USP21 inhibitor, preparation method thereof and application of USP21 inhibitor in medicine

Abstract

The invention relates to the technical field of biological medicine, in particular to a USP21 inhibitor, a preparation method thereof and application of the USP21 inhibitor in medicine. In the invention, a novel pleuromutilin derivative is prepared by a simple and feasible method, and the pleuromutilin derivative has the property of efficiently inhibiting USP21 activity, can be used as a USP21 selective inhibitor, and inhibits growth and diffusion of breast cancer cells by inhibiting the USP21 activity and blocking a mediated signal channel thereof, so that the activity of the USP21 is inhibited, and the activity of the USP21 is inhibited. And new strategies and drug choices are provided for treatment of breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a USP21 inhibitor, a preparation method thereof, and an application in medicine. Background Art

[0002] As the most common malignant tumor among women globally, the incidence of breast cancer continues to rise and shows a significant trend of younger age, and it has become one of the major diseases threatening women's health. Although existing treatment methods (such as surgery, chemotherapy, radiotherapy, endocrine therapy, and emerging immunotherapy / targeted therapy) are constantly expanding, problems such as individual differences in efficacy, drug resistance, and side effects still significantly limit clinical benefits, and there is an urgent need to develop new therapeutic targets and drugs.

[0003] Ubiquitin-specific protease 21 (USP21) has attracted much attention in tumorigenesis and development because it participates in regulating key pathways such as the cell cycle, DNA repair, and signal transduction. Research shows that USP21 is abnormally highly expressed in various tumors such as breast cancer, and drives the proliferation, invasion, and metastasis of tumor cells by stabilizing pro-cancer proteins such as FOXM1. At the same time, it mediates the immune escape mechanism to reshape the tumor microenvironment, which is closely related to the malignant progression and poor prognosis of breast cancer.

[0004] Currently, the research and development of specific inhibitors against USP21 is still in the early stage. Although BAY-805 developed by Bayer has shown in vitro activity and selectivity, its pharmacokinetic properties and safety still need to be optimized, and it has not entered clinical application. The bottlenecks in the existing technology focus on problems such as insufficient inhibitor selectivity, poor in vivo stability, and potential off-target toxicity, which restrict the clinical transformation of USP21-targeted therapy.

[0005] Therefore, developing a structurally novel, highly efficient and low-toxic USP21 selective inhibitor to inhibit breast cancer progression by blocking its oncogenic signaling pathway can provide an innovative strategy to break through the existing treatment limitations and has great clinical value. Summary of the Invention

[0006] In order to selectively inhibit USP21, the present invention provides a USP21 inhibitor, a preparation method thereof, and an application in medicine.

[0007] In the first aspect of the present invention, a pleuromutilin derivative or a pharmaceutically acceptable salt thereof is provided, and the pleuromutilin derivative has the structure shown in Formula 7: ; wherein, R1 is selected from phenyl, substituted phenyl, C1-C4 alkyl, C3-C8 cycloalkyl; R2 is selected from hydrogen, C1-C4 alkyl.

[0008] The pleuromutilin derivative shown in Formula 7 or a pharmaceutically acceptable salt thereof has the property of selectively inhibiting the activity of USP21 and is a USP21 selective inhibitor. The pharmaceutically acceptable salt of the pleuromutilin derivative shown in Formula 7 can be easily prepared from the pleuromutilin derivative shown in Formula 7 as a raw material. For example, reacting the pleuromutilin derivative shown in Formula 7 with hydrochloric acid can prepare the hydrochloride salt of the pleuromutilin derivative shown in Formula 7.

[0009] In some alternative embodiments, R1 selected from the group consisting of phenyl, phenyl substituted with at least one methyl, phenyl substituted with at least one methoxy, methyl, ethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; R2 selected from the group consisting of hydrogen, methyl, ethyl.

[0010] In some alternative embodiments, the pleuromutilin derivative has a structure shown in any one of Formulas 7a to 7e: , , , , .

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned pleuromutilin derivative, which includes the following steps: Obtaining the diol compound shown in Formula 2 by hydrolysis of the pleuromutilin shown in Formula 1; Protecting the hydroxyl group of the diol compound shown in Formula 2 with acetic anhydride to obtain the compound shown in Formula 3; Reacting the compound shown in Formula 3 with ozone to obtain the aldehyde compound shown in Formula 4; Performing the Horner-Wadsworth-Emmons Reaction on the aldehyde compound shown in Formula 4 to obtain the lipid compound shown in Formula 5; Obtaining the carboxylic acid compound shown in Formula 6 by hydrolysis of the lipid compound shown in Formula 5; Performing a condensation reaction on the carboxylic acid compound shown in Formula 6 with R1-NH-R2 to obtain the pleuromutilin derivative shown in Formula 7; , , , , , , .

