18 beta-glycyrrhetinic acid derivative containing diene structure fragment as well as preparation method and application of 18 beta-glycyrrhetinic acid derivative

Abstract

The invention discloses a 18 beta-glycyrrhetinic acid derivative containing a diene structure fragment as shown in a general formula I and a preparation method and application thereof, and belongs to the technical field of medicines, the compound can effectively degrade HDAC3, and further mechanism studies (including Western Blot and quantitative proteomics) prove that a representative compound can effectively degrade the HDAC3 by inducing the degradation of the HDAC3 through specificity. Further, an NLRP3 inflammasome pathway is inhibited, and finally, maturation and secretion of downstream inflammatory factors IL-1beta and caspase-1 are blocked, so that a remarkable anti-inflammatory effect is achieved. A mouse acute shock and colitis model further verifies the excellent anti-inflammatory activity of the compound in vivo. The invention not only provides a novel HDAC3 degradation agent with good activity, but also lays a solid material and theoretical foundation for deep development of anti-inflammatory drugs based on the 18 beta-glycyrrhetinic acid structure.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to 18 fragments containing diene structures. β - Glycyrrhetinic acid derivatives, their preparation methods, and their application in the preparation of drugs for the treatment and / or prevention of various inflammatory diseases. Background Technology

[0002] Given the ever-increasing global burden of inflammatory diseases, the development of new anti-inflammatory treatment strategies is particularly urgent. World Health Organization (WHO) data shows that inflammatory diseases affect over 800 million people worldwide, causing approximately 4 million deaths annually; in Asia, the incidence of inflammatory bowel disease has surged nearly 30-fold in the past 30 years. Exposure to environmental toxins, increased pathogen resistance, and high-stress lifestyles have collectively contributed to the prevalence of these diseases. However, existing mainstream therapies such as glucocorticoids and biologics are far from meeting clinical needs due to limitations including severe side effects, high treatment costs, and inadequate response rates in approximately 40% of patients.

[0003] Histone deacetylase 3 (HDAC3), a core member of the class I HDAC family, has been a promising target for anti-inflammatory therapy in recent years. However, traditional HDAC3 inhibitors (such as hydroxamic acids and benzamides) exhibit poor selectivity for HDAC1 / 2 subtypes and are prone to causing liver damage and hematological toxicity due to their broad-spectrum mechanisms of action, severely limiting their clinical application. In contrast, targeted protein degradation (TPD) technology, with its event-driven catalytic properties and unique mechanism of simultaneously clearing both enzymatic and non-enzymatic functions of the target, offers a new pathway to overcome the shortcomings of traditional inhibitors.

[0004] Natural products and their derivatives exhibit unique therapeutic value in this context. Over 50% of anti-inflammatory clinical candidate molecules are derived from natural lead compounds, whose multi-target mechanisms of action can effectively reshape immune homeostasis, and whose hepatotoxicity and nephrotoxicity are reduced by 3-5 times compared to traditional synthetic drugs. In particular, novel HDAC3 degraders (such as 18...) obtained in recent years based on the structural optimization of natural products... β Glycyrrhetinic acid derivatives not only overcome the problems of poor selectivity and high toxicity of traditional inhibitors, but also achieve precise anti-inflammatory effects by regulating the activation of NLRP3 inflammasomes, providing an important direction for the development of a new generation of highly effective and low-toxic anti-inflammatory drugs. Summary of the Invention

[0005] The technical problem solved by this invention is to provide 18-membered terephthalic acid fragments containing diene structures. β - Glycyrrhetinic acid derivatives and their preparation methods are also provided, along with 18-membered diene-containing fragments. β- Application of glycyrrhetinic acid derivatives in the preparation of drugs for the prevention and / or treatment of various inflammatory diseases.

[0006] This invention relates to 18-membered diene-containing segments of general formula I. β - Glycyrrhetinic acid derivatives and their optical isomers and their pharmaceutically acceptable salts:

[0007] Among them, R1 is -C(O)NR2R3, -NHC(O)R4 or -NHC(O)NR5R6; R2 and R3 may be the same or different, and each is independently selected from hydrogen, substituted or unsubstituted C3-C5 alkyl, substituted or unsubstituted C3-C5 cycloalkane group, or R2 and R3 with the N atom attached to them form a substituted or unsubstituted C4-C6 saturated heterocyclic group or a substituted or unsubstituted C5 bridged ring group; the C4-C6 saturated heterocyclic group includes piperidinyl, aziridine butyl, thiomorpholinyl; the C5 bridged ring group is 3-azirbicyclo[3.1.0]hexyl; the C3-C5 alkyl includes propyl, butyl, pentyl; the substituent is one or more, selected from hydroxyl, halogen atom, hydroxymethyl, cyano.

[0008] R4 is selected from monosubstituted or disubstituted C3-C6 cycloalkane groups, disubstituted C3-C6 alkyl groups, and monosubstituted or disubstituted five- or six-membered heterocyclic groups; the C3-C6 cycloalkane groups include cyclobutyl, cyclopentyl, and cyclohexyl; the C3-C6 alkyl groups include propyl, butyl, and pentyl; the five- or six-membered heterocyclic groups include pyrrolidinyl, piperidinyl, and 2-azaspiro[3.3]heptyl; the substituents are selected from hydroxyl, hydroxymethyl, cyano, halogen atoms, and methyl.

[0009] R5 and R6 may be the same or different, each independently selected from hydrogen, substituted or unsubstituted C3-C5 alkyl groups, or R5 and R6 forming substituted or unsubstituted C3-C6 saturated heterocyclic groups or substituted or unsubstituted C5 bridged ring groups with the N atom attached to them; the C3-C6 saturated heterocyclic groups include azirrobutyl, piperidinyl, thiomorpholinyl, and 3-azibicyclo[3.1.0]hexyl; the C3-C5 alkyl groups include propyl, butyl, and pentyl; the substituents are one or more, selected from hydroxyl, hydroxymethyl, cyano, halogen atom, and 1,1-dioxide (only for thiomorpholinyl).

[0010] The halogen atom described in this invention is selected from fluorine, chlorine, and bromine atoms.

[0011] Particularly preferred compounds include:

[0012] The present invention also provides a pharmaceutical composition comprising 18 segments of general formula I containing a diene structure. β - Glycyrrhetinic acid derivatives or their pharmaceutically acceptable salts and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients refer to any diluent, excipient, and / or carrier that can be used in the pharmaceutical field. The compounds of the present invention can be used in combination with other active ingredients, provided they do not produce other adverse effects, such as allergic reactions. The pharmaceutical compositions of the present invention can be formulated into several dosage forms containing some excipients commonly used in the pharmaceutical field, such as oral formulations (e.g., tablets, capsules, solutions, or suspensions); injectable formulations (e.g., injectable solutions or suspensions, or injectable dry powders that can be used immediately after addition of water for injection); and topical formulations (e.g., ointments or solutions). Carriers used in the pharmaceutical compositions of the present invention are common types available in the pharmaceutical field, including: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, pigments, flavoring agents, etc., for oral formulations; preservatives, solubilizers, stabilizers, etc., for injectable formulations; and matrices, diluents, lubricants, preservatives, etc., for topical formulations. Pharmaceutical formulations can be administered orally or via parenteral route (e.g., intravenously, subcutaneously, intraperitoneally, or locally). If certain drugs are unstable under gastric conditions, they can be formulated into enteric-coated tablets.

