18 beta-glycyrrhetinic acid derivatives as well as preparation method and application thereof

Abstract

The invention discloses 18 beta-glycyrrhetinic acid derivatives as well as a preparation method and application thereof, belongs to the technical field of medicines, and particularly relates to 18 beta-glycyrrhetinic acid derivatives as shown in a general formula I or II or optical isomers or pharmaceutically acceptable salts thereof, the invention also discloses a preparation method of the compound and application of the compound in preparation of medicines for treating and / or preventing inflammatory diseases. According to the present invention, the compound specifically induces the protein degradation of histone deacetylase 3 so as to inhibit the assembly and the activation of NLRP3 inflammasomes, and finally block the maturation and the secretion of downstream inflammatory factors IL-1beta and caspase-1, such that the significant anti-inflammatory effect is provided;

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to 18beta-glycyrrhetinic acid derivatives containing o-phenylenediamine structural fragments, a preparation method of the derivatives, and use of the derivatives in the preparation of drugs for treating and / or preventing various inflammatory diseases. BACKGROUND Environmental toxin exposure, pathogen drug resistance growth, and high-pressure lifestyle jointly drive the prevalence of such diseases. However, existing therapies such as glucocorticoids and biological agents have serious side effects, high treatment costs, and insufficient patient response rates, far from meeting clinical needs. In this context, natural products and their derivatives show unique therapeutic value: more than 50% of anti-inflammatory clinical candidate molecules are derived from natural lead compounds, their multi-target mechanism of action can reshape immune homeostasis, and liver and kidney toxicity is reduced by 3-5 times compared with synthetic drugs. This feature provides an important direction for the development of safe and efficient new anti-inflammatory drugs.

[0002] Licorice, as an important herbal plant with a history of more than 1,000 years of application in traditional Chinese medicine, is widely available, and its compound preparations show unique value in the prevention and treatment of various diseases. The pentacyclic triterpenoid glycyrrhetinic acid (GA) is one of the core active components of licorice, which is abundant in licorice roots and has low inherent toxicity. Based on its pentacyclic structure, chemical modification at the C30 position of GA has been proven to be a key strategy for introducing functional groups and significantly improving its pharmacological activity, especially in enhancing anti-inflammatory, anti-tumor and other activities. SUMMARY

[0003] Based on the above background, the present application designs and synthesizes two 18beta-glycyrrhetinic acid derivatives of general structure, which are characterized by introducing a chemical fragment containing an o-phenylenediamine structure through a piperazine connecting chain at the C30 position of glycyrrhetinic acid.

[0004] The present application also provides a preparation method of the 18beta-glycyrrhetinic acid derivatives and application of the 18beta-glycyrrhetinic acid derivatives in the preparation of drugs for preventing and / or treating inflammatory diseases.

[0005] The present application relates to 18beta-glycyrrhetinic acid derivatives represented by general formula I or II, optical isomers thereof, and pharmaceutically acceptable salts thereof:

[0006] X is fluorine or hydrogen; n is an integer of 5-7; R is cyano or a halogenated hydrocarbon group, preferably trifluoromethyl.

[0007] Particularly preferred compounds include:

[0008] The present application also includes prodrugs of the compounds described herein. According to the present application, a prodrug is a derivative of a compound of Formula I or II, which can have less or even no activity itself, but which, following administration, is converted in the body to a corresponding biologically active form, for example, by metabolic, solvolysis or other processes.

[0009] The present application also includes pharmaceutical compositions comprising an 18β-glycyrrhetinic acid derivative containing a catechol moiety of Formula I or II, and optical isomers thereof, and pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients refer to any diluents, adjuvants and / or carriers that can be used in the pharmaceutical field. The compounds described herein can be used in combination with other active ingredients, provided that they do not produce other adverse effects, such as allergic reactions.

[0010] The pharmaceutical compositions of the present application can be formulated into several dosage forms, each of which contains some excipients commonly used in the pharmaceutical field, for example, oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders, which are dissolved into injection solutions before injection); topical preparations (such as ointments or emulsions).

[0011] The carriers used in the pharmaceutical compositions of the present application are the common types available in the pharmaceutical field, including: binders, lubricants, disintegrants, co-solvents, diluents, stabilizers, suspending agents, pigments, flavoring agents, etc. for oral preparations; preservatives, solubilizers, stabilizers, etc. for injectable preparations; bases, diluents, lubricants, preservatives, etc. for topical preparations. The pharmaceutical preparations can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically), and if certain drugs are not stable in the stomach, they can be formulated into enteric-coated tablets.

[0012] Through activity screening, we found that the compounds described in the present application have good anti-inflammatory activity, and subsequent mechanism study and in vivo activity results show that the compounds can degrade HDAC3 protein and inhibit NLPR3 inflammasome. Therefore, the compounds described in the present application can be used for preparing drugs for treating and / or preventing various inflammatory diseases, such as rheumatoid arthritis (RA), psoriasis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), gout / hyperuricemia, asthma, atopic dermatitis, Alzheimer's disease (AD), multiple sclerosis (MS) or hepatitis.

[0013] The compounds of the present application can be used as the sole anti-inflammatory agent or in combination with one or more other anti-inflammatory agents. Combination therapy is achieved by administering the individual therapeutic components simultaneously, sequentially or separately.

[0014] Methods for preparing the compounds of the embodiments of the present application are provided below.

[0015] Synthetic route:

[0016] X is fluorine or hydrogen; n is an integer from 5 to 7; the dotted circle A represents the structural fragment at the corresponding position of the compounds of Examples 1-18.

[0017] Step a: Glycyrrhetinic acid is oxidized by IBX at 80-90°C to produce intermediate compound 1, using DMSO as solvent; Step b: Intermediate compound 1 is subjected to substitution reaction with I2, pyridine and THF to produce intermediate compound 2; Step c: Intermediate compound 2 is reacted with CuCN at 120-140°C to produce intermediate compound 3, using NMP as solvent; Step d: Glycyrrhetinic acid or intermediate compound 2 or intermediate compound 3 is subjected to amidation reaction with 1-(4-nitrophenyl)piperazine in the presence of EDCI, HOBT, DIEA to produce the corresponding intermediate compound 5, 6 or 4, using DMF as solvent; Step h: Intermediate compound 6 is reacted with FSO2CF2COOCH3 in the presence of HMPA and CuI at 60-70°C to produce intermediate compound 7, using DMF as solvent; Step e: Intermediate compound 4 or 5 or 7 is subjected to reduction reaction in the presence of zinc powder and acetic acid at 50-70°C to produce intermediate compound 8, using ethanol as solvent; Step f: Intermediate compound 8 is reacted with a compound containing the structural fragment of o-phenylenediamine in the presence of HOBT, EDCI and DIEA to produce intermediate compound 9, while the amino group of o-phenylenediamine is protected by tert-butyloxycarbonyl, using DMF as solvent; Step g: Intermediate compound 9 is deprotected to produce the final product.

[0018] The beneficial effects of the present application are as follows: The present application is verified by activity screening experiments that the compounds of the present application can effectively inhibit the release of key inflammatory mediators, nitric oxide. In-depth mechanism research (including Western blot, quantitative proteomics and cell immunofluorescence experiments) has confirmed that some compounds can specifically induce histone deacetylase 3 (HDAC3) protein degradation, thereby inhibiting the assembly and activation of NLRP3 inflammasome, and finally blocking the maturation and secretion of downstream inflammatory factors IL-1β and caspase-1, thereby exerting significant anti-inflammatory effect. Animal experiments such as mouse acute shock model, acute colitis model and acute gout model show that some compounds exhibit excellent in vivo anti-inflammatory activity. Therefore, the present application not only provides a class of glycyrrhetinic acid derivatives with clear anti-inflammatory mechanism and good activity, but also lays a solid material and theoretical foundation for the in-depth development of anti-inflammatory drugs based on glycyrrhetinic acid structure. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The molecular mechanism of HDAC3 regulating NLRP3 inflammasome pathway is verified multidimensionally; wherein A: Western blot analysis shows the expression of HDAC3 protein and NLRP3 inflammasome pathway related proteins; B: immunofluorescence microscope observation shows ASC spot formation; C: quantitative proteomics volcano plot shows differentially expressed proteins; D: mRNA sequencing volcano plot shows differentially expressed genes; E: mRNA sequencing columnar chart shows the expression level of NLRP3 inflammasome pathway related genes.

[0020] Figure 2 The survival curve of mice in different treatment groups of mouse acute shock model.

[0021] Figure 3 The mouse acute colitis model study; wherein A: experimental flowchart; B: representative colon dissection photograph; C: columnar chart shows the statistical results of colon length of mice in different treatment groups; D: colon tissue HE staining micrograph; E: line chart shows the change trend of body weight of mice in different treatment groups during the experiment.

[0022] Figure 4 The mouse acute gout model study; wherein A: experimental flowchart; B: line chart shows the change trend of knee circumference of mice in different treatment groups during the experiment; C: representative mouse knee joint morphology photograph; D: knee joint HE staining micrograph. DETAILED DESCRIPTION

[0023] The following examples are intended to illustrate and not to limit the scope of the present application. Any technology realized based on the above content of the present application belongs to the scope of the present application.

[0024] The nuclear magnetic resonance spectrum of the compound in the present application is determined by using a Bruker ARX-600 nuclear magnetic resonance spectrometer in DMSO-d6 with TMS as an internal standard, and the high-resolution mass spectrum is determined by using a Bruker micro-TOF-Q.

[0025] Example 1 N 1 -aminophenyl- N 7 -(4-(4-(2-cyano-3,11-dioxo-18 β Preparation of 2-(4-(4-(2-cyano-3,11-dioxo-18 Step a: Preparation of 3,11-dioxo-18β-olean-1,12-diene-30-carboxylic acid

[0026] GA (3.5 g, 7.48 mmol) was weighed into 150 mL of DMSO, and IBX (8.37 g, 30 mmol) was added, and stirring was performed at 85°C for 6 h. After cooling to room temperature, it was poured into 300 mL of a 5% NaHCO3 solution, and a white solid was precipitated. Extraction was performed with ethyl acetate (150 mL x 3), and the organic layers were combined. Washing was performed with water, saturated NaCl, and anhydrous Na2SO4 was added for drying. After concentration under reduced pressure, a white solid was obtained, and the yield was 76%.

[0027] Step b: Preparation of 2-iodo-3,11-dioxo-18β-olean-1,12-diene-30-carboxylic acid

[0028] 3,11-dioxo-18β-olean-1,12-diene-30-carboxylic acid (6.0 g, 12.9 mmol) was weighed into a 500 mL flask, and 300 mL of anhydrous THF was added for dissolution. Then, I2 (13.3 g, 51.6 mmol) and pyridine (6.23 mL, 77.4 mmol) were added, and stirring was performed under reflux for 12 h. Then, I2 (6.65 g, 25.8 mmol) and pyridine (3.12 mL, 38.7 mmol) were added, and stirring was continued for 6 h. After cooling to room temperature, THF was evaporated, and dissolution was performed with ethyl acetate. Washing was performed with Na2S2O3 until the solution was golden yellow, and saturated NaCl was added for washing. Anhydrous Na2SO4 was added for drying. After concentration under reduced pressure, silica gel column chromatography was performed with cyclohexane:acetone (V / V) = 50:1 as the eluent, and a white solid was obtained, and the yield was 71%.

