An N-pyrimidinylaniline CDK12 / 13 inhibitor, its preparation and application

Abstract

This invention belongs to the field of pharmaceutical chemistry, specifically relating to an N-pyrimidinylaniline CDK12 / 13 inhibitor or its isomer, a pharmaceutically acceptable salt, as shown in Formula I, its preparation method, and a pharmaceutical composition comprising the same. The compound of Formula I has an aromatic phenyl group linked to the meta-position of the pyridyl group via a flexible amino group, which increases the affinity of the compound for CDK12 / 13 proteins and improves its CDK12 / 13 kinase selectivity. Simultaneously, the compound exhibits significantly improved water solubility, reduced synthetic difficulty, and greatly enhanced drug-likeness.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, specifically relating to an N-pyrimidinylaniline CDK12 / 13 inhibitor or its isomer, a pharmaceutically acceptable salt, methods for preparing them, pharmaceutical compositions containing these compounds, and the use of these compounds or compositions in the preparation of medicaments for treating CDK12 / 13-mediated diseases. Background Technology

[0002] Cyclin-dependent kinases (CDKs) are a family of protein kinases that catalyze the phosphorylation of serine / threonine residues. They are closely related to the cell cycle and transcription and have been identified as oncogenic drivers. Twenty-one CDK subtypes have been identified, each with distinct functions but sharing highly similar ATP-binding pockets. Therefore, while many reported pan-CDK inhibitors exhibit significant antitumor effects, they also suffer from severe toxic side effects, significantly limiting their clinical application. Thus, the development of selective CDK inhibitors is of great importance.

[0003] Studies have shown that CDK12 and CDK13 play a series of important biological functions, including gene transcription regulation, participation in precursor mRNA splicing, promotion of cell proliferation and differentiation, and DNA damage response. With the deepening understanding of the pathogenic mechanisms of tumors such as breast cancer and the ongoing research into genomics, studies have revealed that CDK12 and CDK13 are abnormally expressed or mutated in various cancers, closely related to their occurrence and development. CDK12 / 13 holds promise as a novel biomarker and target for personalized treatment in breast cancer and prostate cancer patients. Inhibiting CDK12 / 13 kinases can enhance the sensitivity of tumor cells to DNA-damaging drugs and increase genomic instability, thereby effectively inhibiting tumor growth. Therefore, developing CDK12 / 13 inhibitors has become a potential new strategy for cancer treatment. Summary of the Invention

[0004] One object of the present invention is to provide an N-pyrimidinylaniline CDK12 / 13 inhibitor or an isomer thereof, or a pharmaceutically acceptable salt thereof, represented by general formula I. The compound of general formula I has an aromatic phenyl group linked to the meta-position of the pyridyl group via a flexible amino group, which increases the affinity of the compound for CDK12 / 13 proteins, improves its CDK12 / 13 kinase selectivity, and simultaneously significantly enhances the water solubility of the compound, reduces the difficulty of synthesis, and greatly improves its drug-likeness.

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

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

[0007] Another object of the present invention is to provide the use of compounds of general formula I of the present invention or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment or prevention of CDK12 / 13-related diseases.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

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

[0010]

[0011] in

[0012] R1 is a halogen;

[0013] R2 is selected from alkyl or cycloalkyl;

[0014] R3 is selected from alkyl or cycloalkyl groups;

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

[0016] In some preferred embodiments, R1 is selected from chlorine or bromine; preferably, R1 is bromine.

[0017] In some preferred embodiments, R2 is selected from C 1-3 Alkyl, C 3-6 Cycloalkyl; preferably, R2 is selected from methane, ethane, propane or cyclopropane.

[0018] In some preferred embodiments, R3 is selected from C 1-3 Alkyl, C 3-6 Cycloalkyl; preferably, R3 is selected from methane, ethane, propane or cyclopropane.

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

[0020] This invention provides the following specific compounds or isomers thereof, and pharmaceutically acceptable salts:

[0021]

[0022]

[0023] The inventors have discovered that linking a single aromatic group to the pyrimidine group with an amino flexible chain at the 4-position can improve its affinity for CDK12 / 13 proteins, thereby increasing its selectivity. Furthermore, the introduction of the amino flexible chain increases water solubility.