[0013] Through target degradation activity screening, we discovered that some compounds of this invention possess good degradation activity. Subsequent mechanism studies and in vivo activity results showed that the compounds can degrade HDAC3 protein, thereby inhibiting the NLPR3 inflammasome pathway. Therefore, this invention also provides the use of the said compounds in the preparation of drugs for diseases related to HDAC3 inhibition or NLRP3 inhibition, and in the preparation of drugs for the treatment and / or prevention of 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.

[0014] The compounds of this invention can be used as the sole anti-inflammatory drug as the active ingredient, or in combination with one or more other anti-inflammatory drugs. Combination therapy is achieved by administering the various therapeutic components simultaneously, sequentially, or separately.

[0015] This invention also provides a method for preparing the compound represented by general formula I: Synthesis Route 1:

[0016] Reagents and reaction conditions: a) NaBH4, NaOH, THF / H2O, reflux; b) HCl, tetrahydrofuran (THF), reflux; c) o-iodobenzoic acid (IBX), dimethyl sulfoxide (DMSO), 85℃; d) iodine, pyridine, THF, reflux; e) CuCN, N-methylpyrrolidone (NMP), 130℃, nitrogen protection; f) substituted amines, N,N-diisopropylethylamine (DIPEA), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), anhydrous N,N-dimethylformamide (DMF), room temperature.

[0017] Synthesis Route 2:

[0018] Reagents and reaction conditions: a) Diphenyl azidophosphate (DPPA), TEA, acetonitrile (MeCN), reflux; b) Concentrated hydrochloric acid, MeCN, room temperature; c) Substituted acids, 1-hydroxybenzotriazole (HOBt) / 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI) (or HATU), N,N-diisopropylethylamine (DIPEA), anhydrous N,N-dimethylformamide (DMF), room temperature.

[0019] Synthesis Route 3:

[0020] Reagents and reaction conditions: a) DPPA, TEA, MeCN, reflux; b) substituted amines, HOBt / EDCI (or HATU), DIPEA, room temperature.

[0021] Wherein, R2, R3, R5, and R6 are the corresponding groups at the corresponding positions of the compounds shown in Formula I.

[0022] The beneficial effects of this invention are as follows: The compounds of this invention can effectively degrade HDAC3. Further mechanistic studies (including Western blotting and quantitative proteomics) have confirmed that the representative compounds specifically induce HDAC3 degradation, thereby inhibiting the NLRP3 inflammasome pathway and ultimately blocking the maturation and secretion of downstream inflammatory factors IL-1β and caspase-1, thus exerting a significant anti-inflammatory effect. Mouse models of acute shock and colitis further validated their excellent anti-inflammatory activity in vivo. This invention not only provides a novel class of HDAC3 degrading agents with good activity but also lays a solid material and theoretical foundation for the in-depth development of anti-inflammatory drugs based on the 18β-glycyrrhetinic acid structure. Attached Figure Description

[0023] Figure 1The anti-inflammatory mechanism of the compound in Example 8 of this invention; A: KEGG enrichment bar chart of integrated proteomics and transcriptomics analysis; B: Western blot analysis of HDAC3 and NLPR3-related proteins in THP-M cell culture supernatant (SN) and cell extract (Lysate).

[0024] Figure 2 This invention relates to a mouse model of acute colitis. A: Experimental flowchart; B: Line graph showing the weight changes of mice in different treatment groups during the experiment; C: Anatomical photographs of representative colon tissues; D: Statistical results of colon length in mice in different treatment groups; E: HE-stained colon tissue micrographs.

[0025] Figure 3 The survival curves of mice in different treatment groups in the acute shock mouse model of this invention are shown.

[0026] Figure 4 The images show representative immunohistochemical (IHC) staining of colon tissue in this invention; A: OCCLUDIN expression; B: ZO-1 expression. Detailed Implementation

[0027] The following examples are intended to illustrate, but not limit, the scope of the invention. All techniques implemented based on the content of this invention fall within its scope. The nuclear magnetic resonance (NMR) spectra of the compounds were determined using a Bruker ARX-600 NMR spectrometer in DMSO-d6 with TMS as an internal standard, and high-resolution mass spectrometry was performed using a Bruker micro-TOF-Q spectrometer. Unless otherwise specified, all raw materials and reagents used in this invention are commercially available.

[0028] Example 1: N -(2-cyano-3-oxo-18) β Preparation of oleanolane-1,9(11),12-triene-30-acyl)-3-hydroxyazacyclobutamine

[0029] Step A: 3 β ,11-Dihydroxy-18 β Preparation of oleanolic acid-12-ene-30-carboxylic acid; Weigh out 18 βGlycyrrhetinic acid (10.00 g, 21.20 mmol) was dissolved in 200 mL of tetrahydrofuran, followed by the addition of 100 mL of sodium hydroxide solution (5.10 g, 0.13 mol), and stirred at room temperature for 10 min. Sodium borohydride (24.13 g, 0.64 mol) was added to the above solution in three portions, and the reaction mixture was kept clear and stirred under reflux for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and the pH was adjusted to 2-3 with 6 N hydrochloric acid in an ice bath. The tetrahydrofuran was distilled off under reduced pressure, and an appropriate amount of saturated sodium chloride solution was added, precipitating a white solid. The solid was filtered, washed with water, and dried to obtain 9.54 g of white solid, with a yield of 95%.

[0030] Step B: 3 β -hydroxy-18 β Preparation of oleanolic acid-9(11),12-diene-30-carboxylic acid; Weigh out 3 β ,11-Dihydroxy-18 β 9.54 g (21.20 mmol) of oleanolic-12-en-30-carboxylic acid was dissolved in 200 mL of dry tetrahydrofuran, followed by the addition of 1 mL of concentrated hydrochloric acid, and the mixture was refluxed and stirred for 3 h. After the reaction was complete, the tetrahydrofuran was distilled off under reduced pressure, and the reaction solution was poured into 250 mL of water and stirred for 15 min, precipitating a white solid. The solid was filtered, washed with water, dried, and recrystallized from 80% ethanol to give 6.10 g of a white solid, with a yield of 63%.

[0031] Step C: 3-Oxo-18 β Preparation of oleanolic acid-1,9(11),12-triene-30-carboxylic acid; Weigh out 3 β -hydroxy-18 β -Oleanane-9(11),12-diene-30-carboxylic acid (6.46 g, 14.20 mmol) was dissolved in 250 mL of dry dimethyl sulfoxide, followed by the addition of IBX (15.92 g, 56.85 mmol), and stirred at 85 °C for 12 h. After the reaction was complete, the reaction solution was poured into 600 mL of water, the pH was adjusted to 7-8 with saturated sodium bicarbonate solution, stirred for 15 min, and extracted with ethyl acetate (500 mL × 3). The combined ethyl acetate layers were washed once with water and once with saturated NaCl aqueous solution, and then dried over anhydrous Na2SO4. The solution was filtered, and the filtrate was evaporated to dryness to give 5.68 g of a pale yellow solid, with a yield of 88%.

