Phenyl ether connection type cyclohexyl pyrimidinamine CDK12 / 13 inhibitor as well as preparation and application thereof

Abstract

The invention belongs to the field of medical chemistry, and particularly relates to a phenyl ether linked cyclohexylpyrimidinamine CDK12 / 13 inhibitor as shown in a formula I or an isomer, a pharmaceutically acceptable salt and a preparation method thereof, and a pharmaceutical composition containing the phenyl ether linked cyclohexylpyrimidinamine CDK12 / 13 inhibitor or the isomer and the pharmaceutically acceptable salt of the phenyl ether linked cyclohexylpyrimidinamine CDK12 / 13 inhibitor. According to the present invention, the compound of the formula I has the ether cyclohexyl amine linking chain group, such that the affinity of the compound with the CDK12 / 13 protein is increased, the selectivity on the CDK12 / 13 kinase is improved, the synthesis difficulty is reduced, the druggability is substantially improved, and the application prospect is broad.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, specifically relating to a phenyl ether-linked cyclohexylpyrimidine amine CDK12 / 13 inhibitor or its isomers, pharmaceutically acceptable salts, methods for their preparation, 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 class of highly conserved serine / threonine protein kinases that play crucial roles in vital processes such as cell cycle, transcriptional regulation, and cellular homeostasis. In various tumors, the CDK signaling pathway is abnormally activated, directly driving the unlimited proliferation and survival of tumor cells. Several CDK subtypes, including CDK7, 9, 12, and 13, have been identified as important oncogenic drivers, leading to a surge in the development of small-molecule inhibitors targeting CDKs. The ATP-binding pockets of CDK family members are highly conserved in their three-dimensional structure. This structural feature has resulted in early pan-CDK inhibitors (such as Flavopiridol and Roscovitine) exhibiting broad-spectrum antitumor activity in preclinical studies. However, their low selectivity has led to widespread toxicity in clinical trials, severely limiting their clinical application potential.

[0003] In recent years, the role of transcription-related CDKs (such as CDK12 / 13) in tumorigenesis and development has gradually attracted attention and become an important direction in anti-tumor drug research. In various tumor types (such as ovarian cancer and prostate cancer), abnormal expression, loss of function, or mutations of CDK12 / 13 are closely related to tumorigenesis and development. Related studies have shown that impaired CDK12 function can lead to downregulation of genes related to various DNA damage repair pathways, thereby increasing genomic instability and enhancing the sensitivity of tumor cells to DNA damage. Since CDK12 and CDK13 have some functional overlap, a dual-target inhibition strategy is considered to have potential advantages in regulating transcriptional homeostasis in tumor cells. Therefore, CDK12 and CDK13 have been considered anti-tumor therapeutic targets of significant research value, and the development of small-molecule inhibitors targeting CDK12 / 13 is of great importance for anti-tumor drug development. CDK12 / 13 specific inhibitors (such as THZ531 and SR-4835) have shown significant inhibitory effects on transcription-dependent tumors in preclinical models and can kill tumor cells by inducing intron retention and transcriptional collapse. However, existing CDK12 / 13 inhibitors suffer from off-target effects, poor in vivo pharmacokinetics, and narrow therapeutic windows, and no selective CDK12 / 13 inhibitors have yet been approved for marketing. Therefore, the development of novel CDK12 / 13 inhibitors holds promise for providing new options for precision clinical treatment of cancer patients. Summary of the Invention

[0004] One object of the present invention is to provide a pharmaceutically acceptable salt of a phenyl ether-linked cyclohexylpyrimidine amine CDK12 / 13 inhibitor represented by general formula I. The ether-cyclohexylamine linking chain group of the compound of general formula I increases its affinity for CDK12 / 13 proteins, improves its selectivity for CDK12 / 13 kinases, and simultaneously significantly enhances the water solubility of the compound, reduces its synthetic difficulty, 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: 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: in: R1 is selected from hydrogen, methyl, methoxy, fluorine, or chlorine; R2 is selected from chlorine, bromine, and trifluoromethyl; R3 is selected from hydrogen, methyl, and methoxy. R4 is selected from alkyl or cycloalkyl groups; R5 is selected from alkyl or cycloalkyl groups.