[0029] Step c: Preparation of 2-cyano-3, 11-dioxo-18β-olean-1, 12-diene-30-carboxylic acid

[0030] Weigh 2-iodo-3, 11-dioxo-18β-olean-1, 12-diene-30-carboxylic acid (5.0 g, 8.4 mmol) into a 200 mL three-necked flask, and dissolve it in methylpyrrolidone (NMP). Add cuprous cyanide (1.51 g, 16.8 mmol), and stir the reaction mixture at 130°C for 3 hours under N2 protection. After cooling the reaction mixture to room temperature, pour it into water, collect the precipitated solid, and purify it by column chromatography on silica gel using cyclohexane:acetone (V / V) = 15:1 as the eluent. Isolate the white solid, and obtain it in a yield of 52%. N - methylpyrrolidone (NMP) into a three-necked flask. Add cuprous cyanide (1.51 g, 16.8 mmol), and stir the reaction mixture at 130°C for 3 hours under N2 protection. After cooling the reaction mixture to room temperature, pour it into water, collect the precipitated solid, and purify it by column chromatography on silica gel using cyclohexane:acetone (V / V) = 15:1 as the eluent. Isolate the white solid, and obtain it in a yield of 52%.

[0031] Step d: Preparation of 1-(4-nitrophenyl)-4-(2-cyano-3, 11-dioxo-18β-olean-1, 12-diene-30- yl) piperazine

[0032] Dissolve 2-cyano-3, 11-dioxo-18β-olean-1, 12-diene-30-carboxylic acid (2 g, 4.07 mmol) in 70 mL of anhydrous DMF, and add HOBT (0.61 g, 4.48 mmol) and EDCI (0.86 g, 4.48 mmol). Stir the mixture at room temperature for 30 min, and then add DIEA (0.58 g, 4.48 mmol) and 1-(4-nitrophenyl)piperazine (0.93 g, 4.48 mmol). Stir the mixture at 70°C for 1 h, and after the reaction is completed, pour the reaction mixture into 300 mL of ice water to obtain a yellow precipitate. Collect the solid by suction filtration under reduced pressure, and obtain the yellow solid in a yield of 82%.

[0033] Step e: Preparation of 1-(4-aminophenyl)-4-(2-cyano-3, 11-dioxo-18β-olean-1, 12-diene-30- yl) piperazine

[0034] Dissolve 1-(4-nitrophenyl)-4-(2-cyano-3, 11-dioxo-18β-olean-1, 12-dien-30- yl)piperazine (2 g, 2.94 mmol) in 40 mL of absolute ethanol, and then add zinc powder (1.93 g, 29.4 mmol) and 5 mL of glacial acetic acid successively. Stir the mixture at 60°C for 45 min. Filter the reaction mixture while hot, and then evaporate the filtrate to dryness. Dissolve the dried solid in 50 mL of dichloromethane, wash with water (30 mL x 1), and then with saturated aqueous sodium chloride (30 mL x 1). Dry the mixture over anhydrous sodium sulfate, and then concentrate under reduced pressure to obtain a yellow-brown solid in a yield of 85%.

[0035] Steps f, g: N 1 -aminophenyl- N 7 -(4-(4-(2-cyano-3, 11-dioxo-18 β Preparation of 1-(4-aminophenyl)-4-(2-cyano-3, 11-dioxo-18β-olean-1, 12-dien-30- yl)piperazine

[0036] Dissolve heptanedioic acid (0.067 g, 0.42 mmol) in 5 mL of dry DMF, and then add HOBT (0.057 g, 0.42 mmol), EDCI (0.081 g, 0.42 mmol), and DIEA (0.054 g, 0.42 mmol) successively. Stir the mixture at room temperature for 30 min, and then add 1-(4-aminophenyl)-4-(2-cyano-3, 11-dioxo-18β- olean-1, 12-dien-30-yl)piperazine (0.25 g, 0.38 mmol). Continue to stir the mixture for 3 h, and then add tert-butyl 2-aminophenylcarbamate (0.088 g, 0.42 mmol). Stir the mixture for 2 h. Pour the reaction mixture into 50 mL of water, and then stir the mixture for 20 min. A yellowish solid is precipitated, which is filtered off, and then dried. Subject the solid to column chromatography on silica gel using dichloromethane:methanol (V / V) = 50:1 as the eluent to obtain a white solid. Treat the obtained intermediate with HCl / EA (30 mL) for 4 h. Adjust the pH of the mixture to 7-8 using a saturated NaHCO3 solution. Filter off the precipitate under reduced pressure to obtain a white solid in a yield of 33%. 1 H NMR (600 MHz, DMSO- d6) δ 9.67 (s, 1H), 9.08(s, 1H), 8.49 (s, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 6.4 Hz, 1H),6.91 – 6.85 (m, 3H), 6.71 (d, J = 6.7 Hz, 1H), 6.55 – 6.49 (m, 1H), 5.64 (s,1H), 4.83 (s, 2H), 3.72 – 3.67 (m, 4H),3.04 – 3.02 (m, 4H), 2.98 (s, 1H),2.34 – 2.29 (m, 3H), 2.27 (t, J = 7.5 Hz, 2H), 2.14 (t, J = 11.3 Hz, 1H),2.02 – 1.98 (m, 1H), 1.87 (d, J = 13.7 Hz, 1H), 1.82 – 1.75 (m, 1H), 1.74 –1.68 (m, 3H),1.67 – 1.56 (m, 6H), 1.56 – 1.44 (m, 2H), 1.40 (s, 1H), 1.38 (s,3H), 1.35 (s, 3H), 1.32(s,2H), 1.24 (d, J = 8.5 Hz, 1H), 1.20 (s, 3H), 1.17(d, J = 7.0 Hz, 1H), 1.14 (s, 3H), 1.09 (s, 3H), 1.07 (s, 3H), 1.03 – 0.93(m, 1H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 198.63 (C-3), 198.60 (C-11), 174.88 (C=O), 173.53 (C-30), 172.87 (C-13), 171.59 (C=O), 171.07 (C-1), 147.18 (Ph-C), 142.35 (Ph-C), 132.52 (Ph-C), 127.38 (C-12), 126.14 (Ph-C), 125.74 (Ph-C), 124.05 (Ph-C), 120.63 (2C, 2´ Ph-C), 116.66 (2C, 2´ Ph-C), 116.63 (Ph-C), 116.34 (Ph-C), 115.96 (-CN), 112.08 (C-2), 53.78, 50.60, 49.87, 48.36, 45.48, 45.07, 45.05, 43.98, 43.88, 43.86, 40.53, 37.89, 36.66, 36.13, 31.93, 31.89, 31.34, 28.82, 28.79, 27.49, 26.66, 26.59, 26.51, 25.59, 25.50, 22.98, 22.56, 21.74, 21.47, 19.75, 18.98, 17.90. HRMS (ESI): calcd for C 54 H 70 N6O5[M+Na] + 905.5300, found: 905.5321.

[0037] Example 2 N 1 -aminophenyl- N 8 -(4-(4-(2-cyano-3,11-dioxo-18 β Preparation of (4-(4-(2-cyano-3,11-dioxo-18 The compound of Example 2 was prepared according to the preparation method of Example 1, using as starting material suberic acid instead of heptanedioic acid of Example 1. Yield 32%. 1H NMR (600 MHz, DMSO-d6) δ 9.66 (s, 1H), 9.07 (s, 1H), 8.49 (s, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 9.4 Hz, 1H), 6.91-6.85 (m, 3H), 6.71 (d, J = 8.0 Hz, 1H), 6.53 (t, J = 7.5 Hz, 1H), 5.66 (s, 1H), 4.80 (s, 2H), 3.71-3.68 (m, 4H), 3.04-3.02 (m, 4H), 2.98 (s, 1H), 2.31 (t, J = 7.5 Hz, 3H), 2.26 (t, J = 7.5 Hz, 2H), 2.17-2.10 (m, 1H), 2.00 (d, J = 10.9 Hz, 1H), 1.87 (d, J = 14.1 Hz, 1H), 1.79 (d, J = 13.7 Hz, 1H), 1.73-1.66 (m, 3H), 1.62-1.56 (m, 6H), 1.56-1.44 (m, 2H), 1.40 (s, 1H), 1.38 (s, 3H), 1.35 (s, 3H), 1.35-1.32 (m, 4H) 1.24 (d, J = 6.7 Hz, 1H), 1.20 (s, 3H), 1.18 (s, 1H), 1.14 (s, 3H), 1.09 (s, 3H), 1.07 (s, 3H), 1.00 (d, J = 18.4 Hz, 1H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 198.62 (C-3), 198.59 (C-11), 174.88 (C=0), 173.51 (C-30), 172.86 (C-13), 171.62 (C=0), 171.11 (C-l), 147.15 (Ph-C), 142.32 (Ph-C), 132.56 (Ph-C), 127.37 (C-12), 126.09 (Ph-C), 125.71 (Ph-C), 124.09 (Ph-C), 120.60 (2C, 2' Ph-C), 116.63 (2C, 2' Ph-C), 116.61 (Ph-C), 116.34 (Ph-C), 115.96 (-CN), 112.08 (C-2), 55.39, 53.77, 50.60, 49.87, 48.34, 45.93, 45.47, 45.06, 43.97, 43.89, 43.85, 40.53, 37.89, 36.76, 36.22, 32.28, 31.89, 31.34, 29.01, 28.99, 28.98, 28.80, 27.49, 26.66, 26.58, 26.50, 25.71, 25.66, 22.98, 21.74, 19.75, 18.98, 17.91. HRMS (ESI): calcd for C 55 H 72 N6O5[M+Na] + 919.5456, found: 919.5487.