[0012] Wherein, R1 selected from the group consisting of phenyl, substituted phenyl, C1-C4 alkyl, C3-C8 cycloalkyl; R2 selected from the group consisting of hydrogen, C1-C4 alkyl.

[0013] In some alternative embodiments, the hydrolysis reaction of pleuromutilin represented by Formula 1 to obtain the diol compound represented by Formula 2 includes: dissolving pleuromutilin represented by Formula 1 in an aqueous solvent, adding sodium hydroxide, and hydrolyzing pleuromutilin represented by Formula 1 to obtain the diol compound represented by Formula 2.

[0014] In some alternative embodiments, protecting the hydroxyl group of the diol compound represented by Formula 2 with acetic anhydride includes: dissolving the diol compound represented by Formula 2 in an organic solvent, adding acetic anhydride, and reacting the diol compound represented by Formula 2 with acetic anhydride.

[0015] In some alternative embodiments, reacting the compound represented by Formula 3 with ozone includes: dissolving the compound represented by Formula 3 and triphenylphosphine in an organic solvent, cooling to -78 °C, and introducing ozone to react the compound represented by Formula 3 with ozone.

[0016] In some alternative embodiments, performing the Horner-Wadsworth-Emmons reaction on the aldehyde compound represented by Formula 4 includes: dissolving the aldehyde compound represented by Formula 4 and trimethyl phosphonoacetate in an organic solvent, cooling to -78 °C, and adding lithium diisopropylamide to react the aldehyde compound represented by Formula 4 with trimethyl phosphonoacetate.

[0017] In some alternative embodiments, the hydrolysis reaction of the lipid compound represented by Formula 5 to obtain the carboxylic acid compound represented by Formula 6 includes: dissolving the lipid compound represented by Formula 5 in an aqueous solvent, adding sodium hydroxide, and hydrolyzing the lipid compound represented by Formula 5 to obtain the carboxylic acid compound represented by Formula 6.

[0018] In some alternative embodiments, performing a condensation reaction of the carboxylic acid compound represented by Formula 6 with R1-NH-R2 includes: under an inert gas atmosphere, dissolving the carboxylic acid compound represented by Formula 6, R1-NH-R2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and triethylamine in an organic solvent, and reacting the carboxylic acid compound represented by Formula 6 with R1-NH-R2.

[0019] The third aspect of the present invention provides the use of the above-mentioned pleuromutilin derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing breast cancer.

[0020] The fourth aspect of the present invention provides a drug for treating and / or preventing breast cancer, which comprises the above-mentioned pleuromutilin derivative or a pharmaceutically acceptable salt thereof. The above-mentioned pleuromutilin derivative or a pharmaceutically acceptable salt thereof is the main active ingredient (main drug) in the above-mentioned drug for treating and / or preventing breast cancer.

[0021] In some alternative embodiments, the above-mentioned drug for treating and / or preventing breast cancer further comprises an excipient. The dosage form of the above-mentioned drug for treating and / or preventing breast cancer is any pharmaceutically acceptable dosage form. The above-mentioned excipient is stable in nature, has no incompatibility with the main drug, does not produce side effects, does not affect the curative effect, is not prone to deformation, cracking, or mildew at room temperature, and is harmless to the human body.

[0022] In some alternative embodiments, the above-mentioned excipient is at least one of gum arabic, syrup, lanolin, and starch.

[0023] The technical solution of the embodiment of the present invention has the following beneficial effects: A new pleuromutilin derivative is prepared by a simple and easy method. The pleuromutilin derivative has the property of highly inhibiting the activity of USP21 and can be used as a USP21 selective inhibitor. By inhibiting the activity of USP21 and blocking the signal pathway mediated by it, the growth and spread of breast cancer cells can be inhibited, providing new strategies and drug options for the treatment of breast cancer. Detailed implementation manners

[0024] The following will combine the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0025] In the following embodiments, the pleuromutilin derivative shown in Formula 7 is synthesized according to the following route: In the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and triethylamine, the carboxylic acid compound shown in Formula 6 reacts with R1-NH-R2 in dichloromethane solvent to undergo a condensation reaction to obtain the pleuromutilin derivative shown in Formula 7;