[0024] On the other hand, the present invention provides a method for preparing compounds of 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 the compound of formula (2);

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

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

[0028] Step 4: The compound of formula (6) and the compound of formula (7) are condensed to obtain the compound of formula (8);

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

[0030]

[0031] Among them, R1, R2, R3 and n have the definitions described in general formula (I).

[0032] Thirdly, the present invention provides pharmaceutical compositions comprising the compounds of the present invention or isomers thereof, or pharmaceutically acceptable salts thereof.

[0033] In some embodiments, the present invention provides compounds or isomers thereof, pharmaceutically acceptable salts thereof, and pharmaceutical compositions comprising compounds or isomers thereof, pharmaceutically acceptable salts thereof, said compounds or pharmaceutical compositions for treating CDK12 / 13-mediated diseases.

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

[0035] The compounds or isomers of the present invention, their pharmaceutically acceptable salts, can be mixed with pharmaceutically acceptable carriers, diluents, or excipients to prepare pharmaceutical formulations suitable for oral or parenteral administration. Administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The formulations can be administered via any route, such as by infusion or bolus, or by absorption through the epithelium or mucous membranes of the skin (e.g., oral mucosa or rectum). Administration can be systemic or local. Examples of oral formulations include solid or liquid dosage forms, specifically including tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions, etc. The formulations can be prepared by methods known in the art and contain carriers, diluents, or excipients conventionally used in the field of pharmaceutical formulations.

[0036] Fourthly, the present invention provides the use of compounds of Formula I or isomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising thereof in the preparation of medicaments for treating CDK12 / 13-mediated diseases.

[0037] In some preferred embodiments, the present invention provides the use of compounds of Formula I or isomers thereof, pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising thereof in the preparation of medicaments 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] Terminology Explanation

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

[0040] In this invention, "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0041] The term "pharmaceutically acceptable salt" in this invention refers to salts of the compounds of this invention that are safe and effective when used in mammals and possess the intended biological activity.

[0042] The term "pharmaceutical composition" as used in this invention refers to a mixture comprising any of the compounds described herein, including corresponding isomers, prodrugs, solvates, pharmaceutically acceptable salts or their chemically protected forms, and one or more pharmaceutically acceptable carriers and / or mixtures of other one or more drugs. The purpose of a pharmaceutical composition is to facilitate the administration of the compound to a living organism. Such compositions are typically used in the preparation of medicaments for the treatment and / or prevention of diseases mediated by one or more kinases.

[0043] The "pharmaceutical-grade carrier" of this invention refers to a carrier that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the administered compound. This includes 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 compounds of this 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 carboxymethyl cellulose, as well as cellulose and cellulose acetate; malt, gelatin, etc.

[0044] In this invention, "excipient" refers to an inert substance added to a pharmaceutical composition to further promote the delivery of the compound. Excipients may include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol. Attached Figure Description

[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 apoptosis in breast cancer cells.

[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 Implementation

[0050] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, all materials used in the following embodiments are commercially available.

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

[0052] Step 1: Synthesis of 2,5-dichloro-N-phenylpyrimidine-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. The reaction was carried out under N2 protection and reflux at 90 °C for 2 h. After the reaction was carried out, the reaction solution was poured into a beaker and diluted with water (H2O) and ethyl acetate (EA) (150 mL × 3). The solution was washed successively with saturated sodium carbonate and saturated brine. The organic phases were combined, dried with anhydrous sodium sulfate powder, and the organic layer was evaporated to dryness to obtain 1.3 g of 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] 2,5-Dichloro-N-phenylpyrimidin-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) were weighed and dissolved in N-methylpyrrolidone (NMP). After reacting under N2 protection and reflux at 135 °C for 3 h, the reaction solution was added to a beaker and diluted with water (H2O) and ethyl acetate (EA) (150 ml × 3). The mixture was washed successively with saturated sodium carbonate and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and the organic layer was evaporated to dryness to obtain a deep 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, with a yield of 48%.