[0038] Example 3 N 1 -aminophenyl- N 9 -(4-(4-(2-cyano-3,11-dioxo-18 β Preparation of (4-(4-(2-cyano-3,11-dioxo-18 The compound of Example 3 was prepared according to the preparation method of Example 1, using as starting material azelaic acid instead of suberic acid of Example 1. Yield 26%. 1 H NMR (600 MHz, DMSO- d6) δ 9.65 (s, 1H), 9.08 (s, 1H), 8.49(s, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 6.4 Hz, 1H), 6.91 – 6.85 (m,3H), 6.70 (d, J = 6.5 Hz, 1H), 6.52 (t, J = 6.8 Hz, 1H), 5.64 (s, 1H), 4.80(s, 2H), 3.71 – 3.68 (m, 4H), 3.04 – 3.02 (m, 4H), 2.98 (s, 1H), 2.34 – 2.27(m, 3H), 2.25 (t, J = 7.4 Hz, 2H), 2.18 – 2.10 (m, 1H), 2.02 – 1.98 (m, 1H),1.87 (d, J = 24.3 Hz, 1H), 1.81 – 1.76 (m,1H), 1.73 – 1.67 (m, 3H), 1.60 –1.57 (m, 6H), 1.52 – 1.44 (m, 2H), 1.40 (s, 1H), 1.38 (s, 3H), 1.35 (s, 3H),1.32 (s, 6H), 1.23 (s, 1H), 1.20 (s, 3H), 1.18 (s, 1H), 1.14 (s, 3H), 1.09(s, 3H), 1.07 (s, 3H), 1.01 (d, J = 5.2 Hz, 1H), 0.76 (s, 3H). 13 C NMR (151MHz, DMSO- d6) δ 196.27 (C-3), 196.24 (C-11), 172.52 (C=0), 171.16 (C-30), 170.51 (C-13), 169.28 (C=0), 168.77 (C-1), 144.81 (Ph-C), 139.93 (Ph-C), 130.18 (Ph-C), 125.03 (C-12), 123.78 (Ph-C), 123.37 (Ph-C), 121.75 (Ph-C), 118.26 (2C, 2' Ph-C), 114.34 (2C, 2' Ph-C), 114.31 (Ph-C), 114.04 (Ph-C), 113.61 (-CN), 109.74 (C-2), 51.43, 48.25, 47.52, 46.00, 43.12, 42.72, 41.62, 41.50, 38.18, 35.55, 34.43, 33.89, 32.32, 29.95, 29.54, 29.09, 28.99, 26.75, 26.72, 26.45, 25.14, 24.47, 24.31, 24.24, 24.15, 23.42, 23.34, 22.91, 20.62, 20.19, 19.40, 17.40, 16.63, 15.55. HRMS (ESI): calcd for C 56 H 74 N6O5[M+Na] + 933.5613, found: 933.5638.

[0039] Example 4 N 1 -(2-aminophenyl)- N 7 -(4-(4-(2-cyano-3,11-dioxo-18 β Preparation of 4-(4-(2-cyano-3,11-dioxo-18 The compound of Example 4 was prepared according to the preparation procedure of Example 1, using 2-amino-4-fluorophenylcarbamic acid tert-butyl ester as the starting material instead of 2-aminophenylcarbamic acid tert-butyl ester in Example 1. Yield 24%. 1 H NMR (600 MHz, DMSO- d6) δ 9.67 (s, 1H), 9.00 (s, 1H), 8.49 (s, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.12 - 7.05 (m, 1H), 6.89 (d, J = 8.6 Hz, 2H), 6.47 (d, J = 11.2 Hz, 1H), 6.28 (t, J = 7.1 Hz, 1H), 5.64 (s, 1H), 5.12 (s, 2H), 3.71 - 3.68 (m, 4H), 3.04 - 3.02 (m, 4H), 2.98 (s, 1H), 2.31 (d, J = 7.3 Hz, 3H), 2.26 (d, J = 7.3 Hz, 2H), 2.13 (t, J = 13.7 Hz, 1H), 2.00 (d, J = 13.6 Hz, 1H), 1.87 (d, J = 16.8 Hz, 1H), 1.78 (t, J = 15.6 Hz, 1H), 1.73 - 1.66 (m, 3H), 1.61 (dt, J = 15.5, 7.7 Hz, 6H), 1.47 (dt, J = 9.7, 6.4 Hz, 2H), 1.40 (s, 1H), 1.38 (s, 3H), 1.35 (s, 3H), 1.34 - 1.31 (m, 2H), 1.24 (d, J = 5.7 Hz, 1H), 1.20 (s, 3H), 1.18 (s, 1H), 1.14 (s, 3H), 1.09 (s, 3H), 1.07 (s, 3H), 1.01 (d, J = 5.9 Hz, 1H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 198.64 (C-3), 198.60 (C-11), 174.88 (C=O), 173.53 (C-30), 172.87 (C-13), 171.84 (C=O), 171.08 (C-1), 160.93 (Ph-C), 147.45 (Ph-C), 144.67 (Ph-C), 144.60 (Ph-C), 132.57 (Ph-C), 127.56 (Ph-C), 127.49 (Ph-C), 127.38 (C-12), 120.62 (Ph-C), 120.08 (Ph-C), 116.70 (Ph-C), 115.97 (-CN), 112.08 (C-2), 102.40 (Ph-C), 101.84 (Ph-C), 53.78, 50.60, 49.90, 48.36, 45.48, 45.07, 43.98, 43.86, 40.53, 37.89, 36.65, 36.00, 34.67, 32.31, 31.89, 31.34, 28.81, 28.79, 28.42, 27.49, 26.81, 26.66, 26.59, 26.51, 25.49, 23.42, 22.97, 21.74, 19.74, 18.98, 18.48, 17.90. HRMS (ESI): calcd for C 54 H 69 FN6O5[M+Na] + 923.5206, found: 923.5244.

[0040] Example 5 N 1 -(2-aminophenyl)- N 8 -(4-(4-(2-cyano-3,11-dioxo-18 β Preparation of (4-(4-(2-cyano-3,11-dioxo-18 The compound of Example 5 was prepared according to the preparation method of Example 1, using raw material suberic acid instead of raw material pimelic acid in Example 1, and using raw material tert-butyl 2-amino-4-fluorophenylcarbamate instead of raw material tert-butyl 2-aminophenylcarbamate in Example 1. Yield 23%. 1 H NMR (600 MHz, DMSO- d6) δ 9.66 (s, 1H), 9.00 (s,1H), 8.49 (s, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.11 – 7.06 (m, 1H), 6.89 (d, J= 8.7 Hz, 2H), 6.48 (d, J = 11.1 Hz, 1H), 6.29 (t, J = 8.5 Hz, 1H), 5.64 (s,1H), 5.12 (s, 2H), 3.72 – 3.67 (m, 4H), 3.03 (d, J = 10.1 Hz, 4H), 2.98 (s,1H), 2.34 – 2.23 (m, 5H), 2.14 (t, J = 13.8 Hz, 1H), 2.00 (d, J = 20.1 Hz,1H), 1.87 (d, J = 10.5 Hz, 1H), 1.81 – 1.74 (m, 1H), 1.73 – 1.66 (m, 3H),1.62 – 1.54 (m, 6H), 1.51 – 1.44 (m, 2H), 1.40 (s, 1H), 1.38 (s, 3H), 1.35(s, 3H), 1.35 – 1.30 (m, 5H), 1.23 (s, 1H), 1.20 (s, 3H), 1.18 (s, 1H), 1.14(s, 3H), 1.09 (s, 3H), 1.07 (s, 3H), 1.02 – 0.96 (m, 1H), 0.75 (s, 3H). 13 C NMR(151 MHz, DMSO- d6) δ 198.62 (C-3), 198.59 (C-11), 174.88 (C=O), 173.51 (C-30), 172.86 (C-13), 171.85 (C=O), 171.09 (C-1), 160.92 (Ph-C), 147.17 (Ph-C), 144.73 (Ph-C), 144.66 (Ph-C), 132.53 (Ph-C), 127.55 (Ph-C), 127.48 (Ph-C), 127.37 (C-12), 120.61 (Ph-C), 120.06 (Ph-C), 116.66 (Ph-C), 115.96 (-CN), 112.08 (C-2), 102.35 (Ph-C), 101.81 (Ph-C), 53.78, 50.60, 49.87, 48.35, 45.47, 45.11, 45.07, 43.98, 43.85, 40.53, 37.89, 36.75, 36.11, 32.30, 31.89, 31.34, 29.50, 29.05, 29.03, 28.79, 27.49, 27.02, 26.81, 26.66, 26.58, 26.50, 25.62, 22.97, 22.57, 21.74, 19.75, 18.98, 17.90. HRMS (ESI): calcd for C 55 H 71 FN6O5[M+Na] + 937.5362, found: 937.5404.

[0041] Example 6 N 1 -(2-aminophenyl)- N 9 -(4-(4-(2-cyano-3,11-dioxo-18 β Preparation of (4-(4-(2-cyano-3,11-dioxo-18 The compound of Example 6 was prepared according to the preparation method of Example 1, using the starting material azelaic acid instead of the starting material of Example 1 suberic acid, and using the starting material tert-butyl 2-amino-4-fluorophenylcarbamate instead of the starting material of Example 1 tert-butyl 2-aminophenylcarbamate. Yield 19%. 1 H NMR (600 MHz, DMSO- d6) δ 9.66 (s, 1H), 9.01 (s,1H), 8.49 (s, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.11 - 7.06 (m, 1H), 6.88 (d, J= 9.1 Hz, 2H), 6.47 (d, J = 8.2 Hz, 1H), 6.29 (t, J = 8.5 Hz, 1H), 5.64 (s,1H), 5.12 (s, 2H), 3.71 - 3.68 (m, 4H), 3.04 - 3.01 (m, 4H), 2.98 (s, 1H),2.29 (t, J = 7.5 Hz, 3H), 2.24 (d, J = 7.3 Hz, 2H), 2.18 - 2.10 (m, 1H), 2.00(dd, J = 13.8, 5.3 Hz, 1H), 1.87 (d, J = 13.5 Hz, 1H), 1.78 (d, J = 8.9 Hz,1H), 1.77 - 1.64 (m, 3H), 1.59 - 1.56 (m, 6H), 1.52 - 1.44 (m, 2H), 1.42 (s,1H), 1.38 (s, 3H), 1.35 (s, 3H), 1.33 - 1.29 (m, 6H), 1.23 (s, 1H), 1.20 (s,3H), 1.18 - 1.16 (m, 1H), 1.14 (s, 3H), 1.09 (s, 3H), 1.07 (s, 3H), 1.01 (s,1H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 197.54 (C-3), 197.51 (C-11), 173.80 (C=0), 172.43 (C-30), 171.78 (C-13), 170.79 (C=0), 170.04 (C-1), 159.82 (Ph-C), 146.08 (Ph-C), 143.61 (Ph-C), 143.53 (Ph-C), 131.46 (Ph-C), 126.41 (Ph-C), 126.33 (C-12), 119.53 (Ph-C), 119.01 (Ph-C), 115.58 (Ph-C), 114.88 (-CN), 111.00 (C-2), 101.26 (Ph-C), 100.74 (Ph-C), 52.70, 49.52, 48.79, 47.26, 44.39, 43.99, 42.89, 42.77, 39.45, 36.81, 35.68, 35.04, 33.72, 30.81, 30.26, 29.92, 28.63, 28.59, 28.03, 28.00, 27.71, 26.41, 25.94, 25.73, 25.57, 25.50, 25.42, 24.60, 21.92, 21.89, 20.66, 18.67, 17.89, 16.82. HRMS (ESI): calcd for C 56 H 73 FN6O5[M+Na] + 951.5519, found: 951.5562.