[0026] Among them, R1-NH-R2 represents an amine substance. R1 can be 2,4-dimethoxyphenyl, methyl, cyclopropyl, cyclopentyl, or cyclohexyl, and R2 can be hydrogen or methyl. The carboxylic acid compound shown in Formula 6 can be synthesized according to the following route: Using the pleuromutilin shown in Formula 1 as the starting material, a hydrolysis reaction is carried out to obtain the diol compound shown in Formula 2; subsequently, it is protected with acetic anhydride to obtain the compound shown in Formula 3; the compound shown in Formula 3 is cleaved by ozone to obtain the aldehyde compound shown in Formula 4; subsequently, the aldehyde compound shown in Formula 4 undergoes a Horner-Wadsworth-Emmons reaction to obtain the lipid compound shown in Formula 5; next, the lipid compound shown in Formula 5 is hydrolyzed to obtain the compound shown in Formula 6;

[0027] Specifically, the pleuromutilin shown in Formula 1 (1.0 g, 2.6 mmol, 1.0 eq) was dissolved in a mixed solvent of 30.0 mL ethanol / water (the volume ratio of ethanol to water was 1:1), sodium hydroxide (156.0 mg, 3.9 mmol, 1.5 eq) was added, and the system was heated to 50 °C in an oil bath for reaction; after detecting the completion of the reaction, 20 mL of saturated ammonium chloride was added, and the mixture was extracted with ethyl acetate. The organic phase was dried and concentrated, and purified by column chromatography to obtain the diol compound shown in Formula 2 (499.2 mg, 60%). The characterization results of the diol compound shown in Formula 2 are as follows: 1 H NMR (400 MHz, CDCl3) δ 6.12 (dd, J = 17.8, 11.1Hz, 1H), 5.41 – 5.19 (m, 2H), 4.31 (dd, J = 7.8, 3.8 Hz, 1H), 3.37 (t, J =6.3 Hz, 1H), 2.25 – 2.09 (m, 3H), 2.06 – 1.98 (m, 1H), 1.87 (dd, J = 15.8,7.7 Hz, 1H), 1.71 (dt, J = 10.5, 3.2 Hz, 2H), 1.66 – 1.53 (m, 3H), 1.49 –1.34 (m, 4H), 1.32 (s, 3H), 1.12 (s, 3H), 1.07 (dd, J = 13.9, 4.5 Hz, 1H),0.93 (d, J = 7.1 Hz, 3H), 0.89 (d, J = 7.1 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ217.9, 139.6, 115.8, 75.1, 66.8, 59.2, 45.4, 45.3, 45.1, 42.4, 36.9, 36.6,34.5, 30.5, 28.7, 27.2, 25.1, 18.3, 13.5, 11.4. The diol compound shown in Formula 2 (400.0 mg, 1.3 mmol, 1.0 eq) was dissolved in 10.0 mL of pyridine, acetic anhydride (291.7 mg, 2.9 mmol, 2.2 eq) was added, and the reaction was carried out at room temperature. After detecting the completion of the reaction, 50 mL of saturated copper sulfate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was dried and concentrated, and purified by column chromatography to obtain the compound shown in Formula 3 (367.6 mg, 70%). The characterization results of the compound shown in Formula 3 are as follows: 1 H NMR (400 MHz, CDCl3) δ 6.32 (ddd, J =17.5, 11.1, 1.9 Hz, 1H), 5.61 (d, J = 8.1 Hz, 1H), 5.22 (d, J = 11.2 Hz, 1H),5.14 (d, J = 17.5 Hz, 1H), 4.85 (d, J = 6.7 Hz, 1H), 2.47 (p, J = 7.1 Hz,1H), 2.27 (dd, J = 19.5, 11.0 Hz, 1H), 2.21 – 2.08 (m, 2H), 2.05 (s, 3H),1.93 (s, 3H), 1.91 – 1.80 (m, 1H), 1.76 – 1.65 (m, 1H), 1.65 – 1.48 (m, 2H),1.42 (d, J = 1.9 Hz, 3H), 1.38 – 1.26 (m, 3H), 1.09 (td, J = 13.9, 4.4 Hz,1H), 1.02 – 0.92 (m, 3H), 0.76 (d, J = 6.9 Hz, 3H), 0.72 – 0.61 (m, 3H). 