[0009] In some preferred embodiments, R4 is methyl.

[0010] In some preferred embodiments, R5 is methyl.

[0011] This invention provides the following specific compounds and pharmaceutically acceptable salts:

[0012]

[0013]

[0014]

[0015]

[0016]

[0017] Secondly, the present invention provides a method for preparing compounds of general formula (I) of the present invention, comprising the following synthetic steps: Step 1: The compound of formula (1) undergoes a nucleophilic substitution reaction with the compound of formula (2) to obtain the compound of formula (3); Step 2: The compound of formula (4) undergoes a nucleophilic substitution reaction with the compound of formula (5) to obtain the compound of formula (6); Step 3: Deprotect the compound of formula (6) to obtain the compound of formula (7); Step 4: The compound of formula (3) reacts with the compound of formula (7) to obtain the compound of formula (8); Step 5: The compound of formula (8) is reduced to obtain the compound of formula (9); Step 6: The compound of formula (9) reacts with the compound of formula (10) to obtain the compound of general formula (I);

[0018] Among them, R1 and R2 have the definition described in general formula (I).

[0019] Thirdly, the present invention provides a pharmaceutical composition comprising the compounds of the present invention and a pharmaceutically acceptable salt.

[0020] In some embodiments, the present invention provides compounds and pharmaceutically acceptable salts of the present invention, as well as pharmaceutical compositions comprising the compounds and pharmaceutically acceptable salts of the present invention, said compounds or pharmaceutical compositions for treating CDK12 / 13-mediated diseases.

[0021] In some embodiments, the present invention provides pharmaceutical compositions comprising the compounds of the present invention and pharmaceutically acceptable salts and pharmaceutically acceptable carriers.

[0022] The compounds of the present invention and 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.

[0023] Fourthly, the present invention provides the use of compounds of Formula I and pharmaceutically acceptable salts, or pharmaceutical compositions comprising them, in the preparation of medicaments for treating CDK12 / 13-mediated diseases.

[0024] In some preferred embodiments, the present invention provides the use of compounds and pharmaceutically acceptable salts of Formula I, or pharmaceutical compositions comprising them, in the preparation of medicaments for treating CDK12 / 13-mediated diseases, including but not limited to breast cancer, ovarian cancer, and prostate cancer. Terminology Explanation

[0025] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0026] 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.

[0027] The term "pharmaceutical composition" as used in this invention refers to a mixture comprising any of the compounds described herein, including a corresponding prodrug, solvate, pharmaceutically acceptable salt or its chemically protected form, and one or more pharmaceutically acceptable carriers and / or other pharmaceutically acceptable mixtures. 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.

[0028] 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.

[0029] 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

[0030] Figure 1 This is a graph showing the effect of M02 on the proliferation capacity of breast cancer and small cell lung cancer cells.

[0031] Figure 2 This is a graph showing the effect of M02 on apoptosis in breast cancer cells.

[0032] Figure 3 This is a graph showing the effect of M02 on the cell cycle of breast cancer.

[0033] Figure 4 and Figure 5 This is a graph showing the effect of M02 on the migration ability of breast cancer and small cell lung cancer cells. Detailed Implementation

[0034] 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.

[0035] Example 1: Preparation of (E)-N-[4-({3-[(5-chloro-4-{(phenyl)amino}pyrimidin-2-yl)amino]cyclohexyl}oxy)phenyl]-4-(dimethylamino)but-2-enamide]

[0036] Step 1: Synthesis of 2,5-dichloro-N-phenylpyrimidine-4-amine 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 25 mL of isopropanol and refluxed at 90 °C for 2 h. After the reaction was complete, the reaction solution was poured into a beaker, diluted with water (H₂O) and ethyl acetate (EA) (150 mL × 3), and extracted. The mixture was washed successively with saturated sodium carbonate and saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 1.32 g of a grayish-white solid, with a yield of 98%.

