CN118324693A - Piperazine compound, preparation method thereof, pharmaceutical composition and application - Google Patents

Abstract

The invention discloses a piperazine compound, a preparation method thereof, a pharmaceutical composition and application thereof. The structure of the compound is shown as a formula I, and the distribution of target tissues and the anti-tumor activity are enhanced by improving the solubility and the permeability, so that the compound is more beneficial to patent medicine. The EZH2 inhibition activity optimally reaches the nanomolar concentration level, is superior to the existing clinical medicines, and has good clinical application prospect.

Description

Piperazine compound and preparation method, pharmaceutical composition and application thereof

Technical Field

The present invention relates to a piperazine compound and a preparation method, a pharmaceutical composition and application thereof, and in particular to a piperazine compound with EZH2 inhibitory activity and a preparation method, a pharmaceutical composition and application thereof.

Background technique

EZH2 is an enzymatic subunit of the polycomb repressive complex 2 (PRC2), which catalyzes the trimethylation of histone H3 lysine 27, leading to chromatin compaction and altered expression of downstream target genes. The functions of EZH2 in cell proliferation, apoptosis and senescence have been identified, and its important role in tumor pathophysiology has also received widespread attention.

Compared with normal tissues, the expression level of EZH2 protein in cancer tissues is abnormally increased, so EZH2 is used as a marker in cancer treatment. Although different types of EZH2 inhibitors have been developed with EZH2 as the target, some reported EZH2 inhibitors have limitations such as poor efficacy, large molecular weight, large dosage, and low bioavailability.

Summary of the invention

Objectives of the invention: The first objective of the present invention is to provide a piperazine compound, the second objective is to provide a method for preparing the compound, the third objective is to provide a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof, and the fourth objective is to provide a pharmaceutical application of the compound or a pharmaceutically acceptable salt thereof and the pharmaceutical composition.

Technical solution: The piperazine compound of the present invention has a structure of formula I,

in:

R is selected from a 6- to 12-membered aromatic group and a 6- to 12-membered aromatic hetero group containing 1 to 3 N atoms, wherein the aromatic group and the aromatic hetero group are substituted by at least one of the following substituents: hydrogen, deuterium, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, cyano, nitro, thiol, and carboxyl.

Preferably, in the structure:

R is selected from phenyl, pyridyl, naphthyl, benzopyridyl, and R is substituted by at least one of the following substituents: hydrogen, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, and cyano.

Preferably, the number of substituents on R is selected from mono-substitution, di-substitution and tri-substitution.

Preferably, in the structure:

R is selected from R' is selected from at least one of hydrogen, halogen, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, and cyano.

Further preferably, the R' is selected from at least one of hydrogen, fluorine, chlorine, bromine, trifluoromethyl, difluoromethyl, methyl, ethyl, isopropyl, n-propyl, methoxy, ethoxy, isopropoxy, n-propoxy, and cyano.

Still more preferably, the number of R' is selected from mono-substitution, di-substitution and tri-substitution.

More preferably, the piperazine compound of the present invention is selected from any of the following compounds:

In order to further improve the inhibitory activity against EZH2, and considering that the solubility of the compound may cause cell penetration problems, the present invention designs and synthesizes a series of piperazine derivatives. The introduction of piperazine can increase the solubility of the compound, improve membrane permeability, improve the physical and chemical properties, and also facilitate interaction with the target. The introduction of different volume groups at the tail, including the introduction of halogen atoms, is conducive to enhancing membrane permeability, enhancing the distribution of drugs in target tissues, and enhancing the anti-tumor activity of the compound.

The pharmaceutically acceptable salt of the piperazine compound of the present invention is a salt formed by the compound and any one of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, and ferulic acid.

"Pharmaceutically acceptable salts" refer to salts of compounds prepared from compounds having specified substituents with relatively nontoxic acids or bases. When the compound contains relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compound contains relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonate or bicarbonate), phosphoric acid (forming phosphate, monohydrogen phosphate, dihydrogen phosphate), sulfuric acid (forming sulfate or bisulfate), hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and the like; organic acid salts also include salts of organic acids such as amino acids (such as arginine, etc.), glucuronic acid, etc. When certain specific compounds contain basic and acidic functional groups, they can be converted into any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

"Pharmaceutically acceptable salts" can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

Preferably, the stereoisomer is an isomer with chiral C and N introduced.

Preferably, the tautomers are isomers formed by conjugated tautomerism of double bonds in unsaturated heterocyclic rings, including carbon-carbon double bond tautomerism, carbon-heteroatom, heteroatom-heteroatom double bond tautomerism, such as tautomers formed by tautomerism of double bonds in imidazole ring systems and pyrazole rings.

Preferably, the prodrug is an ester or amide prodrug with a carboxyl group, a hydroxyl group or an amino group introduced therein, more preferably a C1-C4 alkyl ester, a C1-C4 carboxylate or a C1-C4 alkyl amide.