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

[0057] 1.05 g (2.61 mmol) of tert-butyl 4-((5-chloro-4-(phenylamino)pyrimidin-2-yl)amino)piperidine-1-carboxylic acid was added to 20 mL of 1,4-dioxane solution, followed by 0.33 mL of concentrated hydrochloric acid. The reaction was carried out at room temperature under N2 protection for 3 h, and TLC monitoring showed that the reaction was complete. The reaction was cooled to room temperature, and 100 mL of ethyl acetate (EA) was added to quench the reaction. Then, saturated sodium carbonate (Na2CO3) solution was added to adjust the pH of the solution to alkaline, and the solution was transferred to a separatory funnel. After standing and separating the layers, the organic layer was rotary evaporated, and the residue was purified by silica gel column chromatography to give 0.62 g of a pale yellow solid compound, with a yield of 77%.

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

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

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

[0061] Take compound 5-chloro-N 4 -Phenyl-N 2 1,4-(piperidin-4-yl)pyrimidine-2,4-diamine (0.62 g, 2.04 mmol), compound (E)-4-(3-(dimethylamino)-2-butenamido)benzoic acid (0.73 g, 2.57 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.59 g, 3.09 mmol), (1-hydroxybenzotriazole) (0.40 g, 3.05 mmol), and N,N-diisopropylethylamine (0.49 g, 3.80 mmol) were dissolved in N,N-dimethylformamide in a 50 mL reaction flask and reacted at room temperature for 1.5 h. TLC monitoring showed the reaction was complete. Saturated saline solution, water, and ethyl acetate were added for extraction. After standing and separation, the organic layer was rotary evaporated, and the residue was purified by silica gel column chromatography to give 150 mg of a white solid compound, with a yield of 13.8%. 1HNMR(600MHz,DMSO-d6)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(151MHz,DMSO-d6)δ175.41,169.16,163.85,159.90,156.04,155.62,142.11,139.69,139.52,137.18,129.41,128.69, 126.26,123.59,122.37,121.87,120.42,117.76,106.46,101.84,60.12,55.33,46.70,45.58,41.14,34.78,31.68,29.90.

[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 procedure was the same as in Example 1, and 47 mg of a white powder was obtained by column chromatography. 1H NMR(600MHz,DMSO-d6)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(151MHz,DMSO-d6)δ166.05,162.74,160.03,157.57,154.84,139.39,136.10,132.40,132.27,128.91,128.81,12 4.51,123.63,122.73,122.47,119.42,116.17,99.99,96.37,59.26,56.96,50.72,48.04,47.22,42.06,31.04,30.96.

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

[0065] The procedure was the same as in Example 1, and 76 mg of a white powder was obtained by column chromatography. 1 H NMR(600MHz,DMSO-d6)δ10.45(s,1H),8.88-8.52(m,1H),8.00(s,1H),7.8 6–7.66(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). 13C NMR(151MHz,DMSO-d6)δ163.94,163.77,159.92,156.35,149.53,142.12,141.98,139.82,139.65,137.33,137.09,128 .81,126.47,123.60,122.45,121.76,120.42,117.74,100.73,60.16,48.95,47.71,45.57,42.17,30.73,21.23,14.56.

[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 procedure was the same as in Example 1, yielding 53 mg of a yellowish-white solid powder. 1 H NMR(600MHz,DMSO-d6)δ10.17(d,J=11.6Hz,1H),8.54(s,1H),7.92-7.98(m,1H),7.89(s,1 H),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,2H),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(151MHz,DMSO-d6)δ172.11,168.61,163.88,160.21,156.07,142.13,139.53,137.82,129.11,129.05,128.73,128.67, 128.63,126.25,123.68,122.52,122.39,122.36,120.85,118.46,60.19,54.80,51.99,47.72,45.61,44.81,32.00,29.65.

[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 procedure was the same as in Example 1, yielding 65 mg of a white solid powder. 1 H NMR (600MHz, DMSO-d6) δ10.20(s,1H),8.57(s,1H),8.24(s,1H),8.02(t,J=1.9Hz,1H),7.96(s,1 H),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) ,3.51(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(151MHz,DMSO-d6)δ166.05,163.81,160.10,155.96,155.54,155.26,153.53,145.14,141.92,139.69,139.62,136.18,12 8.93,128.64,126.38,123.41,122.36,122.20,122.08,119.19,69.99,60.16,50.30,48.51,45.56,31.63,22.73,22.27,9.71.