13 CNMR (100 MHz, CDCl3) δ 217.4, 170.6, 169.7, 139.7, 116.2, 76.7, 68.2, 58.7,45.4, 45.2, 43.0, 41.9, 36.8, 36.2, 34.6, 30.4, 27.4, 26.9, 25.1, 22.1, 20.8,16.3, 14.9, 11.9. Dissolve the compound shown in Formula 3 (400.0 mg, 1.0 mmol, 1.0 eq) and triphenylphosphine (262.3 mg, 1.0 mmol, 1.0 eq) in 10.0 mL of dichloromethane. Cool the solution to -78 °C under argon protection and introduce ozone for reaction. After detecting the completion of the reaction, quench the reaction with saturated ammonium chloride and extract with dichloromethane. Dry and concentrate the organic phase, and purify by column chromatography to obtain the aldehyde compound shown in Formula 4 (365.4 mg, 90%). The characterization results of the aldehyde compound shown in Formula 4 are as follows: 1 H NMR(400 MHz, CDCl3) δ 9.61 (s, 1H), 5.29 (d, J = 8.3 Hz, 1H), 4.96 (d, J = 7.3Hz, 1H), 2.77 (p, J = 7.0 Hz, 1H), 2.36 – 2.23 (m, 1H), 2.22 – 2.08 (m, 3H),2.06 (d, J = 1.3 Hz, 3H), 2.01 (d, J = 8.4 Hz, 1H), 1.96 (d, J = 1.4 Hz, 3H),1.94 – 1.83 (m, 1H), 1.77 – 1.58 (m, 4H), 1.44 (d, J = 1.3 Hz, 3H), 1.42 –1.28 (m, 3H), 1.11 (td, J = 14.1, 4.7 Hz, 1H), 1.02 (d, J = 1.3 Hz, 3H), 0.93(dd, J = 7.0, 1.3 Hz, 3H), 0.69 (dd, J = 7.1, 1.3 Hz, 3H). 13 C NMR (100 MHz,CDCl3) δ 216.9, 202.3, 170.6, 170.1, 74.4, 68.7, 58.3, 52.4, 45.3, 41.7,40.4, 36.8, 36.7, 34.5, 30.4, 27.0, 24.8, 22.6, 21.7, 20.7, 16.9, 14.9, 11.5. Dissolve the aldehyde compound shown in Formula 4 (500.0 mg, 1.2 mmol, 1.0 eq) and trimethyl phosphonoacetate (327.6 mg, 1.8 mmol, 1.5 eq) in 10.0 mL of dry tetrahydrofuran. Cool the solution to -78 °C under argon protection and slowly add lithium diisopropylamide (2.0 N in THF, 1.4 mL, 1.2 eq). After detecting the completion of the reaction, quench the reaction with saturated sodium bicarbonate and extract with ethyl acetate. Dry and concentrate the organic phase, and purify it by column chromatography to obtain the lipid compound shown in Formula 5 (304.9 mg, 55%). The characterization results of the lipid compound shown in Formula 5 are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.44(d, J = 16.1 Hz, 1H), 5.94 (d, J = 16.0 Hz, 1H), 5.48 (d, J = 8.0 Hz, 1H),4.90 (d, J = 6.7 Hz, 1H), 3.75 (s, 3H), 2.42 (p, J = 7.0 Hz, 1H), 2.27 (dd, J= 19.5, 10.9 Hz, 1H), 2.20 – 2.09 (m, 3H), 2.07 (s, 3H), 1.94 (s, 3H), 1.84(dt, J = 12.7, 10.1 Hz, 1H), 1.74 – 1.64 (m, 1H), 1.60 (dt, J = 11.6, 7.2 Hz,1H), 1.51 (dd, J = 13.5, 3.4 Hz, 1H), 1.42 (s, 3H), 1.39 (d, J = 5.7 Hz, 1H),1.32 (dtd, J = 13.9, 8.4, 7.1, 2.7 Hz, 2H), 1.09 (td, J = 14.0, 4.4 Hz, 1H),1.01 (s, 3H), 0.78 (d, J = 7.2 Hz, 3H), 0.67 (d, J = 6.8 Hz, 3H). 13 C NMR (100MHz, CDCl3) δ 217.0, 170.4, 169.6, 167.1, 150.5, 122.3, 76.3, 68.1, 58.5,51.7, 45.4, 44.8, 43.6, 41.9, 36.7, 36.6, 34.5, 30.3, 27.6, 26.9, 25.0, 21.9,20.8, 16.4, 14.9, 12.0. Dissolve the lipid compound shown in Formula 5 (500.0 mg, 1.0 mmol, 1.0 eq) in a mixed solvent of 10.0 mL ethanol / water (the volume ratio of ethanol to water is 1:1), add sodium hydroxide (200.0 mg, 5.0 mmol, 5.0 eq), and heat the system to 60 °C in an oil bath for reaction; after detecting the completion of the reaction, add ethyl acetate for extraction, adjust the pH of the aqueous phase to about 4 with 2 N hydrochloric acid, extract with ethyl acetate, and dry and concentrate the organic phase to obtain the carboxylic acid compound shown in Formula 6. The carboxylic acid compound shown in Formula 6 is directly used for the synthesis of the pleuromutilin derivative shown in Formula 7.

