A USP21 inhibitor, its preparation method and its application in medicine

Abstract

The present invention relates to the field of biopharmaceutical technology, and more specifically to a USP21 inhibitor, a preparation method thereof, and its application in medicine. In the present invention, a new pleuromutilin derivative is prepared by a simple and easy method. The pleuromutilin derivative has the property of effectively inhibiting USP21 activity and can be used as a selective USP21 inhibitor. By inhibiting USP21 activity and blocking the signaling pathway mediated by USP21, it can inhibit the growth and spread of breast cancer cells, providing a new strategy and drug option for the treatment of breast cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a USP21 inhibitor, a preparation method thereof, and application in medicine. Background Art

[0002] Breast cancer, the most common malignant tumor in women worldwide, continues to rise in incidence, with a significant trend of younger patients. It has become a major threat to women's health. Despite the continued expansion of existing treatment options (such as surgery, chemotherapy, radiotherapy, endocrine therapy, and emerging immunotherapy / targeted therapies), individual variability in efficacy, drug resistance, and toxic side effects continue to significantly limit clinical benefits, necessitating the development of novel therapeutic targets and drugs.

[0003] Ubiquitin-specific protease 21 (USP21) has attracted considerable attention in tumorigenesis and progression due to its involvement in regulating key pathways such as the cell cycle, DNA repair, and signal transduction. Studies have shown that USP21 is abnormally overexpressed in various tumors, including breast cancer. USP21 drives tumor cell proliferation, invasion, and metastasis by stabilizing oncoproteins such as FOXM1, while mediating immune escape mechanisms to reshape the tumor microenvironment, contributing to the malignant progression and poor prognosis of breast cancer.

[0004] Currently, the development of specific inhibitors targeting USP21 is still in its early stages. Although Bayer's BAY-805 has demonstrated in vitro activity and selectivity, its pharmacokinetic and safety profiles still require optimization, and it has not yet entered clinical use. Current technical bottlenecks, including insufficient inhibitor selectivity, poor in vivo stability, and potential off-target toxicity, have hindered the clinical translation of USP21-targeted therapies.

[0005] Therefore, developing novel, highly effective, and low-toxic USP21 selective inhibitors to inhibit breast cancer progression by blocking its oncogenic signaling pathways can provide an innovative strategy to break through the limitations of existing treatments and has significant 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 application thereof in medicine.

[0007] The first aspect of the present invention provides a pleuromutilin derivative or a pharmaceutically acceptable salt thereof. The pleuromutilin derivative has a structure shown in Formula 7:

[0008] ;

[0009] Wherein, R1 is selected from phenyl, substituted phenyl, C1~C4 alkyl, C3~C8 cycloalkyl; R2 is selected from hydrogen, C1~C4 alkyl.

[0010] The pleuromutilin derivative represented by Formula 7 or a pharmaceutically acceptable salt thereof has the property of selectively inhibiting USP21 activity and is a USP21 selective inhibitor. The pharmaceutically acceptable salt of the pleuromutilin derivative represented by Formula 7 can be easily prepared using the pleuromutilin derivative represented by Formula 7 as a raw material. For example, the hydrochloride salt of the pleuromutilin derivative represented by Formula 7 can be prepared by reacting the pleuromutilin derivative represented by Formula 7 with hydrochloric acid.

[0011] In some optional embodiments, the above 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.

[0012] In some optional embodiments, the pleuromutilin derivative has a structure shown in one of Formulas 7a to 7e:

[0013] 、 、 、

[0014] 、 .

[0015] The second aspect of the present invention provides a method for preparing the above-mentioned pleuromutilin derivative, which comprises the following steps:

[0016] The pleuromutilin shown in Formula 1 is hydrolyzed to obtain the diol compound shown in Formula 2;

[0017] Protecting the hydroxyl group of the diol compound represented by Formula 2 with acetic anhydride to obtain a compound represented by Formula 3;

[0018] reacting the compound represented by Formula 3 with ozone to obtain the aldehyde compound represented by Formula 4;

[0019] subjecting the aldehyde compound represented by Formula 4 to a Horner-Wadsworth-Emmons reaction to obtain an ester compound represented by Formula 5;

[0020] The ester compound represented by Formula 5 is hydrolyzed to obtain the carboxylic acid compound represented by Formula 6;

[0021] allowing the carboxylic acid compound represented by Formula 6 to undergo a condensation reaction with R1-NH-R2 to obtain the pleuromutilin derivative represented by Formula 7;

[0022] 、 、 、 、 、 、 .