[0032] Step D: 2-Iodo-3-oxo-18 β Preparation of oleanolic acid-1,9(11),12-triene-30-carboxylic acid; Weigh out 3-oxo-18β -Oleanane-1,9(11),12-trien-30-carboxylic acid (5.68 g, 12.60 mmol) was dissolved in 200 mL of dry tetrahydrofuran, and iodine (19.20 g, 75.6 mmol) and pyridine (5.98 g, 75.6 mmol) were added. The mixture was refluxed and stirred for 8 h. After the reaction was complete, the tetrahydrofuran was evaporated to dryness, and the resulting oily substance was dissolved in 1000 mL of ethyl acetate. The ethyl acetate layer was washed successively with saturated sodium thiosulfate solution (500 mL × 3), water, 10% hydrochloric acid, and saturated NaCl aqueous solution, and then dried over anhydrous Na2SO4. The filtrate was filtered, and the filtrate was evaporated to dryness. The crude product was precipitated by silica gel column chromatography with petroleum ether:ethyl acetate (V:V) = 50:1 as the eluent to obtain 5.53 g of white solid, with a yield of 76%.

[0033] Step E: 2-Cyano-3-oxo-18 β Preparation of oleanolic acid-1,9(11),12-triene-30-carboxylic acid; Weigh out 2-iodine-3-oxo-18 β -Oleanane-1,9(11),12-trien-30-carboxylic acid (3.70 g, 6.42 mmol) was dissolved in 150 mL of N-methylpyrrolidone, and cuprous cyanide (1.73 g, 19.26 mmol) was added. The mixture was stirred at 130 °C for 2 h under nitrogen protection. After the reaction was completed, the reaction solution was poured into 600 mL of water and stirred for 15 min. A solid precipitated out. The solid was filtered, and the filter cake was dried. The solid was then precipitated by silica gel column chromatography with petroleum ether:ethyl acetate (V:V) = 5:1 as the eluent to obtain 2.23 g of a pale yellow solid, with a yield of 76%.

[0034] Step F: N -(2-cyano-3-oxo-18) β Preparation of oleanol-1,9(11),12-triene-30-acyl)-3-hydroxyazacyclobutamine; Weigh out 2-cyano-3-oxo-18 β -Oleanane-1,9(11),12-trien-30-carboxylic acid (0.2 g, 0.46 mmol) was dissolved in 10 mL of dry DMF, and 3-hydroxyazacyclobutamine (0.04 g, 0.55 mmol), DIPEA (0.24 g, 1.84 mmol), and HATU (0.21 g, 0.55 mmol) were added. After stirring at room temperature for 1 h, the reaction was monitored by TLC (petroleum ether:ethyl acetate = 2:1) and the reaction was complete. 50 mL of water was added, and a solid precipitated. The solid was filtered, washed with water, and dried. PTLC purification gave 0.13 g of white solid, yield: 51%. 1H NMR (600 MHz, DMSO- d 6 ) δ 8.57 (s, 1H, H-1), 6.20 (d, J = 5.7 Hz,1H, H-11), 5.69 (s, 2H), 4.44 – 4.41 (m, 2H, -N(C H2 )2CH-), 4.05 (s, 2H, -N(C H2 )2CH-), 3.62 (s, 1H, -C H OH), 2.11 – 2.06 (m, 1H), 1.85 – 1.79 (m, 2H),1.74 – 1.71 (m, 2H), 1.65 (d, J = 3.9 Hz, 1H), 1.54 (d, J = 7.0 Hz, 3H), 1.41(s, 2H), 1.39 (s, 2H), 1.32 – 1.27 (m, 2H), 1.24 (d, J = 6.2 Hz, 2H), 1.16(s, 3H), 1.15 (s, 3H), 1.07 (s, 3H), 1.01 (s,3H), 0.95 (s, 3H), 0.86 – 0.83(m, 2H), 0.79 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.44 (C-3), 174.94 (C-30), 171.72 (C-1), 149.22 (C-9), 145.45 (C-13), 120.54 (C-12), 118.98 (C-11),115.82 (-CN), 112.78 (C-2), 60.67, 51.68, 48.11, 44.77, 43.83, 42.86, 42.26,40.72, 38.11, 34.67, 31.77, 30.87, 28.79, 28.30, 27.38, 27.25, 26.82, 26.76,25.63, 21.67, 21.59, 19.70, 18.24, 14.45, 11.73. HRMS m / z calcd forC 34 H 46 N2NaO3 + [M+Na] + 553.3401, found 553.3389。

[0035] Example 2: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-30-acyl)-4-hydroxypiperidine

[0036] Following the preparation method of Example 1, the compound of Example 2 was prepared by replacing the 3-hydroxyazacyclobutamine in step F of Example 1 with 4-hydroxypiperidine. Yield: 54%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.58 (s, 1H,H-1), 6.19 (d, J = 5.9 Hz, 1H, H-11), 5.66 (d, J = 5.8 Hz, 1H, H-12), 4.74(d, J = 4.3 Hz, 1H, -CHO H ), 4.02 – 3.88 (m, 2H, -N(C H2 CH2)2CH-), 3.69 (d, J =4.3 Hz, 1H, -C H OH), 3.15 – 3.01 (m, 2H, -N(C H2 CH2)2CH-), 2.29 – 2.22 (m, 1H), 2.00 – 1.93 (m, 1H), 1.87 – 1.81 (m, 2H), 1.77 – 1.73 (m, 2H), 1.65 (q, J =7.5, 6.2 Hz, 2H), 1.58 – 1.54 (m, 1H), 1.41 (s, 3H), 1.40 (s, 3H), 1.37 –1.32 (m, 1H), 1.29 – 1.27 (m, 1H), 1.16 (s, 3H), 1.14 (s, 3H), 1.12 (s, 3H),1.07 (s, 3H), 0.95 (s, 3H), 0.87 – 0.85 (m, 2H), 0.84 (d, J = 2.8 Hz, 2H), 0.83– 0.81 (m, 2H), 0.77 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6) δ 198.44 (C-3), 173.41(C-30), 171.73 (C-1), 149.33 (C-9), 145.27 (C-13), 120.58 (C-12), 119.00 (C-11), 115.81 (-CN), 112.75 (C-2), 66.33, 55.39, 48.14, 46.51, 45.65, 44.77,43.83, 42.96, 42.83, 42.26, 40.77, 38.44, 35.22, 35.16, 32.71, 31.62, 30.89,28.84, 28.25, 27.37, 27.34, 26.91, 25.56, 21.67, 21.50, 19.63, 18.25. HRMS m / z calcd for C 36 H 50 N2NaO3 + [M+Na] + 581.3714, found 581.3709.