[0028] Example 1: Synthesis of the pleuromutilin derivative shown in Formula 7a

[0029] Under nitrogen protection and at 0 °C, dissolve the carboxylic acid compound shown in Formula 6 (364.0 mg, 1.0 mmol, 1.0 eq), 2,4-dimethoxyaniline (183.6 mg, 1.2 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (230.0 mg, 1.2 mmol, 1.2 eq), 4-dimethylaminopyridine (12.2 mg, 0.1 mmol, 0.1 eq) and triethylamine (121.2 mg, 1.2 mmol, 1.2 eq) in dichloromethane and react at room temperature; after detecting the completion of the reaction, add saturated ammonium chloride to quench the reaction, extract with ethyl acetate, dry and concentrate the organic phase, and purify by column chromatography to obtain the pleuromutilin derivative shown in Formula 7a (274.5 mg, 55%).

[0030] The characterization results of the pleuromutilin derivative shown in Formula 7a are as follows: 11H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 9.6 Hz, 1H), 7.73 (s, 1H), 7.30 (d, J = 15.8 Hz, 1H), 6.54 – 6.43 (m, 2H), 6.30 (d, J = 15.7 Hz, 1H), 4.32 (d, J = 7.4 Hz, 1H), 3.85 (s, 3H), 3.79 (s, 3H), 3.49 (d, J = 6.3 Hz, 1H), 2.26 – 2.18 (m, 2H), 2.18 – 2.09 (m, 2H), 2.04 (d, J = 2.8 Hz, 1H), 1.98 (dd, J = 16.1, 7.5 Hz, 1H), 1.77 – 1.63 (m, 4H), 1.61 – 1.53 (m, 1H), 1.45 (ddt, J = 15.8, 10.8, 5.0 Hz, 3H), 1.36 (s, 4H), 1.22 (s, 3H), 1.11 (td, J = 14.0, 4.5 Hz, 1H), 0.96 (dd, J = 7.1, 3.0 Hz, 6H). 13 13C NMR (100 MHz, CDCl3) δ 217.6, 163.7, 156.6, 149.4, 146.9, 124.8, 121.5, 120.9, 103.9, 98.7, 75.3, 67.0, 59.3, 55.8, 55.7, 45.6, 45.2, 42.5, 37.3, 36.9, 34.5, 30.4, 29.0, 27.2, 25.3, 18.3, 13.6, 11.4. Example 2: Synthesis of the pleuromutilin derivative shown in Formula 7b

[0031] The synthesis route and the post-treatment process were the same as those in Example 1, except that the raw material 2,4-dimethoxyaniline (183.6 mg, 1.2 mmol, 1.2 eq) was replaced with cyclohexylamine (118.8 mg, 1.2 mmol, 1.2 eq), and the pleuromutilin derivative shown in Formula 7b (222.7 mg, 50%) was obtained.

[0032] The characterization results of the pleuromutilin derivative shown in Formula 7b are as follows: 11H NMR (400 MHz, DMSO-d6) δ7.64 (d, J = 7.8 Hz, 1H), 6.93 (d, J = 16.0 Hz, 1H), 6.08 (d, J = 16.0 Hz,1H), 4.75 (d, J = 5.5 Hz, 1H), 4.05 (dt, J = 12.7, 6.2 Hz, 2H), 3.59 (tdt, J= 10.9, 7.6, 3.9 Hz, 1H), 3.40 (t, J = 5.6 Hz, 1H), 2.24 – 2.19 (m, 1H), 2.16– 2.11 (m, 1H), 2.06 (t, J = 9.3 Hz, 1H), 1.96 (dd, J = 15.5, 6.9 Hz, 1H),1.88 (t, J = 6.7 Hz, 1H), 1.78 (ddd, J = 11.7, 7.9, 4.2 Hz, 2H), 1.68 (dt, J= 7.9, 3.9 Hz, 3H), 1.58 (dd, J = 12.2, 3.0 Hz, 2H), 1.47 (d, J = 15.8 Hz,2H), 1.34 – 1.20 (m, 9H), 1.17 – 1.12 (m, 2H), 1.04 (s, 3H), 0.97 (dd, J =13.6, 4.1 Hz, 1H), 0.86 (d, J = 6.9 Hz, 3H), 0.80 (d, J = 6.9 Hz, 3H). 13 13C NMR(100 MHz, DMSO-d6) δ 217.9, 164.4, 145.9, 123.7, 72.6, 64.9, 58.3, 47.5,46.2, 45.0, 41.9, 37.1, 34.0, 32.7, 32.6, 30.4, 29.6, 27.1, 25.3, 24.7, 24.7,24.6, 18.2, 13.9, 11.7. Example 3: Synthesis of the pleuromutilin derivative shown in Formula 7c

[0033] The synthetic route and post-treatment process were the same as those in Example 1, except that the raw material 2,4-dimethoxyaniline (183.6 mg, 1.2 mmol, 1.2 eq) was replaced with cyclopropylamine (68.4 mg, 1.2 mmol, 1.2 eq) to obtain the pleuromutilin derivative shown in Formula 7c (229.7 mg, 57%).