Preferably, the solvate is a small molecule binding state formed by the compound and solvent molecules, more preferably a hydrate or an alcoholate; the solvate can further form a salt with a corresponding acid to obtain a salt of the solvate.

Preferably, the isotope compound is a compound in which hydrogen is replaced by deuterium.

Preferably, the crystal is a specific crystal structure formed by the compound during the crystallization process, including different crystal forms of the compound itself, and also includes different crystal forms of its salts, solvates, and salts of solvates.

The preparation method of the piperazine compound of the present invention comprises the following steps:

(1) Compound 1 is salified and cyclized with compound 3 to obtain compound 4;

(2) Compound 4 was subjected to desalination, acetylation, and ester hydrolysis to obtain compound 7;

(3) Compound 7 is acylated and coupled with R to obtain compound I;

Wherein, R is defined as above;

The corresponding acid is reacted with the compound I prepared by the above method to form a salt, thereby obtaining a pharmaceutically acceptable salt of the compound I.

The specific method is as follows:

1. Preparation of Compound 2

Chloroform was selected as the reaction solvent. During the preparation, compound 1 was dissolved in an organic solvent, and thionyl chloride diluted with chloroform was slowly added dropwise, reacted at 0°C for 3 hours, and then condensed and refluxed for 0.5 hours, and the reaction temperature was 60°C.

2. Preparation of Compound 4

The reaction solvent is n-butanol. During the preparation, compound 2 and compound 3 are dissolved in an organic solvent, the reaction time is 72 hours, and the reaction temperature is 120°C.

3. Preparation of Compound 5

The reaction solvent is a sodium hydroxide aqueous solution. During the preparation, compound 4 is dissolved in a sodium hydroxide aqueous solution to adjust the pH to be greater than 11.

4. Preparation of Compound 6

The reaction solvent is dichloromethane; the reaction is carried out in the presence of an acid-binding agent, generally triethylamine. During the preparation, compound 5 is dissolved in an organic solvent, triethylamine is added, ice-bathed at 0°C, acetyl chloride is slowly added dropwise, and the reaction is carried out at room temperature for 1 hour.

5. Preparation of Compound 7

The reaction solvent is methanol; the reaction is carried out under alkaline conditions, generally sodium hydroxide is used. During the preparation, compound 6 is dissolved in an organic solvent, sodium hydroxide aqueous solution is added, condensed and refluxed for 2 hours, and the reaction temperature is 60°C.

6. Preparation of Compound 8

The reaction solvent is N,N-dimethylformamide; the reaction is carried out under alkaline conditions and in the presence of a catalyst, DIPEA provides an alkaline environment, and the catalyst is selected from PyBOP or HATU. During the preparation, compound 7 is dissolved in an organic solvent, a base and a catalyst are added, and after stirring and activating for 0.5 h at room temperature, 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one is added, and the reaction is carried out at room temperature for 12 h.

7. Preparation of Compound I

The reaction solvent is 1,4-dioxane; the reaction solvent is carried out under alkaline conditions and in the presence of a catalyst under nitrogen protection. The base is selected from potassium carbonate, sodium carbonate or cesium carbonate, and the catalyst is selected from Pd(dppf)Cl ₂ or tetrakis(triphenylphosphine)palladium. During the preparation, the base is dissolved in a small amount of water, compound 8 and the corresponding boric acid are dissolved in an organic solvent, stirred, and a catalyst is added. The reaction is carried out under nitrogen protection, the reaction temperature is 100-110°C, and the reaction time is 4-5h.

The pharmaceutical composition of the present invention comprises the piperazine compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Preferably, the preparation form is selected from tablets, capsules, powders, syrups, liquids, suspensions, lyophilized powder injections, and injections.

"Pharmaceutically acceptable carrier" can be an excipient widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

The pharmaceutical composition of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or sustained release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

The piperazine compound, the pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof of the present invention is used in the preparation of EZH2 inhibitor drugs.

Preferably, the drug is an anti-tumor drug.

Further preferably, the tumor is selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, breast cancer, prostate cancer or malignant rhabdoid tumor.

Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

The compounds designed by the present invention enhance target tissue distribution and anti-tumor activity by improving solubility and permeability, and are more conducive to drug development. They have excellent EZH2 inhibitory activity, optimally reaching nanomolar concentration levels, are superior to existing clinical drugs, and have good clinical application prospects.

Detailed ways

The technical solution of the present invention is further described below in conjunction with embodiments.

Example 1: Bis(2-chloroethyl)amine hydrochloride (Compound 2)

Add diethanolamine (2mL, 20.9mmol, 1eq) and chloroform 8mL into a double-necked flask, slowly drop 2mL of chloroform diluted with thionyl chloride (4.56mL, 62.8mmol, 3eq) under 0℃ ice bath condition, react at room temperature for 3h after the dropwise addition. Then reflux at 60℃ for 0.5h. Cool to room temperature, filter, and vacuum dry to obtain 3.18g of white solid bis(2-chloroethyl)amine hydrochloride, yield: 85.2%.