[0037] Step 2: Synthesis of tert-butyl (3-(4-nitrophenoxy)cyclohexyl)carbamate NaH (0.56 g, 23.2 mmol) was slowly added to 50 mL of ultradry N,N-dimethylformamide (DMF) solution under ice bath conditions and stirred for 15 min. Then, tert-butyl (3-hydroxycyclohexyl) carbamate (1 g, 4.64 mmol) was added to the NaH-DMF solution, and the reaction was continued for 30 min. Then, p-nitrofluorobenzene (0.98 g, 6.94 mmol) was added dropwise, and the mixture was transferred to room temperature for 2 h. After the reaction was complete, a small amount of water was added to the reaction solution to quench excess sodium hydrogen. Then, ethyl acetate and water were added, and the mixture was extracted and separated. The organic phase was washed 2-3 times with water. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a yellow solid crude product. Purification was performed by silica gel column chromatography with petroleum ether:ethyl acetate = 15:1 as the eluent, yielding 1.23 g of a white solid, with a yield of 78.8%.

[0038] Step 3: Synthesis of 3-(4-nitrophenoxy)cyclohexane-1-amine tert-butyl(3-(4-nitrophenoxy)cyclohexyl)carbamate (1 g, 2.97 mmol) was dissolved in 10 mL of dichloromethane and placed in an ice bath. 1 mL of TFA (1.49 g, 13.06 mmol) was added, and the reaction was allowed to proceed for 1 h. After the reaction was complete, saturated sodium bicarbonate solution was added to adjust the reaction solution to alkaline. Ethyl acetate and water were added, and the mixture was extracted and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 0.68 g of a yellow solid, with a yield of 97.1%.

[0039] Step 4: 5-Chloro-N²-[3-(4-nitrophenoxy)cyclohexyl]-N 4 Synthesis of 2,4-phenylpyrimidine-2,4-diamine 2,5-Dichloro-N-phenylpyrimidin-4-amine (0.5 g, 2.08 mmol), 3-(4-nitrophenoxy)cyclohexane-1-amine (0.49 g, 2.07 mmol), and DIEA (0.32 g, 2.50 mmol) were added to a solvent containing 20 mL of N-methylpyrrolidone (NMP) and reacted at 125 °C for two hours. After the reaction was complete, the reaction mixture was poured into a separatory funnel, EA and H2O were added, and the mixture was extracted and separated. The organic phase was washed with water 2-3 times to ensure the removal of residual NMP. The organic phases were combined and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a brownish-brown solid crude product. Purification was performed by silica gel column chromatography with petroleum ether:ethyl acetate = 5:1 as the eluent, yielding 0.77 g of an off-white solid product, with a yield of 84.6%.

[0040] Step 5: N²-[3-(4-aminophenoxy)cyclohexyl]-5-chloro-N 4 Synthesis of 2,4-phenylpyrimidine-2,4-diamine 5-Chloro-N²-[3-(4-nitrophenoxy)cyclohexyl]-N 4 2,4-Phenyrimidine-2,4-diamine (0.5 g, 1.14 mmol) was added to a mixed solvent of ethanol:water = 4:1 (20 ml), followed by iron powder (0.64 g, 11.4 mmol) and ammonium chloride (0.61 g, 11.4 mmol). The reaction was carried out at 85 °C for 1 h. After the reaction was complete, saturated sodium bicarbonate solution was added to adjust the pH of the reaction solution to alkaline. Then, dichloromethane and water were added, and the mixture was extracted and separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 0.43 g of a grayish-white solid, with a yield of 93.4%.

[0041] Step 6: Synthesis of (E)-4-dimethylaminobutyronyl chloride Weigh out N,N-dimethyltrans-crotonate (0.5 g, 3.02 mmol) and DMF (0.1 ml) and dissolve them in ultra-dry acetonitrile solution (10 ml). Under ice bath conditions, dissolve oxaloyl chloride (0.4 ml, 4.53 mmol) in ultra-dry acetonitrile solution (2 ml) and slowly add it to the acetonitrile solution containing N,N-dimethyltrans-crotonate. Then continue stirring in an ice bath for 10 minutes, and transfer the reaction system to room temperature for one hour until the color of the reaction solution changes from the initial white turbidity to a pale yellow clear solution, indicating that the reaction is complete.