[0034] The characterization results of the pleuromutilin derivative shown in Formula 7c are as follows: 1 H NMR (400 MHz, DMSO-d6) δ7.86 (d, J = 4.4 Hz, 1H), 6.93 (d, J = 16.0 Hz, 1H), 6.03 (d, J = 16.0 Hz,1H), 4.76 (d, J = 5.5 Hz, 1H), 4.15 – 3.93 (m, 2H), 3.42 – 3.37 (m, 1H), 2.70(dq, J = 7.5, 3.7 Hz, 1H), 2.20 (s, 1H), 2.13 (d, J = 10.8 Hz, 1H), 2.04 (q,J = 9.5 Hz, 1H), 1.95 (dd, J = 16.4, 7.9 Hz, 1H), 1.87 (t, J = 6.8 Hz, 1H),1.60 (t, J = 15.0 Hz, 2H), 1.46 (d, J = 15.9 Hz, 2H), 1.24 (s, 6H), 1.03 (s,3H), 0.97 (dd, J = 13.3, 3.9 Hz, 1H), 0.86 (d, J = 6.8 Hz, 3H), 0.80 (d, J =7.0 Hz, 3H), 0.63 (d, J = 7.5 Hz, 2H), 0.43 (dd, J = 8.1, 3.8 Hz, 2H). 13 C NMR(100 MHz, DMSO-d6) δ 217.9, 166.6, 146.0, 123.3, 72.6, 64.9, 58.3, 45.9,45.0, 44.9, 41.9, 37.2, 37.1, 34.0, 30.4, 29.6, 27.1, 24.7, 22.4, 18.2, 13.9,11.7, 5.9, 5.6. Example 4: Synthesis of the pleuromutilin derivative shown in Formula 7d

[0035] The synthesis route and post-treatment process were the same as in Example 1, except that the raw material 2,4-dimethoxyaniline (183.6 mg, 1.2 mmol, 1.2 eq) was replaced with cyclopentylamine (102.0 mg, 1.2 mmol, 1.2 eq), and the pleuromutilin derivative shown in Formula 7d was obtained (224.2 mg, 52%).

[0036] The characterization results of the pleuromutilin derivative shown in Formula 7d are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.16 (d, J = 15.8 Hz, 1H), 6.09 (d, J = 15.8 Hz, 1H), 5.79 (d, J = 7.6 Hz, 1H), 4.29 (p, J = 7.1 Hz, 2H), 3.46 (d, J = 6.2 Hz, 1H), 2.20 (dt, J = 15.1, 6.2 Hz, 2H), 2.13 – 2.07 (m, 1H), 2.07 – 1.92 (m, 6H), 1.65 (qd, J = 18.9, 18.2, 6.5 Hz, 8H), 1.50 – 1.39 (m, 5H), 1.35 (s, 3H), 1.17 (s, 3H), 1.08 (dd, J = 13.9, 4.4 Hz, 1H), 0.95 (s, 3H), 0.93 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ 217.8, 165.9, 145.8, 124.1, 75.3, 66.9, 59.3, 51.4, 45.6, 45.5, 45.4, 42.5, 37.2, 37.0, 34.6, 33.3, 33.2, 30.4, 29.0, 27.2, 25.3, 24.0, 23.9, 18.3, 13.6, 11.5. Example 5: Synthesis of the pleuromutilin derivative shown in Formula 7e

[0037] The synthesis route and post-treatment process were the same as in Example 1, except that the raw material 2,4-dimethoxyaniline (183.6 mg, 1.2 mmol, 1.2 eq) was replaced with dimethylamine hydrochloride (97.8 mg, 1.2 mmol, 1.2 eq), and the pleuromutilin derivative shown in Formula 7e was obtained (187.7 mg, 48%).