Example 2: Preparation of 5-bromo-2-methyl-3-(piperazin-1-yl)benzoic acid methyl ester hydrochloride (Compound 4)

Add 3-amino-5-bromo-2-methylbenzoic acid methyl ester (4.11 g, 168 mmol, 1 eq), bis(2-chloroethyl)amine hydrochloride (3 g, 168 mmol, 1 eq) and 12 ml of n-butanol into a double-necked flask. Heat to reflux and react at 120°C for 72 h. After the reaction is completed, cool to room temperature, dissolve the mixture slightly with a small amount of methanol, and then pour in an appropriate amount of ether. A large amount of solid precipitates, and 4.49 g of white solid is obtained by suction filtration. The yield is 76.4%.

Example 3: Preparation of methyl 5-bromo-2-methyl-3-(piperazin-1-yl)benzoate (Compound 5)

The compound 4.49 g (128 mmol) obtained in the previous step was adjusted to a pH value greater than 11 with 40% sodium hydroxide solution. After extraction with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and desolventized under reduced pressure to obtain 4.01 g of a yellow oil with a yield of 89.5%.

¹ H NMR (600 MHz, DMSO-d ₆ ) δ: 7.03 (d, J=2.0 Hz, 1H), 6.93 (d, J=2.0 Hz, 1H), 3.81 (s, 3H), 3.52 (s, 4H), 3.17 (s, 4H), 2.21 (s, 3H), 1.24 (s, 1H).

Example 4: Preparation of methyl 3-(4-acetylpiperazine-1-yl)-5-bromo-2-methylbenzoate (Compound 6)

Compound 5 (1.77 g, 5 mmol, 1 eq) was dissolved in 5 mL of dichloromethane solution, triethylamine (0.42 mL, 3 mmol, 1.5 eq) was added and the mixture was cooled to 0°C. Acetyl chloride (0.22 mL, 3 mmol, 1.5 eq) was added dropwise. After 0.5 h, the reaction mixture was allowed to warm to room temperature and reacted for 1 h. The mixture was filtered under reduced pressure and dried in vacuo to obtain 1.15 g of a white solid with a yield of 65.1%.

¹ H NMR (600 MHz, DMSO-d ₆ ) δ: 7.58 (d, J=2.1 Hz, 1H), 7.36 (d, J=2.2 Hz, 1H), 3.83 (s, 3H), 3.65-3.50 (s, 4H), 2.83 (dt, J=42.0, 5.0 Hz, 4H), 2.37 (s, 3H), 2.04 (s, 3H).

Example 5: Preparation of 3-(4-acetylpiperazin-1-yl)-5-bromo-2-methylbenzoic acid (Compound 7)

Compound 6 (1.15 g, 3.37 mmol, 1 eq), 50% sodium hydroxide aqueous solution (2 eq) and 6 mL methanol solution were mixed and refluxed at 60°C for 2 h. After the reaction was completed, the solvent was removed under reduced pressure. The mixture was dissolved in 10 mL of water, acidified with hydrochloric acid to pH = 1.0, and the precipitate was collected by suction filtration and vacuum dried to obtain 0.79 g of a white solid with a yield of 68.7%.

¹ H NMR (600 MHz, DMSO-d ₆ ) δ: 13.23 (s, 1H), 7.56 (d, J=2.1 Hz, 1H), 7.32 (d, J=2.1 Hz, 1H), 3.57 (s, 4H), 2.82 (dt, J=41.6, 5.0 Hz, 4H), 2.38 (s, 3H), 2.04 (s, 3H).

Example 6: Preparation of 3-(4-acetylpiperazin-1-yl)-5-bromo-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methylbenzamide (Compound 8)

Compound 7 (0.57 g, 1.66 mmol, 1.2 eq), PyboP (1.08 g, 2.07 mmol, 1.5 eq), and DIPEA (0.74 mL, 4.14 mmol, 3 eq) were dissolved in 10 mL of anhydrous DMF solution. The reaction mixture was stirred at room temperature for 30 min, and then 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (0.21 g, 1.38 mmol, 1 eq) was added. The reaction was continued at room temperature for 14 h. After the reaction was complete as determined by thin layer chromatography, the reaction mixture was introduced into 100 mL of cold water and stirred for 30 min. The precipitated solid was collected by vacuum filtration, washed with saturated brine (50 mL), and dried. The crude product was purified by methanol: dichloromethane column chromatography to obtain the desired light brown solid 278 mg, with a yield of 42.4%.