[0042] Step 7: (E)-N-[4-({3-[(5-chloro-4-{(phenyl)amino}pyrimidin-2-yl)amino]cyclohexyl}oxy)phenyl]-4-(dimethylamino)but-2-enamide N²-[3-(4-aminophenoxy)cyclohexyl]-5-chloro-N4 2,4-Phenyrimidine-2,4-diamine (0.5 g, 1.21 mmol) was dissolved in 5 ml of NMP solution and placed in an ice bath. Then, the acetonitrile solution of (E)-4-dimethylaminobutyric acid-2-enoyl chloride prepared in step 6 was slowly added dropwise. After the addition was complete, the reaction was continued in an ice bath for another half hour until the reaction was complete. Subsequently, a small amount of saturated sodium bicarbonate solution was added to the reaction solution, followed by EA and H2O. The mixture was extracted and separated. The organic phase was washed with water 2-3 times to ensure the removal of residual NMP. The organic phases were combined and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure to obtain 789 mg of a brown solid crude product. The crude solid product was purified by silica gel column chromatography with dichloromethane:methanol = 30:1 as the eluent, yielding 453 mg of a white solid product, with a yield of 71.3%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.89 (s, 1H), 8.65 – 8.44 (m, 1H), 7.95 (s, 1H), 7.76 (d, J = 7.8 Hz, 2H), 7.49 (s, 2H), 7.31 (t, J = 7.4Hz, 2H), 7.04 (t, J = 7.2 Hz, 2H), 6.95 – 6.74 (m, 2H), 6.68 (dt, J = 15.4, 5.9Hz, 1H), 6.27 – 6.17 (m, 1H), 4.21 (s, 1H), 3.87 – 3.56 (m, 1H), 3.04 (d, J =6.0 Hz, 2H), 2.35 – 2.21 (m, 1H), 2.17 (s, 6H), 2.08 – 1.71 (m, 3H), 1.41 –1.13 (m, 4H). 13 C NMR (100 MHz, CDCl3) δ 163.46, 159.58, 155.63, 154.44,154.26, 141.69, 138.41, 131.41, 128.89, 126.15, 123.89, 121.74, 120.94,116.60, 75.02, 60.37, 45.49, 38.00, 31.87, 31.21, 20.35. Example 2: Preparation of (E)-N-[4-({3-[(5-bromo-4-{(phenyl)amino}pyrimidin-2-yl)amino]cyclohexyl}oxy)phenyl]-4-(dimethylamino)but-2-enamide]

[0043] Using 5-bromo-2,4-dichloropyrimidine (1 g, 4.38 mmol) and aniline (0.56 g, 6.02 mmol) as raw materials, and with other operations the same as in Example 1, silica gel column chromatography was used to separate and purify the target product into a white solid of 415 mg, with a yield of 66.7%. 1 H NMR (400 MHz, DMSO-) d 6) δ 9.89 (s, 1H), 8.39 – 8.16 (m, 1H), 8.02 (s, 1H), 7.72(d, J = 7.8 Hz, 2H), 7.49 (s, 2H), 7.31 (t, J = 7.8 Hz, 2H), 7.04 (t, J = 7.3 Hz,2H), 6.97 – 6.74 (m, 2H), 6.68 (dt, J = 15.3, 6.0 Hz, 1H), 6.26 – 6.18 (m, 1H), 4.18 (s, 1H), 3.86 – 3.53 (m, 1H), 3.05 (d, J = 6.0 Hz, 2H), 2.26 (s, 1H), 2.17(s, 6H), 2.07 – 1.71 (m, 3H), 1.41 – 1.25 (m, 2H), 1.23 – 1.10 (m, 2H). 13 C NMR(100 MHz, CDCl3) δ 162.26, 160.53, 157.56, 156.36, 141.18, 138.46, 126.43,123.84, 123.66, 121.63, 121.09, 120.79, 116.89, 116.51, 73.21, 60.28, 46.37,45.49, 36.75, 32.48, 19.56. Example 3: Preparation of (E)-N-[4-([3-([5-bromo-4-(3-methylphenylamino)pyrimidin-2-yl]amino)cyclohexyl]oxy)phenyl]-4-(dimethylamino)but-2-enamide]