[0037] Example 3: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-30-acyl)-6-hydroxymethyl-3-azabicyclo[3.1.0]hexylamine

[0038] Following the preparation method of Example 1, the starting material 3-hydroxyazacyclobutylamine in step F of Example 1 was replaced with 3-azabicyclo[3.1.0]hex-6-ylmethanol to prepare the compound of Example 3. Yield: 36%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.57 (s, 1H, H-1), 6.19 (d, J = 5.8 Hz, 1H, H-11), 5.63 (d, J = 5.8 Hz,1H, H-12), 4.51 (d, J = 5.5 Hz, 1H, -CH2O H ), 3.78 (t, J = 10.8 Hz, 2H, -N(C H2 CH)2-), 3.49 – 3.34 (m, 2H, -C H2 OH), 3.30 (d, J = 5.6 Hz, 2H, -N(C H2CH)2-),2.10 (d, J = 12.9 Hz, 1H), 1.99 (m, J = 15.7, 10.8 Hz, 2H), 1.83 (m, J =10.9, 5.0 Hz, 2H), 1.73 (dd, J = 12.4, 5.6 Hz, 2H), 1.68 – 1.63 (m, 2H), 1.58– 1.53 (m, 1H), 1.41 (s, 3H), 1.39 (s, 2H), 1.30 – 1.27 (m, 2H), 1.24 (d, J =6.7 Hz, 2H), 1.16 (s, 3H), 1.14 (s, 3H), 1.05 (s, 3H), 1.02 (s, 3H), 0.94 (s,3H), 0.92 – 0.89 (m, 1H), 0.88 – 0.81 (m, 2H), 0.77 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.45 (C-3), 174.41 (C-30), 171.74 (C-1), 149.32 (C-9), 145.34(C-13), 120.45 (C-12), 119.01 (C-11), 115.80 (-CN), 112.75 (C-2), 62.20,48.11, 44.77, 44.52, 42.82, 42.25, 40.76, 38.18, 34.67, 31.80, 31.43, 30.87,28.79, 28.25, 27.42, 27.37, 27.03, 26.81, 26.02, 25.58, 25.26, 24.98, 22.56,22.54, 21.67, 21.55,19.57,18.24. HRMS m / z calcd for C 37 H 50 N2NaO3 + [M+Na] + 593.3714, found 593.3718.

[0039] Example 4: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-triene-30-acyl)-piperidine-4-carboxynitrile

[0040] Following the preparation method of Example 1, the starting material 3-hydroxyazacyclobutamine in step F of Example 1 was replaced with piperidine-4-carboxynitrile to prepare the compound of Example 4. Yield: 26%. 1 H NMR (600 MHz, DMSO- d 6 ) δ 8.58 (s, 1H,H-1), 6.18 (d, J = 5.9 Hz, 1H, H-11), 5.71 (d, J = 5.9 Hz, 1H, H-12), 3.47(d, J = 10.2 Hz, 1H, -C H CN), 3.29 (t, J = 6.1 Hz, 2H, -N( CH2 CH2)2CH-), 3.26–3.18 (m, 2H, -N( CH2 CH2)2CH-), 2.13 (dd, J = 13.3, 4.5 Hz, 2H), 2.04 (dd, J =13.2, 3.3 Hz, 2H), 2.00–1.92 (m, 1H), 1.89 – 1.84 (m, 1H), 1.81 (td, J =13.8, 3.0 Hz, 3H), 1.78–1.65 (m, 2H), 1.66–1.63 (m, 1H), 1.63–1.42 (m, 1H), 1.14 (s, 3H), 1.07 (s, 3H), 1.03 (q, J = 4.3, 3.3 Hz, 3H), 1.01 (s, 3H), 0.94(d, J = 6.7 Hz, 1H), 0.92 (s, 3H), 0.86 (s, 1H), 0.85 (m, 2H), 0.84 (s, 1H), 0.83 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.44 (C-3), 173.73 (C-30), 171.74(C-1), 149.26 (C-9), 145.32 (C-13), 122.50 (-N(CH2CH2)2CH CN), 120.60 (C-12), 118.98 (C-11), 115.81 (-CN), 112.74 (C-2), 48.13, 46.46, 44.76, 43.86, 42.82,42.26, 40.76, 38.33, 34.67, 34.40, 32.60, 31.63, 31.43, 30.89, 29.01, 28.96,28.77, 28.24, 27.35, 26.77, 25.95, 25.54, 25.26, 21.67, 21.50, 19.61, 18.24.HRMS m / z calcd for C 37 H 49 N3NaO2 + [M+Na] + 590.3723, found 590.3736.

[0041] Example 5: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-30-acyl)-2,2-difluoropropylamine

[0042] Following the preparation method of Example 1, the compound of Example 5 was prepared by replacing the 3-hydroxyazacyclobutamine in step F of Example 1 with 2,2-difluoropropylamine. Yield: 59%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.57 (s,1H, H-1), 7.95 (t, J = 6.3 Hz, 1H, -CON H CH2-), 6.20 (d, J = 5.9 Hz, 1H, H-11), 5.65 (d, J = 5.8 Hz, 1H, H-12), 3.54-3.43 (m, 2H, -NHC H2CHF2), 2.12 –2.08 (m, 1H), 1.99 (s, 2H), 1.83 (s, 2H), 1.77 – 1.70 (m, 2H), 1.65 (m, J =3.3 Hz, 2H), 1.56 (s, 3H), 1.41 (s, 3H), 1.37 (d, J = 3.0 Hz, 1H), 1.36 –1.28 (m, 2H), 1.26 (s, 2H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 1.05(m,1H), 1.02 (s, 3H), 0.96 (s, 3H), 0.9 (m, 1H) 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.44 (C-3), 176.45 (C-30), 171.72 (C-1), 148.94 (C-9), 145.34(C-13), 123.62 (- C F2CH3), 120.63 (C-12), 118.90 (C-11), 115.86 (-CN), 112.80(C-2), 55.38, 48.11, 46.33, 44.76, 43.60, 42.89, 40.65, 37.99, HRMS m / z calcd for C 34 H 46 F2N2NaO2 + [M+Na] + 575.3420, found575.3409.

[0043] Example 6: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-triene-30-acyl)-3,3-difluorozahexacyclic butylamine

[0044] Following the preparation method of Example 1, the raw material 3-hydroxyazacyclobutamine in step F of Example 1 was replaced with raw material 3,3-difluoroazacyclobutamine to prepare the compound of Example 6. Yield: 39%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.55(s, 1H), 6.20 (d, J = 5.8 Hz, 1H), 5.62 (d, J = 5.8 Hz, 1H), 4.65 (s, 2H), 4.52 (s, 2H), 2.12 – 2.07 (m, 1H), 1.86 – 1.76 (m, 2H), 1.75 – 1.70 (m, 2H), 1.68 – 1.62 (m, 2H), 1.58 (m, 1H), 1.57 (t, J = 13.5 Hz, 3H), 1.41 (s, 2H), 1.40 (s, 2H), 1.36 – 1.30 (m, 2H), 1.30 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 0.95 (s, 3H), 0.89 (d, J = 55.2 Hz, 2H), 0.80 (s, 3H). 13 C NMR (150 MHz, DMSO-) d 6 ) δ 198.41, 175.41, 171.64, 148.96, 145.48, 120.58, 118.94,116.84, 115.81, 112.76, 48.09, 46.50, 44.76, 44.03, 43.12, 42.84, 42.25,40.72, 37.96, 34.67, 31.77, 30.85, 30.82, 28.66, 28.21, 27.33, 27.17, 26.81,26.58, 25.58, 25.25, 21.66, 21.59, 19.68, 18.23. HRMS m / z calcd forC 34 H 44 F2N2NaO2 + [M+Na] + 573.3263, found 573.3262.