¹ H NMR (600 MHz, DMSO-d ₆ ) δ: 11.49 (s, 1H), 8.24 (t, J=5.0 Hz, 1H), 7.16 (d, J=2.1 Hz, 1H), 7.09 (d, J=2.1 Hz, 1H), 5.86 (s, 1H), 4.25 (d, J=5.0 Hz, 2H), 3.56 (s, 4H), 2.80 (dt, J=42.8, 5.0 Hz, 4H), 2.19 (s, 3H), 2.18 (s, 3H), 2.11 (s, 3H), 2.04 (s, 3H).

Example 7: Preparation of Compound C-1

Compound 8 (238 mg, 0.5 mmol, 1 eq), 4-chlorophenylboronic acid (94 mg, 0.60 mmol, 1.2 eq), anhydrous sodium carbonate (64 mg, 0.6 mmol, 1.2 eq), 1,4-dioxane (10 mL) and a mixed solution of water (1 mL) were added to a round-bottom flask, and tetrakis(triphenylphosphine)palladium (0.56 g, 0.05 mmol) was added and immediately replaced with nitrogen three times. The reaction mixture was reacted at 105°C for 4 h. After the reaction was completed as determined by thin layer chromatography, it was diluted with water, extracted with 10% MeOH/DCM, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by methanol: dichloromethane column chromatography to obtain 91 mg of an off-white product with a yield of 43.1% (mp: 201-203°C).

The test data of compound C-1 are as follows:

¹ H NMR (600 MHz, DMSO-d ₆ )δ:11.49(s,1H),8.22(t,J＝4.9Hz,1H),7.70(d,J＝8.5Hz,2H),7.50(d,J＝7.1Hz,2H),7.29(d,J＝1.5Hz,1H),7.23(d,J＝1.6Hz,1H),5.86(s,1H),4.29(d,J＝4.9Hz,2H),3.58(s,4H),2.95-2.79(m,4H),2.27(s,3H),2.20(s,3H),2.11(s,3H),2.05(s,3H). ¹³ C NMR (150 MHz, DMSO-d ₆ )δ:169.27,168.80,163.49,152.36,150.05,143.26,140.05,137.36,136.84,136.82,129.31(2C),129.12(2C),122.03,120.81,118.72,116.19,107.86,52.24,51.92,46.68,41.80,35.40,21.75,19.43,18.67,14.94.MScalcd for C ₂₈ H ₃₁ ClN ₄ O ₃ [M+H]+m/z:507.21630,found 507.21618.

Example 8: Preparation of Compound C-2

Compound 8 (238 mg, 0.5 mmol, 1 eq), 4-trifluoromethylphenylboronic acid (114 mg, 0.60 mmol, 1.2 eq), anhydrous sodium carbonate (64 mg, 0.6 mmol, 1.2 eq), 1,4-dioxane (10 mL) and a mixed solution of water (1 mL) were added to a round-bottom flask, and tetrakis(triphenylphosphine)palladium (0.56 g, 0.05 mmol) was added and immediately replaced with nitrogen three times. The reaction mixture was reacted at 105°C for 4 h. After the reaction was completed as determined by thin layer chromatography, it was diluted with water, extracted with 10% MeOH/DCM, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by methanol: dichloromethane column chromatography to obtain 94 mg of an off-white product with a yield of 41.8% (mp: 203-205°C).

The test data of compound C-2 are as follows:

¹ H NMR (600 MHz, DMSO-d ₆ )δ:11.48(s,1H),8.21(t,J＝4.9Hz,1H),7.70(d,J＝8.6Hz,2H),7.50(d,J＝8.6Hz,2H),7.29(d,J＝1.7Hz,1H),7.23(d,J＝1.7Hz,1H),5.86(s,1H),4.29(d,J＝4.9Hz,2H),3.59(s,4H),2.87(dt,J＝44.3,4.6Hz,4H),2.27(s,3H),2.20(s,3H),2.11(s,3H),2.05(s,3H). ¹³ C NMR (150 MHz, DMSO-d ₆ )δ:169.11,168.79,163.47,152.50,150.06,144.33,143.27,140.23,136.76,130.52,127.89(2C),126.18(2C),125.76,123.96,121.99,121.15,119.04,107.84,52.21,51.88,46.65,41.77,35.38,21.74,19.41,18.66,15.03.MS calcd for C ₂₉ H ₃₁ F ₃ N ₄ O ₃ [M+H] ⁺ m/z:541.24702,found541.24796.

Example 9: Preparation of Compound C-3

Compound 8 (238 mg, 0.5 mmol, 1 eq), 4-trifluoromethylphenylboronic acid (84 mg, 0.60 mmol, 1.2 eq), anhydrous sodium carbonate (64 mg, 0.6 mmol, 1.2 eq), 1,4-dioxane (10 mL) and a mixed solution of water (1 mL) were added to a round-bottom flask, and tetrakis(triphenylphosphine)palladium (0.56 g, 0.05 mmol) was added and immediately replaced with nitrogen three times. The reaction mixture was reacted at 105°C for 4 h. After the reaction was completed as determined by thin layer chromatography, it was diluted with water, extracted with 10% MeOH/DCM, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by methanol: dichloromethane column chromatography to obtain 109 mg of an off-white product with a yield of 46.4% (mp: 201-203°C).