[0044] Using 5-bromo-2,4-dichloropyrimidine and 3-methylaniline as raw materials, the procedure was the same as in Example 1. The product was purified by silica gel column chromatography to obtain 453 mg of white solid product, with a yield of 68.6%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.87 (s, 1H), 8.28 – 7.96 (m, 2H), 7.61 – 7.39 (m, 4H), 7.19 (t, J = 7.8 Hz, 1H), 7.08 – 6.63(m, 5H), 6.22 (d, J = 15.4 Hz, 1H), 4.44 – 4.07 (m, 1H), 3.90 – 3.56 (m, 1H), 3.04 (d, J = 6.0 Hz, 2H), 2.36 – 2.22 (m, 4H), 2.17 (s, 6H), 2.08 – 1.70 (m,3H), 1.43 – 1.11 (m, 4H). 13 C NMR (100 MHz, DMSO) δ 163.19, 160.24, 158.37,156.51, 153.45, 141.15, 139.44, 137.82, 132.84, 128.52, 126.55, 124.36,122.92, 121.13, 119.48, 116.22, 74.80, 60.22, 45.61, 40.53, 38.45, 31.78,21.71. Example 4: Preparation of (E)-N-[4-([3-([5-bromo-4-(3-chlorophenylamino)pyrimidin-2-yl]amino)cyclohexyl]oxy)phenyl]-4-(dimethylamino)but-2-enamide]

[0045] Using 5-bromo-2,4-dichloropyrimidine and 3-chloroaniline as raw materials, the procedure was the same as in Example 1. The product was purified by silica gel column chromatography to obtain 403 mg of white solid product, with a yield of 64.6%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.88 (s, 1H), 8.56– 8.32 (m, 1H), 8.15 – 7.97 (m, 2H), 7.85 – 7.60 (m, 1H), 7.57 – 7.41 (m, 2H), 7.33 (t, J= 8.1 Hz, 1H), 7.22 – 7.05 (m, 2H), 6.96 – 6.63 (m, 3H), 6.22(d, J = 15.3 Hz, 1H), 4.34 – 4.17 (m, 1H), 3.88 – 3.61 (m, 1H), 3.05 (d, J = 5.7Hz, 2H), 2.36 – 2.23 (m, 1H), 2.17 (s, 6H), 2.10 – 1.69 (m, 3H), 1.43 – 1.16(m, 4H). 13 C NMR (100 MHz, DMSO- D 6) δ 163.26, 160.19, 156.49, 153.53, 141.28,141.16, 133.03, 132.91, 130.33, 126.66, 123.13, 121.67, 121.20, 120.58,116.28, 74.84, 60.26, 45.65, 45.51, 38.43, 31.88, 21.29, 14.61. Example 5: Preparation of (E)-N-[4-([3-([5-bromo-4-(3-fluorophenylamino)pyrimidin-2-yl]amino)cyclohexyl]oxy)phenyl]-4-(dimethylamino)but-2-enamide]