[0045] Example 7: N -(2-cyano-3-oxo-18) βPreparation of oleanolic acid-1,9(11),12-trien-30-acyl)-3,3-difluorocyclobutane

[0046] Following the preparation method of Example 1, the compound of Example 7 was prepared by replacing the 3-hydroxyazacyclobutamine in step F of Example 1 with 3,3-difluorocyclobutamine. Yield: 25%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.57 (s,1H, H-1), 7.91 (d, J = 6.5 Hz, 1H, -CON H -), 6.21 (d, J = 5.9 Hz, 1H, H-11), 5.63 (d, J = 5.8 Hz, 1H, H-12), 4.12 – 4.07 (m, 1H, -CONHC H -), 2.91 – 2.80(m, 2H, -NHCH(C H2 )2-), 2.66 – 2.54 (m, 2H, -NHCH(C H2 )2-), 2.09 – 2.04 (m, 1H), 1.83 (dd, J = 13.7, 4.3 Hz, 2H), 1.74 (p, J = 8.6, 7.5 Hz, 2H), 1.65 (p, J =6.0, 4.7 Hz, 2H), 1.53 (t, J = 13.4 Hz, 1H), 1.41 (s, 3H), 1.39 (s, 2H), 1.28(s, 2H), 1.24 (d, J = 5.3 Hz, 2H), 1.16 (s, 3H), 1.14 (s,3H), 1.07 (s, 3H),1.00 (s, 3H), 0.96 (s, 3H), 0.88 – 0.79 (m, 2H), 0.76 (s, 3H). 13 C NMR (151MHz, DMSO- d 6 ) δ 198.43 (C-3), 176.18 (C-30), 171.69 (C-1), 148.97 (C-9), 145.38 (C-13), 120.63 (C-12), 118.90 (C-11), 118.34 (- CF2), 115.85 (-CN),112.81 (C-2), 48.10, 46.43, 44.76, 43.21, 42.89, 42.78, 42.27, 40.65, 38.05,34.67, 34.46, 34.34, 31.62,30.83,28.93,28.85,28.36,27.42,27.04,26.81,25.62,21.67,21.50,19.78,18.25. 35 H 46 F2N2NaO2 + [M+Na] + 587.3420, found 587.3419.

[0047] Example 8: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-30-acyl)-4,4-difluoropiperidine

[0048] Following the preparation method of Example 1, the compound of Example 8 was prepared by replacing the 3-hydroxyazacyclobutamine in step F of Example 1 with 4,4-difluoropiperidine. Yield: 15%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.57 (s,1H, H-1), 6.19 (d, J = 5.9 Hz, 1H, H-11), 5.65 (d, J = 5.8 Hz, 1H, H-12), 3.67 (s, 2H, -N(C H2 CH2)2-), 3.64 (s, 2H, -N(C H2CH2)2-), 2.24 (dd, J = 13.8, 4.0Hz, 1H), 1.97 (d, J = 5.9 Hz, 2H), 1.92 (s, 2H), 1.86 – 1.79 (m, 2H), 1.73(dd, J = 13.4, 7.2 Hz, 2H), 1.65 (q, J = 5.8 Hz, 2H), 1.59 (d, J = 13.4 Hz,1H), 1.41 (s, 3H), 1.38 (t, J = 3.0 Hz, 2H), 1.35 – 1.30 (m, 2H), 1.26 – 1.18(m, 2H), 1.16 (s, 3H), 1.15 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 0.95 (s, 3H), 0.93 (s, 2H), 0.78 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.43 (C-3), 174.02 (C-30), 171.70 (C-1), 149.19 (C-9), 145.32 (C-13), 123.17 (- C F2),120.62 (C-12), 118.98 (C-11), 115.80 (-CN), 112.73 (C-2), 55.39, 48.13,46.43, 45.52, 44.76, 43.91, 42.25, 40.75, 38.29, 34.53, 34.38, 34.23, 32.57,31.65, 31.43, 30.89, 28.72, 28.22, 27.34, 26.82, 25.54, 22.54, 21.66, 21.52,19.60, 18.24. HRMS m / z calcd for C 36 H 48 F2N2NaO2 + [M+Na] + 601.3576 found 601.3568.

[0049] Example 9: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-30-acyl)-6,6-difluoro-3-azabicyclo[3.1.0]hexylamine

[0050] Following the preparation method of Example 1, the starting material 3-hydroxyazacyclobutamine in step F of Example 1 was replaced with 6,6-difluoro-3-azabicyclo[3.1.0]hexane to prepare the compound of Example 9. Yield: 18%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.57 (s, 1H, H-1), 6.19 (d, J = 5.8 Hz, 1H, H-11), 5.75 (s, 1H, H-12), 3.98 (d, J = 11.7 Hz, 2H, -N(C H2 CH)2-), 3.72 (s, 2H, -N(C H2 CH)2-), 2.06(dd, J = 13.8, 4.0 Hz, 1H), 2.00 (dd, J = 13.7, 4.3 Hz, 1H), 1.96 (d, J = 9.8Hz, 1H), 1.88 – 1.81 (m, 2H), 1.73 (m, J = 9.2, 8.7, 6.0 Hz, 2H), 1.65 (m, J= 7.7, 6.3 Hz, 2H), 1.60 (d, J = 16.8 Hz, 1H), 1.41 (s, 3H), 1.40 – 1.31 (m,2H), 1.29 (d, J = 5.8 Hz, 2H), 1.27 – 1.19 (m, 2H), 1.16 (s, 3H), 1.14 (s,3H), 1.07 (s, 3H), 1.02 (s, 3H), 0.95 (s, 3H), 0.91 (dt, J = 13.5, 2.5 Hz,2H), 0.77 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.42 (C-3), 173.54 (C-30), 171.71 (C-1), 149.21 (C-9), 145.40 (C-13), 120.44 (C-12), 118.98 (C-11), 116.93 (- CF2), 115.79 (-CN), 112.75 (C-2), 55.39, 48.11, 46.73, 44.76, 44.58,42.82, 42.25, 40.73, 38.13, 31.83, 31.43, 30.86, 28.76, 28.21, 27.40, 27.34,25.67, 25.58, 22.54, 21.66, 21.59, 19.55, 18.24, 14.43. HRMS m / z calcd forC 36 H 46 F2N2NaO2 + [M+Na] + 599.3420, found 599.3425.

[0051] Example 10: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-20-yl)-3-hydroxycyclobutane-1-carboxamide

[0052] Steps A and B: 20-amino-3-oxo-18 β Preparation of 1,9(11),12-triene-2-nitrile; Weigh out 2-cyano-3-oxo-18 β -Oleanane-1,9(11),12-trien-30-carboxylic acid (2.23 g, 4.69 mmol) was dissolved in 150 mL of acetonitrile, and diphenyl azidophosphate (1.55 g, 5.63 mmol) and triethylamine (949.0 mg, 9.38 mmol) were added. The mixture was refluxed and stirred for 1 h. After the reaction was complete, the reaction solution was cooled to room temperature, and 1 mL of concentrated hydrochloric acid was added and stirred at room temperature for 10 min. After the reaction was complete, the reaction solution was poured into 300 mL of water, the pH was adjusted to neutral with saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate (200 mL × 1). The organic layer was washed with water and saturated NaCl aqueous solution, and then dried with anhydrous Na2SO4. The mixture was filtered, the filtrate was evaporated to dryness, and the crude product was precipitated by silica gel column chromatography with dichloromethane:methanol (V:V) = 6:1 as the eluent to obtain 1.76 g of white solid, with a yield of 84%.