The test data of compound C-3 are as follows:

¹ H NMR (600 MHz, DMSO-d ₆ )δ:11.48(s,1H),8.20(t,J＝4.9Hz,1H),7.70(d,J＝8.7,5.5Hz,2H),7.27(t,J＝8.8Hz,3H),7.21(d,J＝1.5Hz,1H),5.86(s,1H),4.29(d,J＝4.9Hz,2H),3.58(s,4H),3.01-2.72(m,4H),2.27(s,3H),2.20(s,3H),2.11(s,3H),2.05(s,3H). ¹³ CNMR (150 MHz, DMSO-d ₆ )δ:169.26,168.79,163.47,152.35,150.03,143.25,140.04,137.34,136.83,136.81,129.30,129.05(2C),122.01,120.79,118.70,116.03(2C),107.84,52.23,51.90,46.67,41.78,35.38,21.74,19.41,18.66,14.92.MS calcd for C ₂₈ H ₃₁ FN ₄ O ₃ [M+H] ⁺ m/z:491.24302,found 491.24358.

Example 10: Preparation of Compound C-4

Compound 8 (238 mg, 0.5 mmol, 1 eq), 3-pyridine boronic acid (73.7 mg, 0.60 mmol, 1.2 eq), anhydrous sodium carbonate (64 mg, 0.6 mmol, 1.2 eq), 1,4-dioxane (10 mL) and a mixed solution of water (1 mL) were added to a round-bottom flask, and tetrakis(triphenylphosphine)palladium (0.56 g, 0.05 mmol) was added and immediately replaced with nitrogen three times. The reaction mixture was reacted at 105°C for 4 h. After the reaction was completed as determined by thin layer chromatography, it was diluted with water, extracted with 10% MeOH/DCM, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by methanol: dichloromethane column chromatography to obtain 83.3 mg of an off-white product with a yield of 40.7% (mp: 196-198°C).

The test data of compound C-4 are as follows:

¹ H NMR (600 MHz, DMSO-d ₆ )δ:11.49(s,1H),8.89(d,J＝1.5Hz,1H),8.55(d,J＝3.8Hz,1H),8.22(t,J＝4.8Hz,1H),8.08(d,J＝7.9Hz,1H),7.47(dd,J＝7.8,4.8Hz,1H),7.35(s,1H),7.29(s,1H),5.87(s,1H),4.29(d,J＝4.8Hz,2H),3.59(s,4H),2.89(m,4H),2.29(s,3H),2.21(s,3H),2.11(s,3H),2.05(s,3H). ¹³ C NMR (150 MHz, DMSO-d ₆ )δ:169.13,168.81,163.48,152.55,148.89,148.09,143.30,140.21,135.76,135.25,134.54,130.14,124.30,124.29,121.98,120.98,118.95,107.88,52.20,51.85,46.66,41.78,35.40,21.73,19.42,18.66,15.01.MS calcd for C ₂₇ H ₃₁ N ₅ O ₃ [M+H] ⁺ m/z:474.24769,found474.24753.

Example 11: Preparation of Compound C-5

Compound 8 (238 mg, 0.5 mmol, 1 eq), 5-methylpyridine-3-boric acid (82.2 mg, 0.60 mmol, 1.2 eq), anhydrous sodium carbonate (64 mg, 0.6 mmol, 1.2 eq), 1,4-dioxane (10 mL) and a mixed solution of water (1 mL) were added to a round-bottom flask, and tetrakis(triphenylphosphine)palladium (0.56 g, 0.05 mmol) was added and immediately replaced with nitrogen three times. The reaction mixture was reacted at 105°C for 4 h. After the reaction was completed as determined by thin layer chromatography, it was diluted with water, extracted with 10% MeOH/DCM, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by methanol: dichloromethane column chromatography to obtain 84.9 mg of an off-white product with a yield of 41.5% (mp: 198-202°C).