[0046] Using 5-bromo-2,4-dichloropyrimidine and 3-fluoroaniline as raw materials, the procedure was the same as in Example 1. The product was purified by silica gel column chromatography to obtain 433 mg of white solid product, with a yield of 68.1%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.88 (s, 1H), 8.54– 8.30 (m, 1H), 8.07 (s, 1H), 7.80 (d, J = 12.2 Hz, 1H), 7.62 – 7.41 (m, 3H), 7.38 – 6.75 (m, 5H), 6.68 (dt, J = 15.4, 5.9 Hz, 1H), 6.22 (d, J= 15.3 Hz, 1H),4.34 – 4.15 (m, 1H), 3.89 – 3.59 (m, 1H), 3.09 – 2.99 (m, 2H), 2.34 – 2.22(m, 1H), 2.17 (s, 6H), 2.09 – 1.70 (m, 3H), 1.45 – 1.28 (m, 2H), 1.26 – 1.11(m, 2H). 13 C NMR (100 MHz, DMSO- D 6) δ 163.26, 160.19, 156.49, 153.53, 151.78,146.16, 136.03, 132.91, 130.33, 126.66, 123.13, 121.67, 121.20, 120.58,116.28, 74.84, 60.26, 45.65, 45.51, 38.43, 31.88, 21.29, 14.61. Example 6: Preparation of (E)-N-[4-([3-([5-bromo-4-(3-methoxyphenylamino)pyrimidin-2-yl]amino)cyclohexyl]oxy)phenyl]-4-(dimethylamino)but-2-enamide]

[0047] Using 5-bromo-2,4-dichloropyrimidine and 3-methoxyaniline as raw materials, the procedure was the same as in Example 1. The product was purified by silica gel column chromatography to obtain 423 mg of white solid product, with a yield of 67.6%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.88 (s, 1H),8.29 – 8.09 (m, 1H), 8.03 (s, 1H), 7.56 – 7.27 (m, 4H), 7.25 – 6.73 (m, 4H), 6.72 – 6.60 (m, 2H), 6.21 (d, J = 15.4 Hz, 1H), 4.33 – 4.14 (m, 1H), 3.88 –3.59 (m, 4H), 3.03 (d, J = 6.1 Hz, 2H), 2.34 – 2.21 (m, 1H), 2.16 (s, 6H), 2.08 – 1.71 (m, 3H), 1.39 – 1.14 (m, 4H). 13C NMR (100 MHz, DMSO) δ 163.79, 161.24,158.57, 156.01, 152.45, 141.55, 139.54, 137.82, 131.84, 128.22, 126.35,123.36, 122.82, 120.13, 119.78, 117.22, 75.80, 61.22, 45.91, 40.43, 38.45,30.78, 22.71. Experiment Example 1: Compound Kinase Activity Experiment

[0048] CDK12, CDK13, CDK7, and CDK9 kinases were pre-incubated with different concentrations of compounds THZ531 and MO2 for 30 min. ATP (final concentration 10 μM) and fluorescent substrates were added to initiate the reaction. After the reaction was terminated, substrate phosphorylation levels were detected using a Caliper EZ Reader, and the kinase activity inhibition rate was calculated. The IC50 of the tested compounds for each kinase was determined by fitting dose-response curves using nonlinear regression (GraphPad Prism software). 50 The selectivity of MO2 for the CDK family was evaluated using THZ531 as a control compound. The results are shown in Table 1.

[0049] Table 1. Inhibitory effects of the tested compounds on different CDK kinases

[0050] The experimental results showed that, compared with the control compound THZ531, compound M02 exhibited a significant decrease in CDK9 kinase inhibitory activity, approximately 3.9-fold; however, compared with CDK9, M02 showed increased selectivity for CDK12 and CDK13 kinase inhibition. These results indicate that, compared with the control compound THZ531, compound M02 improved selectivity for CDK12 / 13.

[0051] Experiment Example 2: Tumor Cell Proliferation Inhibition Experiment

[0052] CCK8 assay was used to detect the 48-hour IC50 of the compound on breast cancer and small cell lung cancer cells. 50 Values: The experiment included a solvent treatment control group (DMSO group) and compound treatment experimental groups (THZ531, M01, M02). Each drug experimental group had 8 concentrations, with 3 parallel wells per concentration. Each experiment was repeated three times. In a 96-well plate, 3 × 10⁶ cells were added to each well. 3During inoculation, ensure cells are evenly distributed in each well. To prevent liquid evaporation, leave the outer ring of wells uninoculated and add PBS for hydration. Incubate the 96-well plate overnight at 37°C with 5% CO2. After cell attachment, aspirate and discard the original culture medium from the experimental group. Add 200 μL of culture medium containing the target compound at different final concentrations to each well and continue incubation for 48 h. After 48 h of compound treatment, remove the 96-well plate, add CCK8 solution to each well, and incubate for 2–4 hours in a cell culture incubator. Measure the absorbance (OD) of each well at 450 nm using an automated microplate reader. Calculate cell viability using a formula and fit the IC50 of the compound using GraphpadPrism 10.0 and IBM SPSS Statistics 29.0 software. 50 Values. The results are shown in Table 2.