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

[0071] The procedure was the same as in Example 1, yielding 77 mg of a white solid powder. 1H NMR(600MHz,DMSO-d6)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),7.4 2-7.40(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(151MHz,DMSO-d6)δ166.02,163.76,157.67,152.39,142.37,139.49,137.13,136.03,132.29,128.98,128.79,1 27.46,126.41,124.88,124.72,122.54,122.29,119.21,105.49,103.38,66.82,50.79,47.96,31.05,30.88,27.86.

[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 procedure was the same as in Example 1, yielding 47 mg of a white solid powder. ¹H NMR (600 MHz, Methanol-d₄) values ​​were: δ 8.08 (t, J = 2.0 Hz, 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 = 7.9 Hz, 1H), 7.36 (t, J = 7.8 Hz, 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-d4)δ177.23,167.79,163.12,159.02,156.36,155.55,152.69,138.46,138.34,135.65,132.21,131.8 3,128.71,128.21,123.77,122.90,122.75,122.06,121.85,119.01,57.55,56.47,49.67,48.29,42.07,38.32,31.57,23.01.

[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 procedure was the same as in Example 1, yielding 46 mg of a yellowish-white solid powder. 1 H NMR(600MHz,DMSO-d6)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). 13C NMR (151MHz, DMSO-d6) δ 169.92, 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 procedure was the same as in Example 1, yielding 98 mg of a white solid powder. 1 H NMR(600MHz,DMSO-d6)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 (151MHz, DMSO-d6) 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)piperidin-1-carbonyl)phenyl)-4-(dimethylamino)but-2-enamide (I-10)

[0079] The procedure was the same as in Example 1, yielding 101 mg of a white solid powder. 1 H NMR(600MHz,DMSO-d6)δ11.10(s,1H),10.70(m,1H),10.04(s,1H),8.55(s,1H),8.29( s,1H),7.88(s,1H),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-d6) 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, 117.96, 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 procedure was the same as in Example 1, yielding 74 mg of a white solid powder. 1H NMR(600MHz,DMSO-d6)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=8.2Hz,1H),7.71–7.61(m,1H),7.56(d,J=8.3Hz,1H),7.41–7.25(m,3H),7.18(dd,J =24.3,7.7Hz,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) ,3.61–3.45(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(151MHz,DMSO-d6)δ168.58,163.75,156.03,143.37,141.38,139.49,137.77,129.14,128.73,126.72,125.29,123 .79,123.65,122.51,122.41,120.84,119.01,118.43,110.87,99.99,59.92,54.83,11.87,47.73,45.35,44.84,32.00.

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

[0083] The procedure was the same as in Example 1, yielding 112 mg of a white solid powder. 1 H NMR(600MHz,DMSO-d6)δ10.19(s,1H),8.68-8.52(m,1H),8.00(s,1H),7.88– 7.60(m,3H),7.54(d,J=8.3Hz,1H),7.47–7.23(m,3H),7.13-7.05(m,3H),6.8 2–6.59(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). 13C NMR(151MHz,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, 128.72,125.03,122.64,120.71,119.64,118.31,117.06,114.81,66.79,60.08,57.13,49.08,47.89,36.34,31.29,30.62.

[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 procedure was the same as in Example 1, yielding 107 mg of a white solid powder. 1 H NMR(600MHz,Methanol-d4)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,1H),4.15(s,1H),3.93-4.03(m,2H),3.88(s,1H),3.21-3.19(m,2 H)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-d4)δ174.96,174.26,168.58,164.52,160.77,160.11,159.61,156.56,155.36,150.34,147.02,145.3 8,136.25,135.91,133.53,132.95,130.98,128.37,127.16,123.62,64.92,53.70,52.84,40.35,35.14,34.22,27.14,22.44.

[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 procedure was the same as in Example 1, yielding 94 mg of a white solid powder. 1 H NMR(600MHz,DMSO-d6)δ10.26(d,J=12.0Hz,1H),8.58(s,1H),8.04-7.99(m,1 H),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(151MHz,DMSO-d6)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 .65,123.81,123.72,122.53,122.36,118.89,118.78,111.26,99.99,59.87,54.86,51.99,47.79,45.28,44.95,36.29,29.50.

[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 procedure was the same as in Example 1, yielding 78 mg of a white solid powder. 1 H NMR (600MHz, DMSO-d6) 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,2 H),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). 13C NMR(151MHz,DMSO-d6)δ168.69,163.91,160.55,156.70,142.03,139.39,137.66,132.00,129.21,129.15,128.77,126.35 ,123.87,123.84,122.72,122.56,122.46,120.92,118.41,65.53,60.06,57.16,54.84,51.86,47.75,46.11,45.46,44.88.