[0042] Example 7 N 1 -aminophenyl- N 7 -(4-(4-(2-trifluoromethyl-3,11-dioxo-18 β Preparation of 1 -(4-nitrophenyl)-4-(2-iodo-3,11 -dioxo-18β-olean-1,12-dien-30- yl)piperazin-1 -yl)heptanediamide (Example 7): Step d: Preparation of 1 -(4-nitrophenyl)-4-(2-iodo-3,11 -dioxo-18β-olean-1,12-dien-30- yl)piperazin-1 -yl)heptanediamide (Example 7):

[0043] Obtained according to the preparation method of Step b of Example 1, using 2-iodo-3, 11-dioxo-18β-olean-1, 12-diene-30-carboxylic acid as the starting material instead of 2-cyano-3, 11-dioxo-18β-olean-1, 12-diene-30-carboxylic acid in Step d of Example 1, to give 1-(4-nitrophenyl)-4-(2-iodo-3, 11-dioxo-18β-olean-1, 12-diene-30- yl)piperazine. Yield 79%.

[0044] Step h: Preparation of 1-(4-nitrophenyl)-4-(2-trifluoromethyl-3, 11-dioxo-18β- olean-1, 12-diene-30-yl)piperazine

[0045] Obtained according to the preparation method of Step b of Example 1, using 2-iodo-3, 11-dioxo-18β-olean-1, 12-diene-30-carboxylic acid as the starting material instead of 2-cyano-3, 11-dioxo-18β-olean-1, 12-diene-30-carboxylic acid in Step d of Example 1, to give 1-(4-nitrophenyl)-4-(2-iodo-3, 11-dioxo-18β-olean-1, 12-diene-30- yl)piperazine. Yield 79%.

[0046] Step e: 1-(4-aminophenyl)-4-(2-trifluoromethyl-3, 11-dioxo-18β-olean-1, 12-diene-30- yl)piperazine

[0047] Obtained according to the preparation method of Step b of Example 1, using 2-iodo-3, 11-dioxo-18β-olean-1, 12-diene-30-carboxylic acid as the starting material instead of 2-cyano-3, 11-dioxo-18β-olean-1, 12-diene-30-carboxylic acid in Step d of Example 1, to give 1-(4-nitrophenyl)-4-(2-iodo-3, 11-dioxo-18β-olean-1, 12-diene-30- yl)piperazine. Yield 79%.

[0048] Step f, g: N 1 - amino phenyl - N 7 - (4-(4-(2-trifluoromethyl-3, 11-dioxo-18β-olean-1, 12-dien-30- yl)piperazin-1-yl)phenyl)heptanediamide β Preparation of 1-(4-aminophenyl)-4-(2-trifluoromethyl-3, 11-dioxo-18β- olean-1, 12-dien-30-yl)piperazine

[0049] The compound of Example 7 was prepared according to the procedure of Example 1, Steps f, g, substituting 1-(4-aminophenyl)-4-(2-trifluoromethyl-3, 11-dioxo-18β- olean-1, 12-dien-30-yl)piperazine for the starting material 1-(4-aminophenyl)-4-(2- cyano-3, 11-dioxo-18β-olean-1, 12-dien-30-yl)piperazine in Example 1, Steps f, g. Yield 29%. 1 H NMR (600 MHz, DMSO- d6) δ 9.67 (s, 1H), 9.08 (s, 1H), 8.23 (s, 1H), 7.46 (d, J = 9.1 Hz, 2H), 7.15 (d, J = 6.4 Hz, 1H), 6.91 - 6.84 (m, 3H), 6.71 (d, J = 9.4 Hz, 1H), 6.52 (t, J = 7.5 Hz, 1H), 5.65 (s, 1H), 4.80 (s, 2H), 3.74 - 3.67 (m, 4H), 3.05 - 3.02 (m, 4H), 2.97 (s, 1H), 2.32 (t, J = 7.4 Hz, 3H), 2.27 (t, J = 7.4 Hz, 2H), 2.14 (t, J = 15.9 Hz, 1H), 2.03 - 1.95 (m, 1H), 1.90 (d, J = 16.8 Hz, 1H), 1.83 - 1.74 (m, 1H), 1.73 - 1.66 (m, 3H), 1.66 - 1.56 (m, 6H), 1.47 (t, J = 9.4 Hz, 2H), 1.39 (s, 3H), 1.37 (s, 1H), 1.37 - 1.32 (m, 2H), 1.31 (s, 3H), 1.24 (s, 1H), 1.20 (s, 3H), 1.19 - 1.14 (m, 1H), 1.10 (s, 3H), 1.10 (s, 3H), 1.08 (s, 3H), 1.03 - 0.96 (m, 1H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 199.18 (C-3), 198.71 (C-11), 173.51 (C=O), 172.85 (C-30), 171.58 (C-13), 171.06 (C=O), 164.28 (C-1), 147.16 (Ph-C), 142.31 (Ph-C), 132.54 (Ph-C), 127.46 (C-12), 126.12 (Ph-C), 125.73 (Ph-C), 124.17 (Ph-C), 124.07 (Ph-C), 123.99 (-CF3), 121.92 (C-2), 120.60 (2C, 2´ Ph-C), 116.64 (2C, 2´ Ph-C), 116.36 (Ph-C), 54.08, 50.55, 49.86, 48.35, 45.35, 45.33, 43.97, 43.85, 40.53, 38.25, 37.91, 36.67, 36.13, 34.67, 34.40, 31.88, 31.44, 31.24, 28.82, 28.05, 26.81, 26.67, 26.61, 26.54, 25.59, 25.51, 22.98, 22.54, 21.63, 20.16, 18.93, 18.18. HRMS (ESI): calcd for C 54 H 70 F3N5O5[M+Na] + 948.5221, found: 948.5262.

[0050] Example 8 N 1 -aminophenyl- N 8 -(4-(4-(2-trifluoromethyl-3,11-dioxo-18 β Preparation of (4-(4-(2-trifluoromethyl-3,11-dioxo-18 The compound of Example 8 was prepared according to the procedure of Example 7, using octanedioic acid as starting material instead of heptanedioic acid. Yield 29%. 1 H NMR (600 MHz, DMSO- d6) δ 9.66 (s, 1H), 9.08 (s, 1H), 8.22 (s, 1H), 7.46 (d, J = 9.1 Hz, 2H), 7.15 (d, J = 6.3 Hz, 1H), 6.91 - 6.85 (m, 3H), 6.70 (d, J = 6.4 Hz, 1H), 6.53 (t, J = 7.5 Hz, 1H), 5.64 (s, 1H), 4.80 (s, 2H), 3.74 - 3.68 (m, 4H), 3.05 - 3.02 (m, 4H), 2.97 (s, 1H), 2.34 - 2.28 (m, 3H), 2.27 - 2.23 (m, 2H), 2.18 - 2.10 (m, 1H), 2.03 - 1.98 (m, 1H), 1.92 - 1.86 (m, 1H), 1.82 - 1.75 (m, 1H), 1.73 - 1.64 (m, 3H), 1.61 - 1.57 (m, 6H), 1.51 - 1.44 (m, 1H), 1.42 (s, 1H), 1.39 (s, 3H), 1.37 - 1.33 (m, 5H), 1.31 (s, 3H), 1.24 (d, J = 5.3 Hz, 1H), 1.20 (s, 3H), 1.18 (s, 1H), 1.10 (s, 3H), 1.10 (s, 3H), 1.08 (s, 3H), 1.02 - 0.94 (m, 1H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 198.05 (C-3), 197.57 (C-11), 172.37 (C=O), 171.71 (C-30), 170.47(C-13), 169.95 (C=O), 163.15 (C-1), 146.01 (Ph-C), 141.17 (Ph-C), 131.40 (Ph-C), 126.32 (C-12), 124.98 (Ph-C), 124.58 (Ph-C), 123.03 (Ph-C), 122.95 (Ph-C), 122.84 (-CF3), 120.78 (C-2), 119.46 (2C, 2´ Ph-C), 115.50 (2C, 2´ Ph-C),115.22 (Ph-C), 52.94, 49.41, 48.72, 47.20, 44.94, 44.21, 44.19, 42.83, 42.73,42.70, 39.39, 37.11, 36.77, 35.62, 35.08, 31.18, 30.74, 30.09, 27.87, 27.86,27.68, 26.91, 25.53, 25.47, 25.40, 24.57, 24.50, 21.84, 20.49, 19.02, 17.89,17.79, 17.04. HRMS (ESI): calcd for C 55 H 72 F3N5O5[M-H] - 938.5408, found: 938.5408.

[0051] Example 9 N 1 -aminophenyl- N 9 -(4-(4-(2-trifluoromethyl-3,11-dioxo-18 β Preparation of (4-(4-(2-trifluoromethyl-3,11-dioxo-18 The compound of Example 9 was prepared according to the preparation procedure of Example 7, using sebacic acid as starting material instead of heptanedioic acid. Yield 26%. 1 H NMR (600 MHz, DMSO- d6) δ 9.65 (s, 1H), 9.08 (s, 1H), 8.23 (s, 1H), 7.46 (d, J = 8.9 Hz, 2H), 7.14 (d, J = 6.4 Hz, 1H), 6.91 - 6.85 (m, 3H), 6.70 (d, J = 6.5 Hz, 1H), 6.53 (t, J = 6.8 Hz, 1H), 5.64 (s, 1H), 4.80 (s, 2H), 3.70 (d, J = 5.4 Hz, 4H), 3.05 - 3.02 (m, 4H), 2.97 (s, 1H), 2.33 - 2.27 (m, 3H), 2.25 (t, J = 7.4 Hz, 2H), 2.19 - 2.10 (m, 1H), 2.00 (d, J = 13.0 Hz, 1H), 1.89 (d, J = 12.7 Hz, 1H), 1.83 - 1.75 (m, 1H), 1.74 - 1.64 (m, 3H), 1.58 (s, 6H), 1.47 (d, J = 8.3 Hz, 1H), 1.42 (d, J = 3.1 Hz, 1H), 1.39 (s, 3H), 1.36 - 1.33 (m, 1H), 1.31 (s, 9H), 1.24 (d, J = 6.0 Hz, 1H), 1.20 (s, 3H), 1.18 (s, 1H), 1.10 (s, 3H), 1.10 (s, 3H), 1.08 (s, 3H), 1.03 - 0.97 (m, 1H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 199.20 (C-3), 198.73 (C-11), 173.52 (C=O), 172.86 (C-30), 171.63 (C-13), 171.12 (C=O), 164.32 (C-1), 147.17 (Ph-C), 142.34 (Ph-C), 132.52 (Ph-C), 127.46 (C-12), 126.13 (Ph-C), 125.72 (Ph-C), 124.16 (Ph-C), 124.08 (Ph-C), 123.74 (-CF3), 121.92 (C-2), 120.61 (2C, 2´ Ph-C), 116.64 (2C, 2´ Ph-C), 116.36 (Ph-C), 54.09, 50.55, 49.86, 48.36, 46.13, 45.36, 45.33, 43.98, 43.86, 40.54, 38.25, 37.91, 36.77, 36.24, 34.67, 31.89, 31.43, 31.24, 29.09, 29.06, 28.82, 28.05, 26.81, 26.68, 26.61, 26.55, 25.77, 25.68, 22.98, 22.53, 21.64, 20.16, 18.93, 18.18. HRMS (ESI): calcd for C 56 H 74 F3N5O5[M+Na] + 976.5534, found: 976.5607.