[0053] Table 2. Inhibitory effects of the tested compounds on the proliferation of three types of breast cancer cells and one type of small cell lung cancer cells.

[0054] Experimental results showed that, compared with the control compound THZ531, compound M02 exhibited potent inhibitory activity against the proliferation of two types of breast cancer cells and one type of small cell lung cancer cell, with activity close to that of THZ531. Compound M01, however, showed weaker inhibitory activity, with an IC50 value of less than 48 h in the three cancer cell lines. 50 All values ​​are greater than 1.0.

[0055] Cell cloning assay to detect the effect of compounds on the clonogenic ability of breast cancer and small cell lung cancer cells: Cells in logarithmic growth phase were analyzed at a ratio of 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 MO2 (0, 150, 300, 600 nM) for 14 days, with fresh complete culture medium containing the corresponding concentration of the drug added every 2 days. After 14 days, the supernatant was discarded, and the cells were washed 2-3 times with 1 mL PBS per well. Then, 1 mL of 4% paraformaldehyde was added to fix the cells for 1 hour, after which the paraformaldehyde was discarded. The cells were washed 2-3 times with PBS, and then stained with 1 mL of crystal violet solution per well for 1 hour. The crystal violet solution was discarded, and the 12-well plates were washed 2-3 times with pure water. After air-drying, the plates were photographed. The results were analyzed and statistically processed using ImageJ software and Graphpad Prism 10.0. The experimental results are shown below. Figure 1 This indicates that MO2 can inhibit the cell clone formation of breast cancer and small cell lung cancer in a concentration-dependent manner.

[0056] Experiment 3: M02 promotes apoptosis in breast cancer cells and induces G2 / M phase arrest in breast cancer cells.

[0057] After treating breast cancer cells MFM-223 and MDA-MB-231 with different concentrations of MO2 (0, 150, 300, 600 nM) for 48 h, cells from each group were collected, and flow cytometry was used to detect the level of apoptosis and cell cycle distribution after drug treatment.

[0058] Flow cytometry detection of apoptosis: Breast cancer cells in logarithmic growth phase with a density of approximately 80% were digested, centrifuged, and seeded in 6-well cell culture plates at passage density. Cells were allowed to adhere overnight until stable, then the medium was replaced with the appropriate concentration of MO2. Cells were treated continuously for 48 h, with DMSO added to the control group. After treatment, cells were digested into single cells using trypsin without EDTA, centrifuged at 1000 r / min for 5 min to collect the cell pellet, washed twice with pre-chilled PBS (4°C), and resuspended in 500 μL of pre-chilled PBS. Cells were counted at a rate of 1 × 10⁶ cells / well. 6 Cells were centrifuged, the supernatant was discarded, and the cells were resuspended in 195 μL of Annexin V-FITC binding buffer; 5 μL of Annexin V-FITC was added, and the cells were gently mixed by pipetting; 10 μL of propidium iodide (PI) staining solution was added, and the cells were gently mixed by pipetting; the cells were incubated at room temperature in the dark for 10-20 min, followed by ice bath, and flow cytometry was immediately used for analysis. The distribution of apoptosis in each treatment group was analyzed using ModFit and Graphpad 10.0. Figure 2 ).