[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 procedure was the same as in Example 1, yielding 64 mg of a white solid powder. 1 H NMR(600MHz,DMSO-d6)δ10.26(d,J=13.5Hz,1H),8.57(s,1H),7.95(s,1H),7.77(d,J=8.0H z,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 (151MHz, DMSO-d6) 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)cyclopropaneformamide (I-17)

[0093] The procedure was the same as in Example 1, yielding 116 mg of a white solid powder. 1 H NMR(600MHz,DMSO-d6)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-d6)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-(cyclopropanecarbamate)benzamide (I-18)

[0095] The procedure was the same as in Example 1, yielding 119 mg of a white solid powder. 1 H NMR(600MHz,DMSO-d6)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). 13C NMR(151MHz,DMSO-d6)δ172.24,166.08,159.67,156.01,154.92,139.75,139.56,136.08,128.92,128.63,123.54 ,122.30,122.16,121.98,121.93,118.81,101.55,54.07,50.38,49.57,48.43,36.24,31.60,23.23,14.98,7.68.

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

[0097] The procedure was the same as in Example 1, yielding 89 mg of a white solid powder. 1 H NMR(600MHz,DMSO-d6)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=5.8H z,2H),2.97(s,1H),2.18(s,6H),1.98-1.88(m,2H),1.65-1.50(m,2H). 13 C NMR(151MHz,DMSO-d6)δ166.27,163.83,156.63,144.09,142.98,142.26,156.63,144.09,142.26,130.74,128.73,126 .21,125.93,124.42,123.75,122.76,119.09,106.10,104.67,99.99,70.58,68.46,60.18,52.35,45.64,30.43,29.46.

[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 procedure was the same as in Example 1, yielding 95 mg of a white solid powder. 1H NMR(600MHz,DMSO-d6)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(151MHz,DMSO-d6)δ180.36,168.39,163.87,160.61,160.52,156.70,142.35,141.09,139.47,131.72,128.73, 128.66,126.18,123.86,123.76,122.75,122.58,118.87,118.80,60.17,54.78,51.90,47.76,45.63,44.92,32.04.

[0100] Experiment Example 1: Cell proliferation inhibition activity test

[0101] MTT assay to detect the 48-hour IC50 of drugs in breast cancer cells 50 Values: The experiment included a solvent control group and a drug experimental group. Each drug experimental group had 8 concentrations, with 3 parallel wells per concentration. Each experiment was repeated three times. In a 96-well plate, each well contained 1 × 10⁻⁶ cells. 5 100 μL of cell suspension per cell / mL, i.e., 3 × 10⁶ cells per well. 3 During inoculation, ensure cells are evenly distributed in each well. To prevent liquid evaporation, do not inoculate cells around the perimeter of the 96-well plate; instead, add PBS for hydration. After cell attachment, add 100 μL of the target compound at different concentrations to each well in the drug experimental group. Incubate the 96-well plate at 37°C with 5% CO2 for 72 hours. After 48 hours of drug treatment, remove the 96-well plate, add MTT assay solution to each well, and incubate for another 4 hours in a cell culture incubator. Measure the absorbance (A) of each well at 570 nm using an automated microplate reader. Calculate the inhibition rate using a formula. Perform linear regression analysis of the inhibition rate against the drug concentration, and calculate the IC50 using the linear equation. 50Values. The test results are expressed as mean ± standard deviation. The experimental results were statistically analyzed using SPSS 15.0 software, and a p-value < 0.05 was considered statistically significant. The results are shown in Table 1.

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

[0103]

[0104]

[0105] Experimental results showed that compounds I-1, I-2, I-5, I-13, I-14, I-19, and I-20 exhibited potent inhibitory activity against the proliferation of two types of breast cancer cell lines. In particular, compound I-20 showed an IC50 inhibitory effect on MDA-MB-231 cells. 50 It is 10 times more potent than the positive drug THZ-531.