[0023] Wherein, R1 is selected from phenyl, substituted phenyl, C1~C4 alkyl, C3~C8 cycloalkyl; R2 is selected from hydrogen, C1~C4 alkyl.

[0024] In some optional embodiments, the process of obtaining the diol compound shown in Formula 2 by hydrolyzing the pleuromutilin shown in Formula 1 includes: dissolving the pleuromutilin shown in Formula 1 in an aqueous solvent, adding sodium hydroxide, and hydrolyzing the pleuromutilin shown in Formula 1 to obtain the diol compound shown in Formula 2.

[0025] In some optional embodiments, protecting the hydroxyl group of the diol compound represented by Formula 2 with acetic anhydride comprises: 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.

[0026] In some optional embodiments, reacting the compound of Formula 3 with ozone includes: dissolving the compound of Formula 3 and triphenylphosphine in an organic solvent, cooling to -78°C, introducing ozone, and reacting the compound of Formula 3 with ozone.

[0027] In some optional embodiments, causing the aldehyde compound represented by Formula 4 to undergo a Horner-Wadsworth-Emmons reaction includes: dissolving the aldehyde compound represented by Formula 4 and trimethyl acetate phosphate in an organic solvent, cooling the mixture to -78°C, adding lithium diisopropylamide, and reacting the aldehyde compound represented by Formula 4 with trimethyl acetate phosphate.

[0028] In some optional embodiments, the ester compound represented by Formula 5 is hydrolyzed to obtain the carboxylic acid compound represented by Formula 6, which includes: dissolving the ester compound represented by Formula 5 in an aqueous solvent, adding sodium hydroxide, and hydrolyzing the ester compound represented by Formula 5 to obtain the carboxylic acid compound represented by Formula 6.

[0029] In some optional embodiments, causing the carboxylic acid compound represented by Formula 6 to undergo a condensation reaction 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.

[0030] 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 medicament for treating and / or preventing breast cancer.

[0031] A fourth aspect of the present invention provides a drug for treating and / or preventing breast cancer, comprising the aforementioned pleuromutilin derivative or a pharmaceutically acceptable salt thereof. The aforementioned pleuromutilin derivative or a pharmaceutically acceptable salt thereof is the main active ingredient (primary agent) in the drug for treating and / or preventing breast cancer.

[0032] In some optional embodiments, the drug for treating and / or preventing breast cancer further comprises an excipient. The drug for treating and / or preventing breast cancer can be in any pharmaceutically acceptable dosage form. The excipient is stable, has no incompatibility with the main drug, does not produce side effects, does not affect efficacy, is not easily deformed, cracked, or moldy at room temperature, and is harmless to the human body.

[0033] In some optional embodiments, the excipient is at least one of gum arabic, syrup, lanolin, and starch.

[0034] The technical solution of the embodiment of the present invention has the following beneficial effects:

[0035] A new pleuromutilin derivative was prepared by a simple and easy method. This pleuromutilin derivative has the property of effectively inhibiting the activity of USP21 and can be used as a selective inhibitor of USP21. By inhibiting the activity of USP21, it blocks the signaling pathway mediated by it, thereby inhibiting the growth and spread of breast cancer cells, providing new strategies and drug options for the treatment of breast cancer. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] In the following examples, the pleuromutilin derivatives shown in Formula 7 were synthesized according to the following route:

[0038] In the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and triethylamine, a carboxylic acid compound represented by Formula 6 is subjected to a condensation reaction with R1-NH-R2 in a dichloromethane solvent to obtain a pleuromutilin derivative represented by Formula 7;

[0039]

[0040] Wherein, 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 represented by Formula 6 can be synthesized according to the following route:

[0041] The pleuromutilin of Formula 1 is used as a starting material, and a diol compound of Formula 2 is obtained through a hydrolysis reaction; then, the diol compound is protected with acetic anhydride to obtain a compound of Formula 3; the compound of Formula 3 is subjected to olefin pyrolysis under the action of ozone to obtain an aldehyde compound of Formula 4; then, the aldehyde compound of Formula 4 is subjected to a Horner-Wadsworth-Emmons reaction to obtain an ester compound of Formula 5; and then, the ester compound of Formula 5 is subjected to a hydrolysis reaction to obtain a compound of Formula 6;

[0042]

[0043] Specifically, pleuromutilin (1.0 g, 2.6 mmol, 1.0 eq) represented by Formula 1 was dissolved in 30.0 mL of a mixed solvent of ethanol and water (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 the reaction was complete, 20 mL of saturated ammonium chloride was added, and the mixture was extracted with ethyl acetate. The organic phase was dried, concentrated, and purified by column chromatography to obtain the diol compound represented by Formula 2 (499.2 mg, 60%). Characterization results of the diol compound represented by 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). 13C 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.

[0044] The diol compound represented by Formula 2 (400.0 mg, 1.3 mmol, 1.0 eq) was dissolved in 10.0 mL of pyridine, and acetic anhydride (291.7 mg, 2.9 mmol, 2.2 eq) was added. The mixture was reacted at room temperature. After the reaction was complete, 50 mL of saturated copper sulfate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was dried, concentrated, and purified by column chromatography to obtain the compound represented by Formula 3 (367.6 mg, 70%). Characterization results of the compound represented by 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). 13CNMR (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.

[0045] The compound represented by Formula 3 (400.0 mg, 1.0 mmol, 1.0 eq) and triphenylphosphine (262.3 mg, 1.0 mmol, 1.0 eq) were dissolved in 10.0 mL of dichloromethane. The mixture was cooled to -78°C under argon and ozone was introduced. After completion of the reaction, saturated ammonium chloride was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phase was dried, concentrated, and purified by column chromatography to obtain the aldehyde compound represented by Formula 4 (365.4 mg, 90%). Characterization results of the aldehyde compound represented by 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) . 13C 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.

[0046] The aldehyde compound represented by Formula 4 (500.0 mg, 1.2 mmol, 1.0 eq) and trimethyl phosphate (327.6 mg, 1.8 mmol, 1.5 eq) were dissolved in 10.0 mL of dry tetrahydrofuran. The mixture was cooled to -78°C under argon, and lithium diisopropylamide (2.0 N in THF, 1.4 mL, 1.2 eq) was slowly added dropwise. After the reaction was complete, saturated sodium bicarbonate was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phase was dried, concentrated, and purified by column chromatography to obtain the ester compound represented by Formula 5 (304.9 mg, 55%). Characterization results of the ester compound represented by 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.

[0047] The ester compound represented by Formula 5 (500.0 mg, 1.0 mmol, 1.0 eq) was dissolved in 10.0 mL of an ethanol / water mixture (volume ratio of ethanol to water: 1:1). Sodium hydroxide (200.0 mg, 5.0 mmol, 5.0 eq) was added, and the system was heated to 60°C in an oil bath for reaction. After the reaction was complete, ethyl acetate was added for extraction. The aqueous phase was adjusted to a pH of approximately 4 with 2 N hydrochloric acid, extracted with ethyl acetate, and the organic phase was dried and concentrated to obtain the carboxylic acid compound represented by Formula 6. The carboxylic acid compound represented by Formula 6 was directly used to synthesize the pleuromutilin derivative represented by Formula 7.

[0048] Example 1: Synthesis of the pleuromutilin derivative shown in Formula 7a

[0049]

[0050] Under nitrogen protection at 0°C, the carboxylic acid compound represented by 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) were dissolved in dichloromethane and reacted at room temperature. After the reaction was completed, saturated ammonium chloride was added to quench the reaction, and the product was extracted with ethyl acetate. The organic phase was dried, concentrated, and purified by column chromatography to obtain the pleuromutilin derivative represented by Formula 7a (274.5 mg, 55%).

[0051] The characterization results of the pleuromutilin derivative represented by Formula 7a are as follows: 1 H 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, 3.0Hz, 6H). 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.