[0052] Example 10 N 1 -(2-aminophenyl)- N 7 -(4-(4-(2-trifluoromethyl-3,11-dioxo-18 β Preparation of 4-(4-(2-trifluoromethyl-3,11-dioxo-18 The compound of Example 10 was prepared according to the procedure of Example 7, using 2-amino-4-fluorophenylcarbamic acid tert-butyl ester as starting material instead of 2-aminophenylcarbamic acid tert-butyl ester. Yield 26%. 1 H NMR (600 MHz, DMSO- d6) δ 9.66 (s, 1H), 9.00 (s, 1H), 8.22 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.11 - 7.05 (m, 1H), 6.89 (d, J = 8.6 Hz, 2H), 6.47 (d, J = 8.2 Hz, 1H), 6.28 (t, J = 10.0 Hz, 1H), 5.64 (s, 1H), 5.12 (s, 2H), 3.72 - 3.69 (m, 4H), 3.05 - 3.02 (m, 4H), 2.97 (s, 1H), 2.36 - 2.29 (m, 3H), 2.27 (s, 2H), 2.18 - 2.10 (m, 1H), 2.01 - 1.98 (m, 1H), 1.89 (d, J = 13.5 Hz, 1H), 1.78 (dt, J = 14.1, 7.4 Hz, 1H), 1.69 (q, J = 6.3 Hz, 3H), 1.61 (dt, J = 15.3, 7.4 Hz, 6H), 1.51 - 1.45 (m, 2H), 1.42 (s, 1H), 1.39 (s, 3H), 1.34 (d, J = 6.4 Hz, 2H), 1.31 (s, 3H), 1.23 (s, 2H), 1.20 (s, 3H), 1.12 - 1.05 (m, 9H), 1.02 - 0.97 (m, 1H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 199.19 (C-3), 198.73 (C-11), 173.52(C=O), 172.86 (C-30), 171.83 (C-13), 171.06 (C=O), 164.33 (C-1), 160.13 (Ph-C), 147.18 (Ph-C), 144.73 (Ph-C), 144.66 (Ph-C), 138.25 (C-2), 132.51 (Ph-C), 127.55 (Ph-C), 127.48 (Ph-C), 127.45 (C-12), 123.73 (-CF3), 120.61 (Ph-C), 120.04 (Ph-C), 116.65 (Ph-C), 102.41 (Ph-C), 101.79 (Ph-C), 54.09, 50.55, 49.86, 48.37, 45.36, 45.33, 43.98, 43.86, 40.53, 38.25, 37.91, 36.65, 36.01, 35.34, 31.89, 31.24, 28.82, 28.06, 26.81, 26.68, 26.61, 26.55, 26.30, 26.24, 25.50, 22.98, 22.57, 21.64, 20.79, 20.16, 18.93, 18.18. HRMS (ESI): calcd for C 54 H 69 F4N5O5[M+Na] + 966.5127, found: 966.5220.

[0053] Example 11 N 1 -(2-aminophenyl)- N 8 -(4-(4-(2-trifluoromethyl-3,11-dioxo-18 β Preparation of (4-(4-(2-trifluoromethyl-3,11-dioxo-18 The compound of Example 11 was prepared according to the preparation procedure of Example 7, using suberic acid as starting material instead of pimelic acid, and using 2-amino-4-fluorophenylcarbamic acid tert-butyl ester as starting material instead of 2-aminophenylcarbamic acid tert-butyl ester. Yield 19%. 1 H NMR (600 MHz, DMSO- d6) δ 9.66 (s, 1H), 9.01 (s, 1H), 8.22 (s, 1H), 7.46(d, J = 8.6 Hz, 2H), 7.11 – 7.06 (m, 1H), 6.89 (d, J = 9.2 Hz, 2H), 6.47 (d,J = 11.2 Hz, 1H), 6.29 (t, J = 8.5 Hz, 1H), 5.64 (s, 1H), 5.12 (s, 2H), 3.72– 3.68 (m, 4H), 3.05 – 3.02 (m, 4H), 2.97 (s, 1H), 2.34 – 2.23 (m, 5H), 2.14(t, J = 13.7 Hz, 1H), 2.03 – 1.97 (m, 1H), 1.89 (d, J = 13.1 Hz, 1H), 1.83 –1.74 (m, 1H), 1.69 (d, J = 14.7 Hz, 3H), 1.58 (t, J = 7.1 Hz, 6H), 1.48 (s,1H), 1.42 (s, 1H), 1.39 (s, 3H), 1.36 (s, 1H), 1.33 (s, 4H), 1.31 (s, 3H),1.28 (s, 1H), 1.20 (s, 3H), 1.14 (d, J = 6.4 Hz, 1H), 1.12 – 1.04 (m, 9H),1.04 – 0.96 (m, 1H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 199.20 (C-3), 198.73 (C-11), 173.52 (C=0), 172.86 (C-30), 171.83 (C-13), 171.10 (C=0), 164.31 (C-1), 160.92 (Ph-C), 160.13 (Ph-C), 147.17 (Ph-C), 144.73 (Ph-C), 144.65 (Ph-C), 132.52 (Ph-C), 127.54 (Ph-C), 127.47 (C-2), 124.16 (-CF3), 121.91 (C-12), 120.61 (Ph-C), 120.07 (Ph-C), 116.65 (Ph-C), 102.35 (Ph-C), 101.82 (Ph-C), 54.08, 50.66, 50.55, 49.86, 49.76, 48.36, 45.35, 45.33, 43.98, 43.85, 40.53, 38.25, 37.91, 36.75, 36.11, 32.35, 31.89, 31.24, 29.00, 28.82, 28.12, 28.05, 26.68, 26.61, 26.54, 25.62, 22.98, 21.63, 20.72, 20.54, 20.16, 18.93, 18.18. HRMS (ESI): calcd for C 55 H 71 F4N5O5[M+Na] + 980.5284, found:980.5345.

[0054] Example 12 N 1 -(2-aminophenyl)- N 9 -(4-(4-(2-trifluoromethyl-3,11-dioxo-18 β Preparation of (4-(4-(2-trifluoromethyl-3,11-dioxo-18 The compound of Example 12 was prepared according to the preparation procedure of Example 7, using azelaic acid as the starting material instead of pimelic acid, and using 2-amino-4-fluorophenylcarbamic acid tert-butyl ester as the starting material instead of 2-aminophenylcarbamic acid tert-butyl ester. Yield 20%. 1 H NMR (600 MHz, DMSO- d6) δ 9.65 (s, 1H), 9.00 (s, 1H), 8.22 (s, 1H), 7.45(d, J = 8.6 Hz, 2H), 7.11 – 7.05 (m, 1H), 6.88 (d, J = 8.6 Hz, 2H), 6.47 (d,J = 11.2 Hz, 1H), 6.29 (t, J = 10.0 Hz, 1H), 5.64 (s, 1H), 5.11 (s, 2H), 3.72– 3.69 (m, 4H), 3.05 – 3.02 (m, 4H), 2.97 (s, 1H), 2.34 – 2.22 (m, 5H), 2.14(t, J = 13.6 Hz, 1H), 2.00 (d, J = 10.5 Hz, 1H), 1.89 (d, J = 13.7 Hz, 1H),1.84 – 1.75 (m, 1H), 1.74 – 1.64 (m, 3H), 1.62 – 1.54 (m, 6H), 1.47 (d, J =13.3 Hz, 1H), 1.42 (s, 1H), 1.39 (s, 3H), 1.35 (s, 1H), 1.31 (s, 6H), 1.29(s, 3H), 1.23 (s, 1H), 1.20 (s, 3H), 1.15 (s, 1H), 1.12 – 1.04 (m, 9H), 1.02– 0.97 (m, 1H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 198.63 (C-3), 198.59(C-11), 174.88 (C=O), 173.52 (C-30), 172.86 (C-13), 171.87 (C=O), 171.12 (C-1), 160.92 (Ph-C), 147.17 (Ph-C), 144.72 (Ph-C), 144.64 (Ph-C), 132.52 (Ph-C), 127.52 (Ph-C), 127.46 (C-2), 127.38 (-CF3), 120.62 (C-12), 120.08 (Ph-C), 116.66 (Ph-C), 115.96 (Ph-C), 112.08 (Ph-C), 102.36 (Ph-C), 101.82 (Ph-C), 55.39, 53.78, 50.60, 49.86, 49.71, 48.35, 45.48, 45.07, 43.98, 43.85, 40.53, 37.89, 36.76, 36.12, 32.31, 31.89, 31.34, 29.08, 28.79, 27.49, 26.66, 26.59, 26.50, 25.68, 22.97, 21.74, 21.46, 20.73, 20.24, 20.07, 19.74, 18.98, 18.85, 17.90. HRMS (ESI): calcd for C 56 H 73 F4N5O5[M-H] - 970.5470, found: 970.5390.

[0055] Example 13 N 1 -aminophenyl- N 7 -(4-(4-(3 β -hydroxy-11-oxo-18 β Preparation of 1-(4-nitrophenyl)-4-(3β-hydroxy-11-oxo-18β-olean-12-en-30- yl)piperazin-1-yl)heptanediamide (Example 13): Step d: Preparation of 1-(4-nitrophenyl)-4-(3β-hydroxy-11-oxo-18β-olean-12-en-30- yl)piperazin-1-yl)heptanediamide

[0056] Prepared according to the procedure of Example 1, Step d, substituting starting material GA for the starting material 2-cyano-3,11-dioxo-18β-olean- 1,12-diene-30-carboxylic acid in Example 1, Step d, to give 1-(4-nitrophenyl)- 4-(3β-hydroxy-11-oxo-18β-olean-12-en-30-oyl)piperazine. Yield 69%.

[0057] Step e: Preparation of 1-(4-aminophenyl)-4-(3β-hydroxy-11-oxo-18β- olean-12-en-30-oyl)piperazine

[0058] Prepared according to the procedure of Example 1, Step e, substituting starting material 1-(4-nitrophenyl)-4-(3β-hydroxy-11-oxo-18β-olean-12-en-30- oyl)piperazine for the starting material 1-(4-nitrophenyl)-4-(2-cyano-3,11-dioxo- 18β-olean-1,12-diene-30-oyl)piperazine in Example 1, Step e, to give 1-(4- aminophenyl)-4-(3β-hydroxy-11-oxo-18β-olean-12-en-30-oyl)piperazine. Yield 70%.