[0106] Experiment Example 2: Cell Cloning Experiment

[0107] Cell clonal assay to detect the effect of drugs on the proliferation of breast cancer cells: Cells in logarithmic growth phase were divided into 1×10⁶ cells per well. 3 Breast cancer cells were seeded into 12-well cell culture plates. After overnight cell adhesion, the cells were treated with different final concentrations of I-20 (0 / 25 / 50 / 100 nM) for 14 days, with the medium replaced with fresh complete culture medium containing the corresponding concentration every 2 days. After 14 days, the supernatant was discarded, and each well was washed 2-3 times with 1 mL PBS. Cells were then fixed with 1 mL 4% paraformaldehyde for 1 hour, after which the paraformaldehyde was discarded. The cells were washed 2-3 times with PBS, and each well was stained with 1 mL crystal violet solution for 1 hour. The crystal violet solution was discarded, and the 12-well plates were washed 2-3 times with pure water. The plates were then air-dried before photographing. The results were analyzed and statistically processed using ImageJ 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, indicating that compound I-20 could inhibit breast cancer cell proliferation in a concentration-dependent manner.

[0108] Experiment Example 3: WB Experiment

[0109] After treating breast cancer cells with different concentrations of I-20 (0 / 25 / 50 / 100 nM) for 48 h, cells from each group were collected for Western blotting (WB) experiments. Cells were washed twice with pre-cooled PBS, the supernatant was discarded, and total cell protein was extracted using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using the BCA protein quantification method. Subsequently, SDS-PAGE electrophoresis separation, membrane transfer, room temperature skim milk blocking, overnight incubation with primary antibody at 4 °C, room temperature secondary antibody incubation, and chemiluminescence imaging were performed sequentially 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 below. Figure 2 Compound I-20 exhibits significant selectivity for CDK12 and CDK13, reducing the expression levels of CDK12 and CDK13 proteins in breast cancer cells in a concentration-dependent manner, while having little effect on CDK7.

[0110] Experiment 4: Cell cycle and apoptosis detection

[0111] Breast cancer cells MFM-223 and MDA-MB-231 were treated with different concentrations of I-20 (0 / 25 / 50 / 100 nM) for 48 h. Cells from each group were then collected, and flow cytometry was used to detect apoptosis levels and cell cycle distribution after drug treatment. Results were analyzed and statistically processed using ImageJ software and Graphpad Prism 10. The results showed that I-20 promoted breast cancer cell apoptosis in a concentration-dependent manner and induced G2 / M phase arrest in breast cancer cells in a concentration-dependent manner.

[0112] Experiment Example 5: Cell Scratch Test

[0113] Breast cancer cells were treated with different concentrations of I-20 (0 / 25 / 50 / 100 nM), and cell migration ability was detected by a cell scratch assay: Cells in the logarithmic growth phase were digested with trypsin into a single-cell suspension and seeded into 6-well culture plates. Cells were cultured normally at 37℃ in a 5% CO2 incubator. When the cell density reached 80-90%, scratches were performed using a 200 μL pipette tip. Cells were washed three times with PBS to remove the scratched cells, and then cultured in serum-free medium. Cell migration ability was observed by photographing under a 10x objective lens using a Nikon microscope at 0, 24, and 48 h. The results were analyzed and statistically processed using ImageJ software and Graphpad Prism 10. The experimental results showed that I-20 inhibited breast cancer cell migration in a concentration-dependent manner.

Claims

1. Pyrimidinyl aniline compounds represented by Formula I, and pharmaceutically acceptable salts thereof: in R1 is a halogen; R2 is selected from C 1-3 Alkyl or C 3-6 cycloalkyl; R3 is selected from C 1-3 Alkyl or C 3-6 cycloalkyl; n is selected from 0 or 1. When n is 0, It is a tetrahydropyrrole ring.

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

3. The compound of claim 1, a pharmaceutically acceptable salt, wherein R2 is selected from methane, ethane, propane, or cyclopropyl.

4. The compound of claim 1, a pharmaceutically acceptable salt, wherein R3 is selected from methane, ethane, propane, or cyclopropyl.

5. The compound of claim 1, a pharmaceutically acceptable salt, wherein the compound is selected from the group consisting of:

6. A pharmaceutical composition comprising the compound of any one of claims 1 to 5, a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

7. The use of the compound of any one of claims 1-5, a pharmaceutically acceptable salt, or the pharmaceutical composition of claim 6 in the preparation of a medicament for treating CDK12-related diseases.

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