[0038] The characterization results of the pleuromutilin derivative shown in Formula 7e are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.19(d, J = 15.7 Hz, 1H), 6.67 (d, J = 15.6 Hz, 1H), 4.27 (d, J = 7.3 Hz, 1H),3.46 (d, J = 6.3 Hz, 1H), 3.10 (s, 3H), 3.02 (s, 3H), 2.25 – 2.16 (m, 2H),2.12 – 2.06 (m, 1H), 2.05 (d, J = 2.6 Hz, 1H), 1.95 (dd, J = 16.0, 7.4 Hz,1H), 1.67 (d, J = 16.4 Hz, 3H), 1.62 – 1.53 (m, 2H), 1.50 – 1.42 (m, 3H),1.35 (s, 3H), 1.19 (s, 3H), 1.16 – 1.12 (m, 1H), 1.08 (dd, J = 13.8, 4.8 Hz,1H), 0.95 (d, J = 2.3 Hz, 3H), 0.93 (d, J = 2.3 Hz, 3H). 13 C NMR (100 MHz,CDCl3) δ 217.6, 167.2, 147.0, 121.0, 75.3, 67.2, 59.3, 45.9, 45.6, 45.0,42.4, 37.5, 37.4, 36.8, 35.9, 34.5, 30.4, 29.1, 27.3, 25.3, 18.4, 13.5, 11.4. Example 6: USP kinase inhibition rate test The USP kinase inhibition rate tests of the pleuromutilin derivatives shown in Formulas 7a - 7e prepared by the methods of Examples 1 - 5 were carried out respectively according to the following fluorescence assay method: First, the pleuromutilin derivative was diluted to 10 μM, and then the optimal concentration of deubiquitinating enzyme (DUB) was added. After brief mixing, it was placed in a reaction system with a volume of 200 μL and incubated at room temperature for 1 hour; after adding 200 nM ubiquitin rhodamine 110 (Ub - Rho110, catalog number # M3022, UBPBio), the final fluorescence signal was obtained using a TECAN SPARK multimode microplate reader (excitation wavelength: 485 nm, emission wavelength: 535 nm), and based on this, the inhibition rate of the pleuromutilin derivative on enzyme activity was calculated.

[0039] Among them, the reaction system used contains 50 mM HEPES (pH 8.0), 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, and 0.1 mg / mL bovine serum albumin (BSA).

[0040] The test results are shown in Table 1. All the pleuromutilin derivatives shown in Formulas 7a - 7e can selectively inhibit the kinase activity of USP21 at the protein level, and the inhibition rate is greater than 80%.

[0041] Table 1 Determination results of the inhibition of USPs activity by pleuromutilin derivatives

[0042] In Table 1, +++ represents an inhibition rate greater than 80%, ++ represents an inhibition rate between 50% - 80%, and + represents an inhibition rate less than 50%.

[0043] Example 7: Determination of cell anti - proliferation activity The in vitro anti - proliferation activities of the pleuromutilin derivatives shown in Formulas 7a - 7e prepared by the methods of Examples 1 - 5 against breast cancer cell lines MDA - MB - 231 and BLBC were evaluated by the CCK - 8 assay. The procedure is as follows: The highest concentration was set at 10 μM, diluted in a 3 - fold gradient, and the lowest concentration was set at 0 μM, with a total of 10 concentration gradients. They were co - incubated with MDA - MB - 231 and BLBC cells for 72 h respectively. The activity of the cells was detected by CCK - 8, and the data was processed using GraphPad Prism 9 to calculate the IC 50 value. Meanwhile, BAY - 805 was used as a control to determine its in vitro anti - proliferation activity against breast cancer cell lines MDA - MB - 231 and BLBC.

[0044] The results are shown in Table 2. All the pleuromutilin derivatives shown in Formulas 7a - 7e have excellent in vitro anti - proliferation activities.

[0045] Table 2 Determination results of the anti - proliferation activity of pleuromutilin derivatives against breast cancer cells

[0046] In Table 2, + represents greater than 1000 nM; ++ represents 1000 - 100 nM; +++ represents less than 100 nM.

[0047] Example 9: Animal tumor inhibition experiment Animal experiments were conducted on xenograft tumor models of two breast cancer cell lines (MDA-MB-231 and BLBC) in BALB / c mice to evaluate the anti-tumor ability of pleuromutilin derivatives in vivo. A subcutaneous tumor-bearing mouse model was constructed, and the anti-tumor effects of the pleuromutilin derivatives represented by Formula 7a to Formula 7e prepared by the methods of Examples 1 to 5 were evaluated respectively. The process is as follows: MDA-MB-231 and BLBC were cultured in an incubator at 37 °C with 5% carbon dioxide; all mice were raised under standard specific pathogen-free (SPF) conditions. MDA-MB-231 and BLBC cells were subcutaneously injected into 6- to 8-week-old female BALB / c mice. The mice were examined for tumor formation every day to determine the development of the tumors. When the tumors grew to a certain size, the mice were randomly divided into a control group and a drug administration group, with 5 mice in each group. The drug administration group was given intraperitoneal injection, and the dosage was 50 mpk. The tumor diameter was measured using a vernier caliper every day, and the body weight of the mice was recorded. After 21 days, the tumor tissues were removed, and the tumor volume change was used to evaluate the tumor inhibitory effect of the pleuromutilin derivative. At the same time, BAY-805 was used as a control, and the dosage was also 50 mpk.