[0059] Steps f, g: N 1 -aminophenyl- N 7 -(4-(4-(3 β -hydroxy-11-oxo-18 β -olean-12-en-30-oyl)piperazin-1-yl)phenyl)heptanediamide

[0060] Prepared according to the procedure of Example 1, Steps f, g, substituting starting material 1-(4-aminophenyl)-4-(3β-hydroxy-11-oxo-18β-olean-12-en-30- oyl)piperazine for the starting material 1-(4-aminophenyl)-4-(2-cyano-3,11-dioxo- 18β-olean-1,12-diene-30-oyl)piperazine in Example 1, to give the compound of Example 13. Yield 21%. 1 HNMR (600 MHz, DMSO- d6) δ 9.67 (s, 1H), 9.10 (s, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 7.9 Hz, 1H), 6.92 - 6.85 (m, 3H), 6.72 (d, J = 7.8 Hz, 1H), 6.54 (t, J = 7.9 Hz, 1H), 5.49 (s, 1H), 4.29 (s, 2H), 3.71 - 3.66 (m, 4H), 3.04 - 3.00 (m, 5H), 2.57 (d, J = 13.3 Hz, 1H), 2.34 - 2.29 (m, 3H), 2.29 - 2.23 (m, 2H), 2.21 (d, J = 11.7 Hz, 1H), 2.12 (t, J = 13.8 Hz, 1H), 1.99 (d, J = 9.5 Hz, 1H), 1.87 (d, J = 14.3 Hz, 1H), 1.78 - 1.73 (m, 1H), 1.68 (s, 1H), 1.64 - 1.59 (m, 4H), 1.55 - 1.48 (m, 2H), 1.40 (s, 1H), 1.35 (s, 6H), 1.34 - 1.29 (m, 2H), 1.24 (t, J = 5.1 Hz, 1H), 1.19 (s, 3H), 1.14 (dt, J = 5.1, 3.1 Hz, 1H), 1.02 (s, 6H), 0.97 (s, 1H), 0.95 (s, 1H), 0.91 (s, 3H), 0.89 - 0.78 (m, 3H), 0.73 (s, 3H), 0.69 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 199.57 (C-11), 173.52 (C-30), 171.59 (C=0), 171.06 (C=0), 170.43 (C-13), 147.17 (Ph-C), 142.34 (Ph-C), 132.52 (Ph-C), 127.93 (C-12), 126.13 (Ph-C), 125.74 (Ph-C), 124.05 (Ph-C), 120.60 (2C, 2' Ph-C), 116.65 (2C, 2' Ph-C), 116.63 (Ph-C), 116.34 (Ph-C), 77.06, 61.57, 54.59 (2C), 49.87, 48.23, 45.22 (2C), 45.21, 45.13, 43.81, 43.74, 43.45, 40.52, 39.24, 38.98, 37.93, 37.14, 36.67, 36.13, 32.66, 31.88, 28.83, 28.75, 28.61, 27.44, 26.70, 26.48, 25.60, 25.51, 23.15, 18.82, 17.62, 16.67, 16.47. HRMS (ESI): calcd for C 53 H 75 N5O5[M+Na] + 884.5660, found: 884.5734.

[0061] Example 14 N 1 -aminophenyl- N 8 -(4-(4-(3 β -hydroxy-11-oxo-18 β Preparation of (4-(4-(3,4-dihydroxy-2-hydroxymethylcyclohexyl)- 1 -oxo- 1 - (piperazin- 1 -ylcarbonyl)phenyl)octanedioic acid (Example 14) Example 14 was prepared according to the procedure of Example 13, using octanedioic acid as starting material instead of heptanedioic acid. Yield 24%. 1 H NMR (600 MHz, DMSO- d6) δ 9.66 (s, 1H), 9.09 (s, 1H), 7.46 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 7.2 Hz, 1H), 6.87 (d, J = 7.8 Hz, 3H), 6.71 (d, J = 7.9 Hz, 1H), 6.53 (t, J = 7.5 Hz, 1H), 5.49 (s, 1H), 4.81 (s, 2H), 4.29 (d, J = 5.0 Hz, 1H), 3.68 (s, 4H), 3.03 - 3.00 (m, 5H), 2.58 (d, J = 12.9 Hz, 1H), 2.34 - 2.30 (m, 3H), 2.28 - 2.23 (m, 2H), 2.21 (d, J = 13.3 Hz, 1H), 2.11 (t, J = 13.9 Hz, 1H), 1.98 (d, J = 12.9 Hz, 1H), 1.87 (d, J = 13.4 Hz, 1H), 1.78 - 1.70 (m, 1H), 1.67 (t, J = 13.5 Hz, 1H), 1.61 - 1.58 (m, 4H), 1.54 - 1.49 (m, 2H), 1.39 (s, 1H), 1.35 (s, 8H), 1.24 (s, 2H), 1.19 (s, 3H), 1.16 (s, 1H), 1.13 (s, 1H), 1.02 (s, 6H), 0.97 (s, 1H), 0.96 - 0.93 (m, 1H), 0.91 (s, 3H), 0.85 (t, J = 11.7 Hz, 3H), 0.73 (s, 3H), 0.69 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 199.57 (C-11), 173.52 (C-30), 171.61 (C=O), 171.09 (C=O), 170.43(C-13), 147.17 (Ph-C), 142.34 (Ph-C), 132.53 (Ph-C), 127.93 (C-12), 126.14(Ph-C), 125.74 (Ph-C), 124.07 (Ph-C), 120.59 (2C, 2´ Ph-C), 116.36 (2C, 2´Ph-C), 116.64 (Ph-C), 116.36 (Ph-C), 77.06, 61.57, 54.59 (2C), 49.87, 48.23,45.22 (2C), 45.11, 43.81, 43.74, 43.45, 40.53, 39.24, 38.99, 37.93, 37.14,36.76, 36.23, 32.66, 32.59, 31.88, 29.02, 29.00, 28.75, 28.61, 27.44, 26.70,26.48, 25.71, 25.64, 23.15, 18.82, 17.61, 16.67, 16.47. HRMS (ESI): calcd for C 54 H 77 N5O5[M+Na] + 898.5817, found: 898.5881.

[0062] Example 15 N 1 -aminophenyl- N 9 -(4-(4-(3 β -hydroxy-11-oxo-18 β Preparation of (4-(4-(3 The compound of Example 15 was prepared according to the procedure of Example 13, using sebacic acid as starting material instead of heptanedioic acid. Yield 23%. 1 H NMR (600 MHz, DMSO- d6) δ 9.65 (s, 1H), 9.08 (s, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 7.9 Hz, 1H), 6.89 - 6.85 (m, 3H), 6.70 (d, J = 7.8 Hz, 1H), 6.53 (t, J = 7.6 Hz, 1H), 5.49 (s, 1H), 4.80 (s, 2H), 4.29 (d, J = 5.1 Hz, 1H), 3.70 - 3.67 (m, 4H), 3.03 - 3.00 (m, 5H), 2.57 (d, J = 13.6 Hz, 1H), 2.34 - 2.28 (m, 3H), 2.27 - 2.23 (m, 2H), 2.21 - 2.18 (m, 1H), 2.12 (t, J = 14.4 Hz, 1H), 1.98 (d, J = 12.7 Hz, 1H), 1.87 (d, J = 13.4 Hz, 1H), 1.76 - 1.72 (m, 1H), 1.68 (s, 1H), 1.59 - 1.56 (m, 4H), 1.54 - 1.49 (m, 2H), 1.44 (d, J = 3.2 Hz, 1H), 1.35 (s, 6H), 1.32 (s, 4H), 1.23 (s, 1H), 1.19 (s, 3H), 1.15 - 1.12 (m, 3H), 1.02 (s, 6H), 0.97 (s, 1H), 0.95 - 0.93 (m, 1H), 0.91 (s, 3H), 0.85 (q, J = 8.1, 7.6 Hz, 3H), 0.73 (s, 3H), 0.69 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 199.51 (C-11), 173.48 (C-30), 171.57 (C=O), 171.04 (C=O), 170.37 (C-13), 147.12 (Ph-C), 142.29 (Ph-C), 132.48 (Ph-C), 127.88 (C-12), 126.08 (Ph-C), 125.69 (Ph-C), 124.03 (Ph-C), 120.56 (2C, 2´ Ph-C), 116.60 (2C, 2´ Ph-C), 116.59 (Ph-C), 116.31 (Ph-C),77.02, 61.53, 54.55,49.83, 48.19, 46.09, 45.18, 43.78, 43.41, 40.50, 39.21, 38.95, 37.89, 37.11,36.73, 36.19, 32.62, 31.85, 31.39, 28.97, 28.71, 28.57, 27.41, 26.78, 26.66,26.45, 25.70, 25.68, 25.60, 25.22, 23.12, 22.93, 22.50, 18.79, 17.58, 16.63,16.43. HRMS (ESI): calcd for C 55 H 79 N5O5[M+Na] + 912.5973, found: 912.6060.

[0063] Example 16 N 1 -(2-amino-4-fluorophenyl)- N 7 -(4-(4-(3 β -hydroxy-11-oxo-18 β Preparation of oleanol-12-en-30-acyl)piperazine-1-yl)phenyl)heptanediamide (Example 16) Following the preparation method of Example 13, the compound of Example 16 was prepared by replacing tert-butyl 2-aminophenylcarbamate with tert-butyl 2-amino-4-fluorophenylcarbamate. Yield: 17%. 1 H NMR (600 MHz, DMSO- d6) δ 9.65 (s, 1H), 9.00 (s, 1H), 7.45 (d, J = 9.0 Hz, 2H), 7.08 (dd, J = 8.7, 6.3 Hz, 1H), 6.88 (d, J = 9.1 Hz, 2H), 6.47 (dd, J = 11.1, 3.0 Hz, 1H), 6.29 (td, J = 8.5, 2.9 Hz, 1H), 5.49 (s, 1H), 5.11 (s, 2H), 4.30 (d, J = 5.1 Hz, 1H), 3.69 (s, 4H), 3.02 (s, 5H), 2.63 - 2.54 (m, 1H), 2.31 - 2.20 (m, 5H), 2.12 (d, J = 31.9 Hz, 1H), 1.99 (d, J = 12.5 Hz, 1H), 1.90 - 1.85 (m, 1H), 1.78 - 1.71 (m, 1H), 1.68 (t, J = 13.5 Hz, 1H), 1.58 (t, J = 7.1 Hz, 5H), 1.51 (d, J = 3.1 Hz, 2H), 1.46 - 1.44 (m, 1H), 1.35 (s, 3H), 1.35 (s, 1H), 1.31 (s, 5H), 1.27 (s, 1H), 1.23 (s, 1H), 1.19 (s, 3H), 1.17 (s, 1H), 1.09 (s, 1H), 1.02 (d, J = 2.0 Hz, 6H), 0.95 (dd, J = 11.0, 6.7 Hz, 1H), 0.91 (s, 3H), 0.73 (s, 3H), 0.69 (d, J = 2.6 Hz, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 199.58 (C-11), 173.53 (C-30), 171.87 (C=0), 171.11 (C=0), 170.43 (C-13), 160.92 (Ph-C), 147.18 (Ph-C), 144.72 (Ph-C), 144.64 (Ph-C), 132.51 (Ph-C), 127.92 (C-12), 127.52 (Ph-C), 127.46 (Ph-C), 120.60 (Ph-C), 120.08 (Ph-C), 116.66 (Ph-C), 102.36 (Ph-C), 101.82 (Ph-C), 77.06, 61.57, 54.58, 49.87, 48.24, 45.22, 43.82, 43.46, 40.54, 39.24, 38.98, 37.92, 37.15, 36.77, 36.13, 32.66, 31.89, 31.43, 29.08, 29.06, 28.75, 28.64, 28.61, 27.56, 27.44, 26.81, 26.70, 26.49, 25.68, 23.15, 22.54, 18.82, 17.61, 16.67, 16.47. HRMS (ESI): calcd for C 53 H 74 FN5O5[M+Na] + 902.5566, found: 902.5653.