[0059] Cell cycle analysis by flow cytometry: Breast cancer cells in logarithmic growth phase with a density of approximately 80% were seeded in 6-well cell culture plates at passage density. Cells were allowed to adhere overnight until stable. Cells were digested into individual cells using trypsin without EDTA, centrifuged at 1000 rpm for 5 min, and the cell pellet was collected. The cells were washed three times with pre-chilled PBS (4°C) under the same conditions. After washing, the cells were resuspended in 100 μL of pre-chilled PBS. 900 μL of pre-chilled 70% ethanol (4°C) was slowly added to 100 μL of the cell suspension while shaking, and the cells were fixed overnight at 4°C. The fixed cells were centrifuged at 1000 rpm for 5 min, washed twice with pre-chilled PBS (4°C), and 0.5 mL of PI staining solution was added, gently mixed, and incubated at 37°C in the dark for 30 min. Fluorescence was detected by flow cytometry at an excitation wavelength of 488 nm. The cell cycle distribution of each group was analyzed using ModFit and Graphpad 10.0 software. Figure 3 ).

[0060] Experimental results showed that MO2 could promote apoptosis in breast cancer cells in a concentration-dependent manner and could induce G2 / M phase arrest in breast cancer cells in a concentration-dependent manner.

[0061] Experimental Example 4: M02 Inhibits Breast Cancer Cell Migration

[0062] Different concentrations of MO2 (0, 150, 300, 600 nM) were used to treat breast cancer and small cell lung cancer cells, and cell scratch assay and Transwell migration assay were used to detect cell migration ability.

[0063] 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 cell confluence reached ≥90%, scratches were performed using a 200 μL pipette tip. Cells were washed three times with PBS to remove the scratched cells, and then serum-free medium was added for further culture. Cell migration ability was observed by photographing under a 10x objective lens using a Nikon inverted microscope at 0, 24, and 48 h. Results were analyzed and statistically processed using ImageJ software and Graphpad Prism 10. Figure 4 ).

[0064] Transwell migration assay: Cells in the logarithmic growth phase with a density of approximately 80% were seeded in the upper chamber of a 24-well Transwell plate at a cell density of 2 × 10⁻⁶ cells / well. 4 For each well, add 200 μL of culture medium to the upper chamber for incubation. After cells adhere overnight, replace the upper chamber with 200 μL of FBS-free medium and add 500 μL of medium containing 20% ​​FBS to the lower chamber as a chemotactic medium. After 48 h of incubation, remove the Transwell chambers from the original 24-well cell culture plates. Gently wash the upper chamber with PBS to remove the liquid, then place the plates in new 24-well cell culture plates and fix the cells with 1 mL of paraformaldehyde per well at room temperature for 1 h. After fixation, remove the Transwell chambers from the 24-well cell culture plates and gently shake them again in PBS wells to remove excess paraformaldehyde. Stain the Transwell chambers thoroughly with 0.1% crystal violet for 1 h. Then wash away any remaining crystal violet on the surface of the Transwell chambers with PBS, and gently absorb any remaining crystal violet from the upper chamber mesh surface with a PBS-soaked swab. The cells were allowed to dry completely overnight at room temperature. The Transwell cells were then placed on a slide, and photographed and stained under a 10x objective lens using a Nikon inverted microscope. The results were analyzed using ImageJ software and Graphpad 10.0. Figure 5 Experimental results showed that MO2 could inhibit the migration ability of breast cancer and small cell lung cancer cells in a time- and concentration-dependent manner.

Claims

1. A phenyl ether-linked cyclohexylpyrimidineamine compound of Formula I, or a pharmaceutically acceptable salt thereof. in: R1 is selected from hydrogen, methyl, methoxy, fluorine, or chlorine; R2 is selected from chlorine, bromine, or trifluoromethyl; R3 is selected from hydrogen, methyl, and methoxy. R4 is selected from C 1-6 Alkyl or C 3-6 cycloalkyl; R5 is selected from C 1-6 Alkyl or C 3-6 Cycloalkyl.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is hydrogen.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R2 is bromine.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R3 is hydrogen.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R4 is selected from methyl, ethyl, propyl or cyclopropyl; preferably, R4 is methyl.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R5 is selected from methyl, ethyl, propyl or cyclopropyl; preferably, R5 is methyl.

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

8. A pharmaceutical composition comprising the compound of any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

9. The use of the compound of any one of claims 1-7 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 8 in the preparation of a medicament for treating CDK12 / 13-related diseases.

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