The test data of compound C-5 are as follows:

¹ H NMR (600 MHz, DMSO-d ₆ )δ:11.49(s,1H),8.68(d,J＝1.9Hz,1H),8.39(d,1H),8.21(t,J＝4.9Hz,1H),7.91(s,1H),7.35(d,J＝1.6Hz,1H),7.28(d,J＝1.6Hz,1H),5.87(s,1H),4.29(d,J＝4.9Hz,2H),3.59(s,4H),2.88(dt,J＝43.6,5.0Hz,4H),2.36(s,3H),2.28(s,3H),2.21(s,3H),2.11(s,3H),2.05(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ:11.49(s,1H),8.68(d,J＝1.9Hz,1H),8.39(d,1H),8.21(t,J＝4.9Hz,1H), ^7.91 (s,1H),7.35(d,J＝1.6Hz,1H),7.28(d,J＝1.6Hz,1H),5.87(s,1H),4.29(d,J＝4.9Hz,2H),3.59(s,4H),2.88(dt,J＝43.6,5.0Hz,4H),2.36(s,3H),2.28(s,3H),2.21(s,3H),2.11(s,3H),2.05(s,3H). ₆ )δ:169.15,168.78,163.48,152.52,150.05,149.23,145.27,143.26,140.22,135.29,135.21,134.85,133.51,130.03,122.00,120.89,118.91,107.85,52.24,51.91,46.67,41.78,35.40,21.74,19.43,18.67,18.33,15.00.MS calcd for C ₂₈ H ₃₃ N ₅ O ₃ [M+H] ⁺ m/z:488.26834,found488.26365.

Example 12: Preparation of Compound C-6

Compound 8 (238 mg, 0.5 mmol, 1 eq), 4-cyanoboric acid (88.16 mg, 0.60 mmol, 1.2 eq), anhydrous sodium carbonate (64 mg, 0.6 mmol, 1.2 eq), 1,4-dioxane (10 mL) and a mixed solution of water (1 mL) were added to a round-bottom flask, and tetrakis(triphenylphosphine)palladium (0.56 g, 0.05 mmol) was added and immediately replaced with nitrogen three times. The reaction mixture was reacted at 105°C for 4 h. After the reaction was completed as determined by thin layer chromatography, it was diluted with water, extracted with 10% MeOH/DCM, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by methanol: dichloromethane column chromatography to obtain 95.6 mg of an off-white product with a yield of 46.7% (mp: 203-205°C).

The test data of compound C-6 are as follows:

¹ H NMR (600 MHz, DMSO-d ₆ )δ:11.49(s,1H),8.24(s,1H),7.93(s,4H),7.91(s,1H),7.32(s,1H),5.86(s,1H),4.29(s,2H),3.61(s,4H),2.99-2.91(m,4H),2.28(s,3H),2.20(s,3H),2.11(s,3H),2.06(s,3H). ¹³ C NMR (150 MHz, DMSO-d ₆ )δ:169.20,168.78,163.48,152.43,150.01,143.25,140.14,139.14,137.01,132.71,129.77,129.27(2C),128.87(2C),122.02,120.78,118.67,107.83,52.23,51.91,46.67,41.79,35.40,21.74,19.42,18.67,14.96.MS calcd for C ₂₉ H ₃₁ N ₅ O ₃ [M+H]+m/z:498.25269,found498.25219.

Example 13: Preparation of Compound C-7

Compound 8 (238 mg, 0.5 mmol, 1 eq), 4-methoxyphenylboronic acid (91.2 mg, 0.60 mmol, 1.2 eq), anhydrous sodium carbonate (64 mg, 0.6 mmol, 1.2 eq), 1,4-dioxane (10 mL) and a mixed solution of water (1 mL) were added to a round-bottom flask, and tetrakis(triphenylphosphine)palladium (0.56 g, 0.05 mmol) was added and immediately replaced with nitrogen three times. The reaction mixture was reacted at 105°C for 4 h. After the reaction was completed as determined by thin layer chromatography, it was diluted with water, extracted with 10% MeOH/DCM, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by methanol: dichloromethane column chromatography to obtain 91.5 mg of an off-white product with a yield of 43.4% (mp: 201-202°C).

The test data of compound C-7 are as follows:

¹ H NMR (600 MHz, DMSO-d ₆ )δ:11.49(s,1H),8.19(t,J＝4.9Hz,1H),7.63(d,J＝8.8Hz,1H),7.59(d,J＝7.1Hz,2H),7.27(d,J＝1.7Hz,1H),7.24(d,J＝1.6Hz,1H),5.87(s,1H),4.29(d,J＝4.9Hz,2H),3.79(s,3H),3.59(s,4H),2.92-2.81(m,4H),2.26(s,3H),2.21(s,3H),2.11(s,3H),2.05(s,3H). ¹³ C NMR (150 MHz, DMSO-d ₆ )δ:169.40,168.78,163.48,159.46,159.34,152.27,149.98,143.21,140.02,138.07(2C),132.72,128.16,122.05,120.36,118.19,114.75(2C),107.83,55.65,52.25,51.94,46.68,41.80,35.38,21.74,19.41,18.66,14.85.MS calcd for C ₂₉ H ₃₄ N ₄ O ₄ [M+H]+m/z:503.25801,found 503.25733.