[0064] Example 17 N 1 -(2-aminophenyl)- N 8 -(4-(4-(3 β -oxo-18 β Preparation of (2-(4-(4-(3 The compound of Example 17 was prepared according to the preparation procedure of Example 13, using suberic acid as starting material instead of pimelic acid, and using 2-amino-4-fluorophenylcarbamic acid tert-butyl ester as starting material instead of 2-aminophenylcarbamic acid tert-butyl ester. Yield 18%. 1 H NMR (600 MHz, DMSO- d6) δ 9.65 (s, 1H), 9.01 - 8.98 (m, 1H), 7.45 (d, J = 9.0 Hz, 2H), 7.12 - 7.06 (m, 1H), 6.88 (d, J = 9.0 Hz, 2H), 6.48 (dd, J = 11.1, 2.8 Hz, 1H), 6.29 (t, J = 8.5 Hz, 1H), 5.49 (s, 1H), 5.11 (s, 2H), 4.29 (d, J = 5.0 Hz, 1H), 3.70 - 3.67 (m, 4H), 3.02 (t, J = 5.3 Hz, 5H), 2.58 (d, J = 13.4 Hz, 1H), 2.34 - 2.23 (m, 5H), 2.21 (d, J = 13.6 Hz, 1H), 2.15 - 2.07 (m, 1H), 1.99 (d, J = 10.9 Hz, 1H), 1.88 (d, J = 10.8 Hz, 1H), 1.77 - 1.71 (m, 1H), 1.70 - 1.66 (m, 1H), 1.61 - 1.56 (m, 4H), 1.52 (d, J = 12.8 Hz, 2H), 1.43 - 1.39 (m, 3H), 1.36 - 1.32 (m, 8H), 1.31 (s, 1H), 1.27 (s, 1H), 1.24 (s, 1H), 1.19 (s, 3H), 1.15 (d, J = 13.3 Hz, 1H), 1.10 - 1.07 (m, 1H), 1.02 (s, 6H), 1.00 - 0.93 (m, 2H), 0.91 (s, 3H), 0.73 (s, 3H), 0.69 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 199.57 (C-11), 173.52 (C-30), 171.85 (C=O), 171.08 (C=O), 170.42 (C-13), 160.92 (Ph-C), 147.17 (Ph-C), 144.73 (Ph-C), 144.65 (Ph-C), 132.53 (Ph-C), 127.93 (C-12), 127.54 (Ph-C), 127.47 (Ph-C), 120.59 (Ph-C), 120.07 (Ph-C), 116.65 (Ph-C), 102.35 (Ph-C), 101.82 (Ph-C), 77.06, 61.57,54.59 (2C), 49.87, 48.23, 45.22 (2C),, 43.81, 43.45, 40.53, 39.24, 39.00,38.99, 37.93, 37.14, 36.76, 36.11, 34.75, 32.65, 31.88, 29.01, 28.75, 28.61,27.44, 26.69, 26.48, 25.63, 24.31, 23.15, 19.35, 19.00, 18.81, 17.61, 16.67,16.47. HRMS (ESI): calcd for C 54 H 76 FN5O5[M+Na] + 916.5723, found: 916.5816.

[0065] Example 18 N 1 -(2-amino-4-fluorophenyl)- N 9 -(4-(4-(3 β -hydroxy-11-oxo-18 β Preparation of -oleanol-12-en-30-acyl)piperazine-1-yl)phenyl)nonadiamide (Example 18) Following the preparation method of Example 13, azelaic acid was used instead of pimelic acid, and tert-butyl 2-aminophenylcarbamate was used instead of tert-butyl 2-aminophenylcarbamate to prepare the compound of Example 18. Yield: 26%. 1 H NMR (600 MHz, DMSO- d6) δ 9.65 (s, 1H), 9.00 (s, 1H), 7.45 (d, J = 9.0 Hz, 2H), 7.11 - 7.06 (m, 1H), 6.88 (d, J = 9.1 Hz, 2H), 6.47 (dd, J = 11.2, 2.9 Hz, 1H), 6.29 (t, J = 8.5 Hz, 1H), 5.49 (s, 1H), 5.11 (s, 2H), 4.29 (d, J = 5.0 Hz, 1H), 3.70 - 3.67 (m, 4H), 3.04 - 3.00 (m, 5H), 2.57 (d, J = 13.4 Hz, 1H), 2.34 - 2.23 (m, 5H), 2.24 - 2.18 (m, 1H), 2.16 - 2.07 (m, 1H), 2.02 - 1.95 (m, 1H), 1.90 - 1.84 (m, 1H), 1.76 (d, J = 10.7 Hz, 1H), 1.68 (t, J = 13.4 Hz, 1H), 1.60 - 1.55 (m, 4H), 1.52 (s, 1H), 1.51 (s, 1H), 1.45 (s, 1H), 1.40 (s, 1H), 1.35 (s, 4H), 1.31 (s, 6H), 1.27 (s, 1H), 1.24 (s, 1H), 1.19 (s, 3H), 1.16 - 1.10 (m, 1H), 1.09 (s, 1H), 1.02 (s, 6H), 0.99 - 0.92 (m, 2H), 0.91 (s, 3H), 0.89 - 0.77 (m, 2H), 0.73 (s, 3H), 0.70 (s, 1H), 0.69 (s, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 199.58 (C-11), 173.54 (C-30), 171.90 (C=O), 171.15 (C=O), 170.43 (C-13), 160.87 (Ph-C), 144.43 (Ph-C), 144.35 (Ph-C), 132.76 (Ph-C), 130.12 (Ph-C), 127.93 (C-12), 127.49 (Ph-C), 127.42 (Ph-C), 120.58 (Ph-C), 120.23 (Ph-C), 116.81 (Ph-C), 102.55 (Ph-C), 101.98 (Ph-C), 77.06, 61.57, 54.58, 50.01, 48.22, 45.22, 43.81, 43.73, 43.45, 40.52, 39.24, 38.98, 37.92, 37.14, 36.77, 36.69, 36.12, 32.65, 36.57, 31.88, 29.51, 29.09, 29.07, 28.88, 28.74, 28.66, 28.60, 27.44, 26.84, 26.69, 26.48, 25.68, 23.15, 18.81, 17.61, 16.66, 16.47. HRMS (ESI): calcd for C 55 H 78 FN5O5[M-H] - 906.5909, found: 906.5889.

[0066] Pharmacological action research of the compound of the embodiment of the present application: 1. Cell anti-inflammatory activity experiment For various inflammations, the release of NO is considered to be a sign of inflammation. The present application uses the Griess method to preliminarily evaluate the ability of the compound to inhibit the release of NO, and the results are shown in Table 1. Examples 1-18 show better ability to inhibit the release of NO than glycyrrhetinic acid.

[0067] The specific experimental procedure includes: 1) Cell culture RAW 264.7 cells were cultured in DMEM medium and verified by STR genotyping. The medium was supplemented with 10% (V / V) fetal bovine serum, 100 U / mL penicillin-100 μg / mL streptomycin. The cells used in the experiment were cultured in a culture box at 37℃, 5% CO2 saturated humidity.

[0068] 2) Drawing of NO standard curve Griess Reagent I and II were taken out and allowed to return to room temperature. The standards (NaNO2 solution) were diluted with DMEM complete medium at concentrations of 0, 1, 2, 5, 10, 20, 40, 60 and 100 μM. The standards were added to a 96-well plate at 50 μL / well, and Griess Reagent I was added to each well at 50 μL, followed by shaking for 10 min in the dark, and then Griess Reagent II was added to each well at 50 μL, and the absorbance was measured at 540 nm.

[0069] 3) Anti-inflammatory activity determination results A single RAW 264.7 cell suspension was prepared using a culture medium containing 10% fetal bovine serum, and the cell suspension was adjusted to a concentration of 5 x 10 5 After 24 h of culture, 100 μL of a solution containing LPS (at a final concentration of 1 μg / mL) and different concentrations of the drug was added to each test well, and an LPS group (containing LPS and no drug) and a blank control group (containing no LPS and no drug) were set up, with 3 parallel wells for each sample, and the plate was incubated in a constant-temperature and constant-humidity incubator at 37°C and 5% CO2 for 24 h. Color development: after 24 h of incubation at 37°C, 50 μL of supernatant was transferred to a 96-well plate, and Griess Reagent I was added to each well at 50 μL, followed by shaking for 10 min in the dark, and then Griess Reagent II was added to each well at 50 μL, followed by shaking for 10 min in the dark, and the absorbance was measured at 540 nm. The results were recorded, and the NO concentration was calculated according to the NO standard curve and the OD value obtained from the drug concentration, and the relative release rate of NO and the inhibition rate of drug administration were calculated.