[0052] Example 2: Synthesis of the pleuromutilin derivative shown in Formula 7b

[0053]

[0054] The synthetic route and post-treatment process were the same as those in Example 1, except that the starting material was replaced by cyclohexylamine (118.8 mg, 1.2 mmol, 1.2 eq) instead of 2,4-dimethoxyaniline (183.6 mg, 1.2 mmol, 1.2 eq) to obtain the pleuromutilin derivative represented by Formula 7b (222.7 mg, 50%).

[0055] The characterization results of the pleuromutilin derivative represented by Formula 7b are as follows: 1 H NMR (400 MHz, DMSO- d 6) 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 C NMR (100 MHz, DMSO- d 6) 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.

[0056] Example 3: Synthesis of the pleuromutilin derivative shown in Formula 7c

[0057]

[0058] The synthetic route and post-treatment process were the same as those in Example 1, except that the starting material was replaced by cyclopropylamine (68.4 mg, 1.2 mmol, 1.2 eq) from 2,4-dimethoxyaniline (183.6 mg, 1.2 mmol, 1.2 eq) to obtain the pleuromutilin derivative represented by Formula 7c (229.7 mg, 57%).

[0059] The characterization results of the pleuromutilin derivative represented by Formula 7c are as follows: 1 H NMR (400 MHz, DMSO- d 6) 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- d 6) 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.

[0060] Example 4: Synthesis of the pleuromutilin derivative shown in formula 7d

[0061]

[0062] The synthetic route and post-treatment process were the same as those in Example 1, except that the starting material was replaced by cyclopentylamine (102.0 mg, 1.2 mmol, 1.2 eq) instead of 2,4-dimethoxyaniline (183.6 mg, 1.2 mmol, 1.2 eq) to obtain the pleuromutilin derivative represented by Formula 7d (224.2 mg, 52%).

[0063] The characterization results of the pleuromutilin derivative represented by 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, 6.2Hz, 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) δ217.8, 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.

[0064] Example 5: Synthesis of the pleuromutilin derivative represented by formula 7e

[0065]

[0066] The synthetic route and post-treatment process were the same as those in Example 1, except that the starting material was replaced by dimethylamine hydrochloride (97.8 mg, 1.2 mmol, 1.2 eq) instead of 2,4-dimethoxyaniline (183.6 mg, 1.2 mmol, 1.2 eq) to obtain the pleuromutilin derivative represented by Formula 7e (187.7 mg, 48%).

[0067] The characterization results of the pleuromutilin derivative represented by 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.

[0068] Example 6: USPs kinase inhibition rate test

[0069] The pleuromutilin derivatives represented by Formula 7a to Formula 7e prepared by the methods of Examples 1 to 5 were tested for USPs kinase inhibition rate according to the following fluorescence assay:

[0070] The pleuromutilin derivative was first diluted to 10 μM, and then the optimal concentration of deubiquitinase (DUB) was added. After brief mixing, the mixture was placed in a 200 μL reaction system 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 (excitation wavelength: 485 nm, emission wavelength: 535 nm) was obtained using a TECAN SPARK multi-function microplate reader to calculate the inhibition rate of the pleuromutilin derivative on the enzyme activity.

[0071] The reaction system used contained 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).

[0072] The test results are shown in Table 1. The pleuromutilin derivatives represented by Formula 7a to Formula 7e can selectively inhibit the kinase activity of USP21 at the protein level, and the inhibition rate is greater than 80%.

[0073] Table 1 Determination results of the inhibitory activity of pleuromutilin derivatives on USPs

[0074]

[0075] In Table 1, +++ represents an inhibition rate greater than 80%, ++ represents an inhibition rate between 50% and 80%, and + represents an inhibition rate less than 50%.