[0048] The results are shown in Table 3. The pleuromutilin derivatives represented by Formula 7a to Formula 7e all have excellent in vivo anti-proliferative activity.

[0049] Table 3 Results of animal experiments

[0050] In Table 3, + represents that the tumor inhibition rate is less than 50%, ++ represents that the tumor inhibition rate is between 50% and 80%; +++ represents that the tumor inhibition rate is greater than 80%.

[0051] The above are only the preferred embodiments of the present invention, and do not impose any formal or substantial limitations on the present invention. It should be noted that for those of ordinary skill in the art of this technology, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any equivalent changes, such as slight modifications, decorations, and evolutions made by those skilled in the art without departing from the spirit and scope of the present invention using the technical content disclosed above, are equivalent embodiments of the present invention; at the same time, any equivalent changes, such as modifications, decorations, and evolutions made to the above embodiments based on the essential technology of the present invention, still fall within the scope of the technical solution of the present invention.

Claims

1. A pleuromutilin derivative or a pharmaceutically acceptable salt thereof, characterized in that: The pleuromutilin derivative has a structure shown in Formula 7: ； Wherein, R1 is selected from phenyl, substituted phenyl, C1~C4 alkyl, C3~C8 cycloalkyl; R2 is selected from hydrogen, C1~C4 alkyl.

2. The pleuromutilin derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The R1 is selected from phenyl, at least one methyl-substituted phenyl, at least one methoxy-substituted phenyl, methyl, ethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl; R2 is selected from hydrogen, methyl, ethyl.

3. The pleuromutilin derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The pleuromutilin derivative has a structure shown in one of Formula 7a to Formula 7e: 、 、 、 、 。 4. A method for preparing a pleuromutilin derivative, characterized in that: The following steps are involved: The diol compound shown in Formula 2 is obtained by hydrolyzing the pleuromutilin shown in Formula 1; Protecting the hydroxyl group of the diol compound represented by Formula 2 with acetic anhydride to obtain a compound represented by Formula 3; Reacting the compound represented by Formula 3 with ozone to obtain the aldehyde compound represented by Formula 4; The aldehyde compound represented by Formula 4 is subjected to Horner-Wordsworth-Emmons reaction to obtain the lipid compound represented by Formula 5; The lipid compound shown in Formula 5 is subjected to a hydrolysis reaction to obtain a carboxylic acid compound shown in Formula 6; A carboxylic acid compound represented by Formula 6 is subjected to a condensation reaction with R1-NH-R2 to obtain a pleuromutilin derivative represented by Formula 7; 、 、 、 、 、 、 ； Wherein, R1 is selected from phenyl, substituted phenyl, C1~C4 alkyl, C3~C8 cycloalkyl; R2 is selected from hydrogen, C1~C4 alkyl.

5. The method according to claim 4, characterized in that The step of protecting the hydroxyl group of the diol compound of Formula 2 with acetic anhydride comprises: dissolving the diol compound of Formula 2 in an organic solvent, adding acetic anhydride, and reacting the diol compound of Formula 2 with acetic anhydride.

6. The method according to claim 4, characterized in that The step of reacting the compound of Formula 3 with ozone comprises: dissolving the compound of Formula 3 and triphenylphosphine in an organic solvent, cooling the mixture to -78°C, introducing ozone, and reacting the compound of Formula 3 with ozone.

7. The method according to claim 4, characterized in that The method of causing the aldehyde compound of formula 4 to undergo a Horner-Wordsworth-Emmons reaction comprises: dissolving the aldehyde compound of formula 4 and trimethyl phosphate acetate in an organic solvent, cooling the mixture to -78°C, adding lithium diisopropylamide, and causing the aldehyde compound of formula 4 to react with trimethyl phosphate acetate.

8. The method according to claim 4, characterized in that The method of causing the carboxylic acid compound represented by Formula 6 to undergo a condensation reaction with R1-NH-R2 comprises: dissolving the carboxylic acid compound represented by Formula 6, R1-NH-R2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and triethylamine in an organic solvent under an inert gas atmosphere, and causing the carboxylic acid compound represented by Formula 6 to react with R1-NH-R2.

9. Use of the pleuromutilin derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating and / or preventing breast cancer.

10. A drug for treating and / or preventing breast cancer, characterized in that: The invention comprises the pleuromutilin derivative according to claim 1 or a pharmaceutically acceptable salt thereof.

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