Example 14: Preparation of Compound C-8

Compound 8 (238 mg, 0.5 mmol, 1 eq), quinoline-6-boric acid (103 mg, 0.60 mmol, 1.2 eq), anhydrous sodium carbonate (64 mg, 0.6 mmol, 1.2 eq), 1,4-dioxane (10 mL) and a mixed solution of water (1 mL) were added to a round-bottom flask, and tetrakis(triphenylphosphine)palladium (0.56 g, 0.05 mmol) was added and immediately replaced with nitrogen three times. The reaction mixture was reacted at 105°C for 4 h. After the reaction was completed as determined by thin layer chromatography, it was diluted with water, extracted with 10% MeOH/DCM, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by methanol: dichloromethane column chromatography to obtain 94.1 mg of an off-white product with a yield of 42.8% (mp: 199-201°C).

The test data of compound C-8 are as follows:

¹ H NMR (600 MHz, DMSO-d ₆ )δ:11.47(s,1H),8.95-8.89(m,3H),8.28(d,J＝2.8Hz,1H),8.08(d,J＝8.6Hz,2H),7.45(d,J＝4.2Hz,2H),7.21(s,1H),5.84(s,1H),4.28(s,2H),3.58(s,4H),2.94(m,4H),2.30(s,3H),2.18(s,3H),2.09(s,3H),2.02(s,3H). ¹³ C NMR (150 MHz, DMSO-d ₆ )δ:169.37,168.78,163.48,152.31,149.98,143.22,140.08,139.09,138.30,137.45,137.15,135.04,129.96,129.93,128.96,126.99,126.92,126.89,122.05,120.57,118.41,107.83,52.26,51.96,46.69,41.81,35.40,21.14,19.41,18.67,14.88.MScalcd for C ₃₁ H ₃₃ N ₅ O ₃ [M+H]+m/z:524.25334,found 524.25321.

Example 15: Preparation of Compound C-9

Compound 8 (238 mg, 0.5 mmol, 1 eq), 4-methylphenylboronic acid (81.6 mg, 0.60 mmol, 1.2 eq), anhydrous sodium carbonate (64 mg, 0.6 mmol, 1.2 eq), 1,4-dioxane (10 mL) and a mixed solution of water (1 mL) were added to a round-bottom flask, and tetrakis(triphenylphosphine)palladium (0.56 g, 0.05 mmol) was added and immediately replaced with nitrogen three times. The reaction mixture was reacted at 105°C for 4 h. After the reaction was completed as determined by thin layer chromatography, it was diluted with water, extracted with 10% MeOH/DCM, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by methanol: dichloromethane column chromatography to obtain 96.9 mg of an off-white product with a yield of 47.4% (mp: 201-203°C).

The test data of compound C-9 are as follows:

¹ H NMR (600 MHz, DMSO-d ₆ )δ:11.50(s,1H),8.20(t,J＝4.9Hz,1H),7.55(d,J＝8.1Hz,2H),7.26(d,J＝3.8Hz,2H),7.25(s,1H),7.21(s,1H),5.86(s,1H),4.29(d,J＝4.9Hz,2H),3.59(s,4H),2.91-2.76(m,4H),2.33(s,3H),2.27(s,3H),2.21(s,3H),2.11(s,3H),2.05(s,3H). ¹³ C NMR (150 MHz, DMSO-d ₆ )δ:169.37,168.78,163.48,152.28,150.00,143.50,143.23,140.04,138.37,137.77,128.95,128.75(2C),127.00(2C),122.03,120.63,118.47,107.83,52.25,51.94,46.68,41.79,35.37,22.96,21.75,19.41,18.66,16.13.MS calcd for:C ₂₉ H ₃₄ N ₄ O ₃ [M+H]+m/z:487.26309,found 487.26319.

Example 16: Preparation of Compound C-10

Compound 8 (238 mg, 0.5 mmol, 1 eq), 4-ethylphenylboronic acid (89.9 mg, 0.60 mmol, 1.2 eq), anhydrous sodium carbonate (64 mg, 0.6 mmol, 1.2 eq), 1,4-dioxane (10 mL) and a mixed solution of water (1 mL) were added to a round-bottom flask, and tetrakis(triphenylphosphine)palladium (0.56 g, 0.05 mmol) was added and immediately replaced with nitrogen three times. The reaction mixture was reacted at 105°C for 4 h. After the reaction was completed as determined by thin layer chromatography, it was diluted with water, extracted with 10% MeOH/DCM, and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by methanol: dichloromethane column chromatography to obtain 96.1 mg of an off-white product with a yield of 45.7% (mp: 203-204°C).