[0053] Step C: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-20-yl)-3-hydroxycyclobutane-1-carboxamide; Weigh out 20-amino-3-oxo-18 β -Oleanane-1,9(11),12-trien-2-nitrile (0.2 g, 0.45 mmol) was dissolved in 10 mL of dry DMF, and 3-hydroxycyclobut-1-ylcarboxylic acid (0.06 g, 0.54 mmol), DIPEA (0.24 g, 1.84 mmol), and HATU (0.21 g, 0.54 mmol) were added. After stirring at room temperature for 1 h, the reaction was monitored by TLC (DCM:MeOH = 20:1) and the reaction was complete. 50 mL of water was added, and a solid precipitated. The solid was filtered, washed with water, and dried. PTLC purification yielded 0.048 g of a white solid, yield: 19%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.57 (s, 1H, H-1), 7.17 (s, 1H, -N H CO-),6.18 (d, J = 5.8 Hz, 1H, H-11), 5.57 (d, J = 5.8 Hz, 1H, H-12), 3.10 – 3.05(m, 1H, -C H OH), 2.67 (dd, J = 9.8, 4.8 Hz, 1H, - NHCOC H -), 2.33 (s, 2H), 2.30(td, J = 9.6, 7.1 Hz, 2H), 2.14 – 2.09 (m, 1H), 2.00 – 1.97 (m, 2H), 1.86 –1.80 (m, 2H), 1.77 – 1.73 (m, 2H), 1.59 (d, J = 7.6 Hz, 1H), 1.40 (s, 3H), 1.40 (s, 2H), 1.38 – 1.33 (m, 2H), 1.30 (s, 2H), 1.21 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 0.94 (s, 3H), 0.84 (d, J = 6.9 Hz, 2H), 0.83 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.45 (C-3), 173.30 (-NH CO-), 171.79 (C-1), 148.82 (C-9), 145.13 (C-13), 120.50 (C-12), 118.99 (C-11), 115.86 (-CN), 112.77 (C-2), 62.02, 52.92, 48.12, 44.76, 44.44, 43.29, 42.85, 42.25, 40.70,37.09, 36.79, 36.32, 31.53, 30.88, 30.31, 28.84, 28.36, 28.12, 27.41, 27.01,26.81, 25.54, 21.67, 21.48, 19.72, 18.25. HRMS m / z calcd for C 35 H 48 N2NaO3 + [M+Na] + 567.3557, found 567.3550.

[0054] Example 11: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-20-yl)-4-hydroxycyclohexane-1-formamide

[0055] Following the preparation method of Example 10, the compound of Example 11 was prepared by replacing the 3-hydroxycyclobut-1-carboxylic acid in Example 10 with the 4-hydroxycyclohexyl-1-ylcarboxylic acid. Yield: 15%. 1 H NMR (600 MHz, DMSO- d 6) δ8.58 (s, 1H, H-1), 7.02 (s, 1H, -N H CO-), 6.19 (d, J = 5.9 Hz, 1H, H-11), 5.70 (d, J = 5.9 Hz, 1H, H-12), 4.52 (d, J = 4.5 Hz, 1H, -CHO H ), 2.89 (s, 1H, -C HOH), 2.73 (s, 1H), 2.15 (d, J = 4.2 Hz, 1H), 2.12 (s, 2H), 2.02 – 1.91 (m,2H), 1.85 (s, 2H), 1.81 (s, 2H), 1.75 – 1.71 (m, 2H), 1.66 (s, 2H), 1.65 –1.63 (m, 2H), 1.52 (s, 1H), 1.40 (s, 3H), 1.39 (s, 3H), 1.36 (d, J = 3.2 Hz,2H), 1.24 (t, J = 7.5 Hz, 2H), 1.22 – 1.17 (m, 2H), 1.16 (s, 3H), 1.15 (s,3H), 1.07 (s, 3H), 0.92 (s, 3H), 0.87 – 0.84 (m, 2H), 0.81 (s, 3H). 13 C NMR (151 MHz, DMSO-) d 6 ) δ 198.44 (C-3), 175.25 (-NH C O-), 171.79 (C-1), 148.82 (C-9), 145.09 (C-13), 120.49 (C-12), 119.00 (C-11), 115.85 (-CN), 112.76 (C-2), 68.91, 52.81, 48.13, 44.76, 44.44, 44.06, 43.38, 42.84, 42.25, 40.70, 36.35,36.25, 35.35, 31.55, 31.24, 30.88, 30.74, 28.91, 28.48, 28.43, 28.35, 28.12,27.03, 25.55, 21.67, 21.49, 19.72, 18.26. HRMS m / z calcd for C 37 H 52 N2NaO3 + [M+Na] + 595.3870, found 595.3855.

[0056] Example 12: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-20-yl)-4-hydroxymethylcyclohexane-1-carboxamide

[0057] Following the preparation method of Example 10, the compound of Example 12 was prepared by replacing the 3-hydroxycyclobut-1-carboxylic acid in Example 10 with 4-hydroxymethylcyclohexyl-1-ylcarboxylic acid. Yield: 26%. 1 H NMR (600 MHz, DMSO- d 6) δ8.58 (s, 1H, H-1), 6.99 (s, 1H, -N H CO-), 6.20 (s, 1H, H-11), 5.71 (d, J = 5.8Hz, 1H, H-12), 4.35 (d, J = 5.2 Hz, 1H, -CH2O H ), 3.20 (t, J = 5.7 Hz, 2H, -C H2 OH), 2.17 (dd, J = 12.7, 4.2 Hz, 1H), 2.10 (m, J = 38.3, 13.6, 3.3 Hz, 2H), 1.81 (s, 2H), 1.77 (d, J = 2.2 Hz, 2H), 1.74 (d, J = 3.2 Hz, 2H), 1.73(s, 2H), 1.69 (d, J = 3.8 Hz,2H), 1.65 (d, J = 3.4 Hz, 2H), 1.52 (s,1H), 1.40(s, 3H), 1.39 (s, 2H), 1.34 (dd, J = 13.5, 2.8 Hz, 2H), 1.24 (d, J = 3.9 Hz,2H), 1.16 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 1.04 (m, 1H), 1.02 (m, 1H), 0.93 (s, 3H), 0.85 (t, J=4.4Hz, 2H), 0.82 (s,3H). 13 C NMR (151MHz, DMSO- d 6 ) δ 198.44 (C-3), 175.54 (-NH CO-), 171.79 (C-1), 148.82 (C-9),145.08 (C-13), 120.68 (C-12), 119.00 (C-11), 115.85 (-CN), 112.76 (C-2),67.04, 55.38, 52.77, 48.13, 45.08, 44.76, 44.43, 43.42, 42.84, 42.24, 40.70,36.37, 31.55, 30.88, 30.73, 29.52, 29.18, 28.91, 28.35, 28.13, 27.41, 27.04,26.25, 25.55, 22.57, 21.67, 21.49, 19.73, 18.26. HRMSm / z calcd for C 38 H 54 N2NaO3 + [M+Na] + 609.4027, found 609.4014.