[0070] Table 1 Inhibition rate (%) of NO release from RAW264.7 cells at 5 μM of the compound

[0071] 2. Mechanism of action of Example 1 Studies have shown that HDAC3 not only has the classic deacetylase activity, but also plays a key role in innate immune response by regulating mitochondrial metabolism and promoting the activation of NLRP3 inflammasome. In macrophages, HDAC3 promotes mitochondrial remodeling, promotes the maturation and secretion of IL-1β through NLRP3 activation. In order to study whether the compound of Example 1 exerts its anti-inflammatory effect by inhibiting the activation of NLRP3 inflammasome through HDAC3 degradation, we conducted a mechanism study in THP-1 derived macrophages. As shown in Figure 1, the compound of Example 1 significantly inhibited the activation of NLRP3 inflammasome in a dose-dependent manner. Figure 1As shown in Figure A, LPS / ATP stimulation significantly increased the level of extracellular mature IL-1β and caspase-1, while the compound of Example 1 intervened to induce HDAC3 degradation in a dose-dependent manner, and simultaneously reduced the release of IL-1β and caspase-1. As shown in Figure B, the compound of Example 1 significantly inhibited ASC speck formation, directly blocking the inflammasome assembly. Further confirmed by transcriptome (mRNA-seq) and quantitative proteomics analysis, the compound of Example 1 did not affect the gene or protein expression of the core components of the NLRP3 inflammasome (Figure C). Figure 1 B, the compound of Example 1 significantly inhibited ASC speck formation, directly blocking the inflammasome assembly. Further confirmed by transcriptome (mRNA-seq) and quantitative proteomics analysis, the compound of Example 1 did not affect the gene or protein expression of the core components of the NLRP3 inflammasome (Figure C). Figure 1 C~ Figure 1 E). Therefore, the compound of Example 1 specifically blocks the NLRP3 inflammasome activation process by degrading HDAC3, providing precise evidence of the mechanism of action for targeted therapy.

[0072] The specific experimental procedures include: 1) Western Blot After incubating the cells with a specific concentration of the compound for a period of time, the cells were centrifuged and collected. The cells were lysed with a strong lysis solution RIPA for 30 min, followed by centrifugation at 12000 rpm for 5 min at 4°C. The supernatant was determined for protein concentration by BCA method, and the loading amount was determined according to the protein concentration. 5x loading buffer was added to the lysate at a ratio of 1:4, and denatured at 95°C for 5 min. The protein samples were separated by non-continuous SDS polyacrylamide gel electrophoresis. After electrophoresis, the proteins on the gel were transferred to a 0.45 μm PVDF membrane by an electrotransfer device. The membrane was washed with TBST solution, then blocked with TBST solution containing 5% skim milk powder at room temperature for 1.5 h, and the specific antibody was incubated with the target protein band at 4°C overnight. After washing the membrane with TBST, the membrane was incubated with the secondary antibody at room temperature for 1.5 h. After washing the membrane with TBST, the membrane was wiped dry with filter paper, and equal volumes of A and B liquids in the ECL luminescence kit were mixed and evenly added to the membrane. The full-automatic luminescence fluorescence image system was used for processing.

[0073] 2) Quantitative proteomics analysis After the sample cells were taken out from the -80 °C refrigerator, an appropriate amount of lysis buffer (8 M urea, 1 mM PMSF, 2 mM EDTA) was added, and the cells were lysed on ice for 5 min, and then the supernatant was collected by centrifugation at 15000 g at 4 °C for 10 min. The protein concentration was determined by BCA kit. 100 μg of protein solution was taken and the volume was adjusted to 200 μL with 8 M urea, and then DTT (final concentration 5 mM) was added and reduced at 37 °C for 45 min. Then, in the dark, iodoacetamide (final concentration 11 mM) was added at room temperature for alkylation for 15 min. 800 μL of 25 mM ammonium bicarbonate solution and 2 μL of trypsin were added, and the enzyme was digested overnight at 37 °C. After the digested peptides were adjusted to pH 2-3 with 20% TFA, desalting treatment was performed using C18 resin. Finally, the peptide concentration was determined using a quantitative peptide detection kit.

[0074] 3) Liquid chromatography-mass spectrometry detection and mRNA sequencing analysis The peptide sample was separated by an ultra-high performance liquid chromatography (UHPLC) system. Data-independent acquisition mass spectrometry analysis was performed on a high-resolution mass spectrometer. The obtained mass spectrometry raw data was analyzed using DIA-NN software with a library-free method. At the same time, total RNA was extracted by the phenol-guanidine isothiocyanate method and dissolved in DEPC water. The RNA concentration was accurately determined using a fluorometer, and the RNA integrity value (RIN value) was detected by a high-throughput biological fragment analyzer to ensure that the sample quality meets the library construction standard. The qualified sample was used to construct the mRNA library, and after quality inspection, the libraries were mixed according to the effective concentration and target sequencing data volume, and then sequenced to generate 150 bp double-end reads. This sequencing technology is based on the principle of synchronous synthesis sequencing: four kinds of fluorescently labeled dNTPs, DNA polymerase and adapter primers are added to the sequencing flow cell for amplification. When the complementary strand is extended in each sequencing cluster, a corresponding fluorescent signal is released every time a fluorescently labeled dNTP is incorporated; the sequencer captures these signals, and computer software converts them into sequencing peak charts, thereby obtaining the sequence information of the target fragment.

[0075] 3. Evaluation of the anti-inflammatory activity of the compound of Example 1 in vivo To verify the anti-inflammatory efficacy of the compound of Example 1 in vivo, a LPS-induced endotoxin shock model was used to evaluate systemic inflammatory response: treatment with the compound of Example 1 (20 mg / kg, i.p.) improved the survival rate of mice to 80% at 84 h and 70% at 120 h, significantly improving lethal inflammatory injury (P < 0.05). Figure 2 Further, in the DSS-induced acute colitis model, treatment with the compound of Example 1 (15 mg / kg, i.p.) significantly inhibited disease progression, and the colon length was restored to 6.10 cm compared to 4.64 cm in the model group, and histopathology showed repair of mucosal integrity and an increase in the number of goblet cells.Figure 3 ). To expand its indications in NLRP3 inflammasome-mediated inflammation, the acute gouty arthritis model was induced by MSU: the compound of Example 1 (15 mg / kg) treatment for 24 h inhibited the joint swelling rate by 76.27%, and histology confirmed that it significantly reduced intra-articular crystal deposition, inflammatory infiltration and synovial hyperplasia (Fig. 4). Figure 4 ].

[0076] The specific experimental procedures include: The animals used in the experiment were male C57Bl / 6 mice (SPF level, body weight 20±2 g, 8 weeks old) purchased from Liaoning Changsheng Biological Technology Co., Ltd. (Experimental animal production license). The mice were raised in the barrier environment of Shenyang Pharmaceutical University SPF animal experiment center with temperature 22±1℃, humidity 50±10%, 12 h light / dark cycle.

[0077] 1) Acute shock model After 7 days of adaptive feeding, the mice were randomly divided into groups, 10 mice in each group. The drug was administered by intraperitoneal injection, and 2 h after intraperitoneal injection (20 mg / kg), LPS saline solution was injected intraperitoneally at a dose of 30 mg / kg. After injection of LPS aqueous solution, the survival status and clinical symptoms (such as piloerection, respiratory distress, decreased activity, etc.) of the mice were observed every 12 h, and the survival data of each group of mice were plotted as Kaplan-Meier curves for analysis.

[0078] 2) Acute colitis model After 7 days of adaptive feeding, the mice were randomly divided into groups (n=5), and 2.5% (w / v) dextran sulfate sodium (DSS) was used to induce acute colitis. On days 1-2, the mice were allowed to drink ordinary drinking water; on days 3-9, the mice were replaced with 2.5% DSS solution; on days 10-11, the mice were given ordinary drinking water. The compound (15 mg / kg) or vehicle control group was administered intraperitoneally for 11 consecutive days. Daily assessment indicators include body weight change, fecal character score, and benzidine occult blood test. On day 11, the mice were sacrificed to measure the colon length, and a representative colon segment was fixed with 4% paraformaldehyde for hematoxylin-eosin (H&E) histopathological analysis.

[0079] 3) Acute gout model Mice were randomized into vehicle, MSU model, and compound (7.5 / 15 mg / kg) groups (n = 6) 7 days after acclimation. The drugs were administered intraperitoneally at 1 hour before and 12 hours after modeling. Acute gout was induced by intra-articular injection of 1 mg of MSU crystals into the right knee joint. The circumference of the knee joint was measured using a measuring tape at 1 hour before modeling (-1 h) and 0, 1, 3, 6, 12, and 24 hours after modeling, and the gait abnormality score was recorded simultaneously. The joint tissue was collected 24 hours after modeling and fixed with 4% paraformaldehyde for synovitis H&E pathological evaluation.

Claims

1. An 18β-glycyrrhetinic acid derivative represented by the general formula I or II: ###0001### or an optical isomer thereof or a pharmaceutically acceptable salt thereof. ； X is fluorine or hydrogen; n is an integer from 5 to 7; R is cyano or halogenated hydrocarbon group.

2. The 18β-glycyrrhetinic acid derivative or its optical isomer or a pharmaceutically acceptable salt thereof according to claim 1, characterized by, The 18β-glycyrrhetinic acid derivative is selected from the following compounds: ###0002### 。 3. A pharmaceutical composition, characterized by, A pharmaceutical composition comprising the 18β-glycyrrhetinic acid derivative or an optical isomer thereof or a pharmaceutically acceptable salt thereof according to claim 1 or 2, and a pharmaceutically acceptable diluent, adjuvant and / or carrier.

4. The pharmaceutical composition of claim 3, wherein, The dosage form of the pharmaceutical composition includes oral preparations, injection preparations and topical preparations; wherein the oral preparations include tablets, capsules, solutions and suspensions; the injection preparations include injectable solutions, suspensions and injectable dry powders which are dissolved into injection solutions by adding water for injection before injection; the topical preparations include ointments and emulsions.

5. A process for preparing the 18β-glycyrrhetinic acid derivative or its optical isomer or a pharmaceutically acceptable salt thereof according to claim 2, characterized by, The synthesis comprises the following steps: ； wherein: X is fluorine or hydrogen; n is an integer from 5 to 7; the dotted circle A represents a structural fragment at the corresponding position of the compound of Example 1-18; Reagents and reaction conditions: step a: o-iodoxybenzoic acid, 80-90°C; step b: iodine, pyridine; step c: cuprous cyanide, 120-140°C; step d: 1-(4-nitrophenyl)piperazine, EDCI, HOBT, DIEA; step e: zinc powder, acetic acid, 50-70°C; step f: HOBT, EDCI, DIEA; step g: HCl / EA; step h: FSO2CF2COOCH3, HMPA, CuI, 60-70°C.

6. Use of the 18β-glycyrrhetinic acid derivative or an optical isomer thereof or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or the pharmaceutical composition according to claim 3 or 4, in the preparation of a medicament for treating and / or preventing an inflammatory disease.

7. Use according to claim 6, characterized in that, The inflammatory disease includes rheumatoid arthritis, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, gout / hyperuricemia, asthma, atopic dermatitis, Alzheimer's disease, multiple sclerosis or hepatitis.

8. Use of the 18β-glycyrrhetinic acid derivative or an optical isomer thereof or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or the pharmaceutical composition according to claim 3 or 4, in the preparation of a medicament for treating a disease associated with HDAC3 inhibition or NLRP3 inhibition.

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