every 2 days. After 14 days, the supernatant was discarded, and the cells were washed 2-3 times with 1 mL of PBS in each well. After that, 1 mL of 4% paraformaldehyde was added to fix the cells for 1 hour, and then the paraformaldehyde was discarded. The cells were washed with PBS 2-3 times, and 1 mL of crystal violet solution was added to each well for staining for 1 hour. The crystal violet solution was discarded, and the 12-well plate was washed with pure water 2-3 times. It was placed in a dry place to dry naturally and photographed. The results were analyzed and statistically analyzed using Image J software and Graphpad Prism 10. The experimental results are attached. Figure 1 As the concentration of compound I-20 increased, the number of MFM-223 cell clones decreased significantly, and compound I-20 could inhibit the proliferation of breast cancer cells in a concentration-dependent manner.

[0108] Experimental Example 3: WB Experiment

[0109] After treating breast cancer cells with different concentrations of I-20 (0 / 25 / 50 / 100 nM) for 48 hours, cells from each group were collected for WB experiments: rinsed twice with pre-cooled PBS and the supernatant was discarded, RIPA lysis buffer containing protease and phosphatase inhibitors was added to extract total cell protein, and the protein concentration was determined by BCA protein quantification method, followed by SDS-PAGE electrophoresis separation, membrane transfer, room temperature skim milk powder blocking, 4°C overnight primary antibody incubation, room temperature secondary antibody incubation and chemiluminescence development operations, to detect the effects of different concentrations of I-20 on the expression levels of CDK12, CDK13, and CDK7 proteins in breast cancer cells. Results are shown in Figure 2 Compound I-20 has obvious selectivity for CDK12 and CDK13, and can reduce the expression levels of CDK12 and CDK13 proteins in breast cancer cells in a concentration-dependent manner, while having little effect on CDK7.

[0110] Experimental Example 4 Cell cycle and apoptosis detection

[0111] After treating breast cancer cells MFM-223 and MDA-MB-231 with different concentrations of I-20 (0 / 25 / 50 / 100 nM) for 48 h, cells in each group were collected and flow cytometry was used to detect the level of cell apoptosis and cell cycle distribution after drug treatment. The results were analyzed and statistically analyzed using Image J software and Graphpad Prism 10. The experimental results showed that I-20 could promote breast cancer cell apoptosis in a concentration-dependent manner and induce G2 / M phase arrest in breast cancer cells in a concentration-dependent manner.

[0112] Experimental Example 5 Cell scratch test

[0113] Breast cancer cells were treated with different concentrations of I-20 (0 / 25 / 50 / 100 nM) and the cell migration ability was detected by cell scratch assay: cells in the logarithmic growth phase were taken, digested with trypsin to form a single cell suspension, and inoculated into a 6-well culture plate. Cells were normally cultured at 37°C and 5% CO 2 Incubator. When the cell density is 80-90%, use a 200μL pipette tip to scratch. Add PBS to wash the cells 3 times, remove the scratched cells, and then add serum-free medium for culture. Take pictures under a 10x objective lens of a Nikon microscope at 0, 24, and 48 hours to observe the cell migration ability. The results were analyzed and counted using Image J software and Graphpad Prism 10. The experimental results showed that I-20 can inhibit the migration of breast cancer cells in a concentration-dependent manner.

Claims

1. A pyrimidinylaniline compound represented by formula I or its isomers or pharmaceutically acceptable salts: in R1 is halogen; R2 is selected from alkyl or cycloalkyl; R3 is selected from alkyl or cycloalkyl; n is selected from 0 or 1. When n is 0, It is a tetrahydropyrrole ring.

2. The compound according to claim 1 or its isomer or pharmaceutically acceptable salt, wherein R1 is selected from chlorine or bromine.

3. The compound according to claim 1 or its isomer, pharmaceutically acceptable salt, wherein R2 is selected from methane, ethyl, propane or cyclopropyl; preferably, R2 is methane.

4. The compound according to claim 1 or its isomer, pharmaceutically acceptable salt, wherein R3 is selected from methane, ethyl, propane or cyclopropyl; preferably, R3 is methane.

5. The compound according to claim 1 or its isomer or pharmaceutically acceptable salt, Selected from a tetrahydropyrrole ring or a piperidine ring.

6. The compound according to claim 1 or its isomer, pharmaceutically acceptable salt, wherein the compound is selected from the following compounds:

7. A pharmaceutical composition comprising the compound according to any one of claims 1 to 6 or an isomer thereof, a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

8. Use of the compound according to any one of claims 1 to 6 or its isomer, pharmaceutically acceptable salt or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating CDK12-related diseases.

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