[0058] Example 13: N -(2-cyano-3-oxo-18) β Preparation of oleanolane-1,9(11),12-trien-20-yl)-4-cyanocyclohexane-1-formamide

[0059] Following the preparation method of Example 10, the compound of Example 13 was prepared by replacing the 3-hydroxycyclobut-1-carboxylic acid in Example 10 with 4-cyanocyclohexyl-1-ylcarboxylic acid. Yield: 19%. 1 H NMR (600 MHz, DMSO- d 6) δ8.58 (s, 1H, H-1), 7.08 (s, 1H, -N HCO-), 6.19 (d, J = 5.9 Hz, 1H, H-11), 5.69(d, J = 5.9 Hz, 1H, H-12), 2.67 (tt, J = 11.8, 3.6 Hz, 1H), 2.23 (m, J =11.5, 7.8, 3.7 Hz, 1H), 2.16 – 2.12 (m, 1H), 2.08 – 2.03 (m, 2H), 1.97 (m, J= 27.4, 13.0, 3.6 Hz, 2H), 1.82 (m, J = 13.8, 3.6 Hz, 2H), 1.76 – 1.72 (m,2H), 1.71 (s, 2H), 1.65 (m, J = 11.0, 5.4 Hz, 2H), 1.56 – 1.49 (m, 2H), 1.47(d, J = 3.0 Hz, 1H), 1.40 (s, 3H), 1.39 – 1.37 (m, 2H), 1.30 – 1.26 (m, 2H),1.26 – 1.24 (m, 2H), 1.16 (s, 3H), 1.15 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H),0.92 (s, 3H), 0.86 (s, 2H), 0.81 (s, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 198.45(C-3), 174.55 (-NH C O-), 171.78 (C-1), 148.81 (C-9), 145.16 (C-13), 123.55 (-N(CH2CH2)2CH C N), 120.51 (C-12), 118.97 (C-11), 115.86 (-CN), 112.77 (C-2),52.90, 48.11, 44.76, 44.52, 43.20, 42.96, 42.28, 40.70, 36.34, 31.56, 31.43,30.87, 30.84, 28.91, 28.89, 28.37, 28.31, 28.17, 28.07, 27.41, 27.02, 25.55,22.54, 21.67, 21.49, 19.71,18.25,14.44. HRMS m / z calcd for C 38 H 51 N3NaO2+ [M+Na] + 604.3873, found 604.3879.

[0060] Example 14: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-20-yl)-2,2-difluoropropionamide

[0061] Following the preparation method of Example 10, the compound of Example 14 was prepared by replacing the raw material 3-hydroxycyclobut-1-carboxylic acid in Example 10 with the raw material 2,2-difluoropropionic acid. Yield: 25%. 1 H NMR (600 MHz, DMSO- d 6) δ 8.59 (s,1H, H-1), 7.71 (s, 1H, -N H CO-), 6.20 (d, J = 6.0 Hz, 1H, H-11), 5.76 (d, J =5.9 Hz, 1H, H-12), 2.16 (m, J = 9.8, 2.6 Hz, 1H), 2.00 (m, J = 14.1, 4.1 Hz, 2H), 1.84 – 1.77 (m, 2H), 1.75 (s, 3H), 1.65 (m, J = 7.1, 4.9, 4.2 Hz, 2H), 1.58 (t, J = 13.6 Hz, 1H), 1.41 (s, 3H), 1.38 (d, J = 11.2 Hz, 2H), 1.33 (d,J = 9.7 Hz, 2H), 1.23 (q, J = 4.1, 3.4 Hz, 2H), 1.20 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 0.94 (s, 3H), 0.93 – 0.87 (m, 2H), 0.82 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.45 (C-3), 171.80 (C-1), 164.05 (-NH C O-), 163.86 (- CF2CH3), 148.30 (C-9), 145.24 (C-13), 120.80 (C-12), 118.99 (C-11), 115.99 (-CN), 112.77 (C-2), 54.40, 48.13, 44.76, 44.58, 42.85, 42.53, 42.25,31.48, 30.89, 30.38, 28.83, 28.37, 27.41, 27.04, 26.81, 25.49, 21.91, 21.74,21.67, 21.57, 21.45, 19.69, 18.25. HRMS m / z calcd for C 33 H 44 F2N2NaO2 + [M+Na] + 561.3263, found 561.3268.

[0062] Example 15: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-20-yl)-3,3-difluorocyclobutane-1-carboxamide

[0063] Following the preparation method of Example 10, the compound of Example 15 was prepared by replacing the 3-hydroxycyclobutane-1-carboxylic acid in Example 10 with the raw material 3,3-difluorocyclobutane-1-ylcarboxylic acid. Yield: 19%. 1 H NMR (600 MHz, DMSO- d 6) δ8.57 (s, 1H, H-1), 7.33 (s, 1H, -N HCO-), 6.20 (d, J = 6.0 Hz, 1H, H-11), 5.69(d, J = 5.9 Hz, 1H, H-12), 2.97 (td, J = 8.5, 3.6 Hz, 1H), 2.71 – 2.66 (m,2H), 2.66 – 2.60 (m, 2H), 2.17 – 2.11 (m, 1H), 1.83 – 1.77 (m, 2H), 1.73 (d,J = 6.5 Hz, 2H), 1.65 (d, J = 3.2 Hz, 2H), 1.57 (s, 1H), 1.41 (s, 3H), 1.40(s, 2H), 1.38 – 1.31 (m, 2H), 1.26 – 1.22 (m, 2H), 1.18 (s, 3H), 1.16 (s,3H), 1.14 (s, 3H), 1.07 (s, 3H), 0.93 (s, 3H), 0.86 – 0.83 (m, 2H), 0.82 (s,3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.44 (C-3), 171.75 (C-1), 171.62 (-NH C O-),148.71 (C-9), 145.22 (C-13), 122.28 (- C F2), 120.53 (C-12), 120.39, 118.97 (C-11), 115.86 (-CN), 112.77 (C-2), 53.38, 48.10, 44.76, 44.48, 43.26, 42.85,42.25, 40.70, 38.31, 36.28, 34.67, 31.57, 30.87, 30.73, 28.78, 28.33, 27.94,27.41, 26.96, 26.81, 25.53, 21.67, 21.51, 19.74, 18.25. HRMS m / z calcd forC 35 H 46 F2N2NaO2 + [M+Na] + 587.3420, found 587.3435。

[0064] Example 16: N-(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-20-yl)-4,4-difluorocyclohexane-1-formamide

[0065] Following the preparation method of Example 10, the compound of Example 16 was prepared by replacing the 3-hydroxycyclobut-1-carboxylic acid in Example 10 with 4,4-difluorocyclohexyl-1-ylcarboxylic acid. Yield: 26%. 1 H NMR (600 MHz, DMSO- d 6) δ8.58 (s, 1H, H-1), 7.16 (s, 1H, -N H CO-), 6.20 (d, J = 5.9 Hz, 1H, H-11), 5.69(d, J = 5.9 Hz, 1H, H-12), 2.38 – 2.32 (m, 1H), 2.18 – 2.11 (m, 1H), 2.06(dq, J = 9.7, 3.1 Hz, 2H), 1.82 (s, 2H), 1.78 (s, 2H), 1.76 – 1.74 (m, 2H), 1.72 (t, J = 7.4 Hz, 2H), 1.67 – 1.63 (m, 2H), 1.63 – 1.59 (m, 2H), 1.54 (t,J = 13.5 Hz, 1H), 1.40 (s, 3H), 1.40 (s, 2H), 1.28 (dd, J = 13.6, 3.7 Hz,2H), 1.25 – 1.23 (m, 2H), 1.16 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s,3H), 0.93 (s, 3H), 0.87 – 0.83 (m, 2H), 0.82 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.43 (C-3), 174.01 (-NH C O-), 171.76 (C-1), 148.78 (C-9), 145.15 (C-13), 124.25 (- CF2), 120.51 (C-12), 118.98 (C-11), 115.85 (-CN), 112.77 (C-2), 53.02, 48.13, 44.76, 44.57, 43.21, 42.84, 42.24, 41.82, 40.71, 36.35, 32.97,32.81, 32.65, 31.57, 30.86, 28.87, 28.33, 28.04, 27.41, 27.01, 26.42, 26.27,25.54, 21.67, 21.56, 19.72, 18.26. HRMSm / z calcd for C 37 H 50 F2N2NaO2 + [M+Na] + 615.3733, found 615.3727.