[0076] Example 7: Determination of cell anti-proliferative activity

[0077] The in vitro anti-proliferative activity of the pleuromutilin derivatives represented by Formula 7a to Formula 7e prepared by the methods of Examples 1 to 5 against breast cancer cell lines MDA-MB-231 and BLBC was evaluated by CCK-8 assay, and the process is as follows:

[0078] The highest concentration was set to 10 μM, and the dilution was performed in a 3-fold gradient, with the lowest concentration set to 0 μM. A total of 10 concentration gradients were used, and the cells were incubated with MDA-MB-231 and BLBC cells for 72 h. Cell activity was detected by CCK-8, and data were processed using GraphPad Prism 9 to calculate the IC 50 At the same time, BAY-805 was used as a control to determine the in vitro anti-proliferative activity of BAY-805 against breast cancer cell lines MDA-MB-231 and BLBC.

[0079] The results are shown in Table 2. The pleuromutilin derivatives represented by Formula 7a to Formula 7e all have excellent in vitro anti-proliferative activity.

[0080] Table 2 Results of the antiproliferative activity assay of pleuromutilin derivatives against breast cancer cells

[0081]

[0082] In Table 2, + represents greater than 1000 nM; ++ represents 1000-100 nM; and +++ represents less than 100 nM.

[0083] Example 9: Animal tumor inhibition experiment

[0084] Animal experiments were conducted using two breast cancer cell xenograft models (MDA-MB-231 and BLBC) in BALB / c mice to evaluate the in vivo anti-tumor activity of pleuromutilin derivatives. A subcutaneous tumor-bearing mouse model was constructed to evaluate the anti-tumor effects of the pleuromutilin derivatives represented by Formulas 7a to 7e, prepared by the methods of Examples 1 to 5, in BALB / c mice. The following procedures were used:

[0085] MDA-MB-231 and BLBC cells were cultured in a 37°C, 5% CO2 incubator. All mice were housed under standard specific pathogen-free (SPF) conditions. Six- to eight-week-old female BALB / c mice were subcutaneously injected with MDA-MB-231 and BLBC cells. Tumor formation was assessed daily to determine tumor progression. When tumors reached a certain size, mice were randomly divided into a control group and a treatment group (n=5). The treatment group received 50 mpk of the drug via intraperitoneal injection. Tumor diameters were measured daily with a vernier caliper, and mouse weights were recorded. Tumor tissue was harvested 21 days later, and changes in tumor volume were used to evaluate the anti-tumor effect of the pleuromutilin derivative. BAY-805 was also administered at 50 mpk as a control.

[0086] 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.

[0087] Table 3 Animal experiment results

[0088]

[0089] In Table 3, + represents a tumor inhibition rate of less than 50%, ++ represents a tumor inhibition rate of 50% to 80%, and +++ represents a tumor inhibition rate of more than 80%.

[0090] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still 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, at least one methyl-substituted phenyl, at least one methoxy-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, and cyclohexyl; and R2 is selected from hydrogen, methyl, and 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 Formulas 7a to 7e: 、 、 、 、 。 4. A method for preparing a pleuromutilin derivative, characterized in that: The following steps are involved: The pleuromutilin shown in Formula 1 is hydrolyzed to obtain the diol compound shown in Formula 2; 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 a Horner-Wadsworth-Emmons reaction to obtain an ester compound represented by Formula 5; The ester compound represented by Formula 5 is hydrolyzed to obtain the carboxylic acid compound represented by Formula 6; allowing the carboxylic acid compound represented by Formula 6 to undergo a condensation reaction with R1-NH-R2 to obtain the pleuromutilin derivative represented by Formula 7; 、 、 、 、 、 、 ； Wherein, R1 is selected from phenyl, at least one methyl-substituted phenyl, at least one methoxy-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 protecting the hydroxyl group of the diol compound represented by Formula 2 with acetic anhydride comprises: 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 the acetic anhydride.

6. The method according to claim 4, characterized in that Said 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 represented by Formula 4 to undergo a Horner-Wadsworth-Emmons reaction comprises: dissolving the aldehyde compound represented by Formula 4 and trimethyl acetate phosphate in an organic solvent, cooling the mixture to -78°C, adding lithium diisopropylamide, and reacting the aldehyde compound represented by Formula 4 with trimethyl acetate phosphate.

8. The method according to claim 4, characterized in that The condensation reaction of the carboxylic acid compound represented by Formula 6 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 reacting the carboxylic acid compound represented by Formula 6 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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