The test data of compound C-10 are as follows:

¹ HNMR (600 MHz, DMSO-d ₆ )δ: 11.48 (s, 1H), 8.18 (t, J = 5.0 Hz, 1H), 7.56 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 7.27 (s, 1H), 7.21 (d, J = 1.7 Hz, 1H), 5.86 (s, 1H), 4.30 (d, J = 4.9 Hz, 2H), 3.59 (s, 4H), 2.93-2.82 (m, 4H), 2.68-2.60 (m, 2H), 2.27 (s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 2.05 (s, 3H), 1.22-1.18 (m, 3H). ¹³ C NMR (150 MHz, DMSO-d ₆ ) δ: 169.36, 168.78, 163.47, 152.28, 150.00, 143.50, 143.22, 140.04, 138.37, 137.76, 128.95, 128.75 (2C), 127.00 (2C), 122.03, 120.63, 118.47, 107.83, 52.25, 51.94, 46.68, 41.79, 35.37, 28.28, 21.75, 19.41, 18.66, 16.13, 14.90. MS calculated _{for C 30} H ₃₆ N ₄ O ₃ [M+H] ⁺ m/z:501.27874,found501.27975.

Example 17: Evaluation of in vitro EZH2 inhibitory activity of compounds

1. Experimental methods

(1) Cell distribution: The cultured NCI-H1975 cells were digested and evenly seeded in a 96-well plate. 150 μL of DMEM culture medium was added to each well and the plate was gently shaken to ensure uniform distribution of the cells.

(2) Cultivating cells: Place cells in a standard cell culture incubator and culture for 3 days (observe the cell status and make appropriate adjustments) until the cells are fully confluent as a monolayer.

(3) Drug administration: Add drugs prepared with DMSO in a concentration gradient, 100 μL per well.

(4) Color development: After 24 hours of culture, take out the cells and observe their status under a microscope. Add 10 μL of 5 mg/mL MTT solution to each well. After incubation for another 3 hours, terminate the culture and take out the cells. Rinse along the plate wall with PBS three times. Add 80 μL of DMSO solution to each well and shake on a shaker for 20 minutes.

(5) Colorimetry: Measure the absorbance of each well using a colorimeter and record the absorbance value. Use software to calculate the _IC50 value.

2. Experimental results

Table 1. Inhibitory activity of compounds on NCI-H1975 tumor cell proliferation

Compound IC ₅₀ (μM) Tazemetostat 3.42 C-1 1.06 C-2 0.39 C-3 0.53 C-4 1.63 C-5 1.51 C-6 0.92 C-7 1.05 C-8 2.32 C-9 1.67 C-10 2.56

As can be seen from Table 1, the compounds of the present invention have better inhibitory activity than Tazemetostat, and the optimal IC ₅₀ value is lower than 1 μM, which shows the feasibility of the present invention to use Tazemetostat as a lead compound and to optimize and transform it.

Claims (10)

1. A piperazine compound, characterized in that it has a structure of formula I, in: R is selected from a 6- to 12-membered aromatic group and a 6- to 12-membered aromatic hetero group containing 1 to 3 N atoms, wherein the aromatic group and the aromatic hetero group are substituted by at least one of the following substituents: hydrogen, deuterium, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, cyano, nitro, thiol, and carboxyl. 2. The piperazine compound according to claim 1, wherein in the structure: R is selected from phenyl, pyridyl, naphthyl, benzopyridyl, and R is substituted by at least one of the following substituents: hydrogen, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, and cyano. 3. The piperazine compound according to claim 1, wherein in the structure: R is selected from R' is selected from at least one of hydrogen, halogen, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, and cyano. 4. The piperazine compound according to claim 3, characterized in that in the structure: The R' is selected from at least one of hydrogen, fluorine, chlorine, bromine, trifluoromethyl, difluoromethyl, methyl, ethyl, isopropyl, n-propyl, methoxy, ethoxy, isopropoxy, n-propoxy, and cyano. 5. The piperazine compound according to claim 1, characterized in that it is selected from any one of the following compounds: 6. A pharmaceutically acceptable salt of a piperazine compound according to claim 1, characterized in that the salt is formed by the compound and any one of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, and ferulic acid. 7. A method for preparing the piperazine compound according to claim 1, characterized in that it comprises the following steps: (1) Compound 1 is salified and cyclized with compound 3 to obtain compound 4; (2) Compound 4 was subjected to desalination, acetylation, and ester hydrolysis to obtain compound 7; (3) Compound 7 is acylated and coupled with R to obtain compound I; Wherein, R is defined as in claim 1; The corresponding acid is reacted with the compound I prepared by the above method to form a salt, thereby obtaining a pharmaceutically acceptable salt of the compound I. 8. A pharmaceutical composition, characterized in that it comprises the piperazine compound according to claim 1 or the pharmaceutically acceptable salt according to claim 6 and a pharmaceutically acceptable carrier. 9. Use of the piperazine compound according to claim 1, the pharmaceutically acceptable salt according to claim 6 or the pharmaceutical composition according to claim 8 in the preparation of an EZH2 inhibitor. 10. The use according to claim 9, characterized in that the drug is an anti-tumor drug.