[0066] Example 17: N -(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-20-yl)-1-methylpyrrolidine-2-carboxamide

[0067] Following the preparation method of Example 10, using raw materials N The compound of Example 17 was prepared by replacing the starting material 3-hydroxycyclobut-1-methylcarboxylic acid in Example 10 with methyltetrahydropyrrole-2-ylcarboxylic acid. Yield: 25%. 1 H NMR (600 MHz, DMSO- d 6)δ 8.56 (s, 1H, H-1), 7.91 (d, J = 6.5 Hz, 1H, -N H -NHCOC H-), 2.96 – 2.78 (m, 3H), 2.66 – 2.54 (m, 2H), 2.51 (m, 2H), 2.05(dd, J = 13.4, 4.6 Hz, 1H), 1.98 (td, J = 13.6, 4.5 Hz, 2H), 1.91 – 1.85 (m,2H), 1.82 (m, J = 13.4, 8.2, 5.1 Hz, 2H), 1.73 (q, J = 7.0 Hz, 2H), 1.65 (dt,J = 10.7, 5.4 Hz, 2H), 1.53 (t, J = 13.5 Hz, 1H), 1.41 (s, 3H), 1.40 – 1.36(m, 1H), 1.28 (s, 1H), 1.27 (s, 2H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s,3H), 1.00 (d, J = 2.6 Hz, 3H), 0.95 (s, 3H), 0.86 – 0.82 (m, 2H), 0.76 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.44 (C-3), 172.70 (-NH C O-), 171.74 (C-1), 148.46 (C-9), 145.45 (C-13), 120.54 (C-12), 118.90 (C-11), 115.86 (-CN), 112.81 (C-2), 69.61, 56.47, 52.44, 48.10, 45.47, 44.77, 42.88, 42.26, 41.63,40.76, 40.41, 36.42, 31.90, 31.62, 31.10, 30.87, 28.84, 28.43, 27.82, 27.44,26.82, 25.52, 24.22, 21.68, 21.47, 19.70, 18.24. HRMS m / z calcd for C 36 H 52 N3O2 + [M+H] + 558.4054, found 558.4044.

[0068] Example 18: 1-(2-cyano-3-oxo-18) βPreparation of -oleanolane-1,9(11),12-trien-20-yl)-3-(3-hydroxyazacyclobutane-1-yl)urea

[0069] 2-cyano-3-oxo-18 β -Oleanane-1,9(11),12-trien-30-carboxylic acid (150.0 mg, 0.32 mmol) was dissolved in 15 mL of acetonitrile. Diphenyl azidophosphate (105.0 mg, 0.38 mmol) and triethylamine (64.0 mg, 0.63 mmol) were added, and the mixture was refluxed and stirred for 30 min. After the reaction was complete, 3-hydroxyazacyclobutylamine (0.04 g, 0.51 mmol), DIPEA (0.24 g, 1.84 mmol), and HATU (0.21 g, 0.54 mmol) were added. The mixture was stirred at room temperature for 1 h, and TLC monitoring (DCM:MeOH = 20:1) showed that the reaction was complete. 50 mL of water was added, and a solid precipitated. The solid was filtered, washed with water, and dried. PTLC purification yielded 0.048 g of a white solid, yield: 27%. 1 H NMR (600 MHz, DMSO- d 6 ) δ 8.58 (s, 1H, H-1), 6.19 (d, J =5.9 Hz, 1H, H-11), 5.72 (d, J = 5.9 Hz, 1H, H-12), 5.51 (d, J = 6.3 Hz, 1H, -CHO H ), 5.35 (s, 1H, -N H CO-), 4.36–4.32 (m, 1H, -C H OH), 3.97 (t, J = 7.6 Hz,2H, -N(C H2 )2CH-), 3.57 (t, J = 7.7 Hz, 2H, -N(C H2)2CH-), 2.16 (dd, J = 13.4,4.6 Hz, 2H), 2.00–1.93 (m, 1H), 1.89 (d, J = 13.5 Hz, 1H), 1.85–1.78 (m, 2H),1.73 (t, J = 6.8 Hz, 2H), 1.54–1.44 (m, 3H), 1.40 (s, 3H), 1.24 (m, 2H), 1.16 (s, 6H), 1.15 (s, 3H), 1.07 (s, 3H), 1.05 – 0.96 (m, 2H), 0.93 (s, 3H), 0.84(s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.47 (C-3), 171.83 (C-1), 160.19 (-NH C O-), 148.87 (C-9), 145.06 (C-13), 120.61 (C-12), 118.97 (C-11), 115.88 (-CN), 112.76 (C-2), 60.02, 59.80, 55.39, 52.44, 48.13, 44.76, 44.58, 43.49,42.86, 42.25, 40.74, 36.45, 31.61, 31.35, 30.90, 28.96, 28.87, 28.41, 27.43,27.15, 25.55, 21.68, 21.44, 19.73, 18.26. HRMS m / z calcd for C 34 H 47 N3O3 + [M+H] + 546.3695, found 546.3701.

[0070] Example 19: 1-(2-cyano-3-oxo-18) β Preparation of -oleanolane-1,9(11),12-trien-20-yl)-3-(4-hydroxypiperidin-1-yl)urea

[0071] Following the preparation method of Example 18, the starting material 3-hydroxyazacyclobutamine in Example 18 was replaced with 4-hydroxypiperidine to prepare the compound of Example 19. Yield: 26%. 1H NMR (600 MHz, DMSO- d 6 ) δ 8.58 (s, 1H, H-1), 6.19 (d, J = 5.9 Hz, 1H, H-11), 5.72 (d, J = 5.8 Hz, 1H, H-12), 5.60 (s,1H, -N H CO-), 4.66 (d, J = 4.2 Hz, 1H, -CHO H ), 3.71 (dd, J = 11.3, 7.0 Hz, 2H,-N(C H2 CH2)2CH-), 3.62–3.56 (m, 1H, -C H OH ), 2.87–2.78 (m, 2H, -N(C H2 CH2)2CH-),2.14 (dd, J = 13.2, 4.4 Hz, 1H), 2.06–2.02 (m, 1H), 2.02–1.91 (m, 2H), 1.85–1.77 (m, 1H), 1.77–1.69 (m, 2H), 1.68 (d, J = 4.0 Hz, 1H), 1.67–1.63 (m, 3H),1.54(m, 1H), 1.46(m, 1H),1.40(m, 3H), 1.39 – 1.31 (m, 1H), 1.30 (t, J = 13.4Hz, 2H), 1.29–1.25 (m, 1H), 1.25 (s, 1H), 1.23 (s, 3H), 1.18 (s, 3H), 1.16(s, 3H), 1.14 (s, 3H), 1.07 (s, 1H), 1.02 (dd, J = 14.0, 3.6 Hz, 3H), 0.93(s, 1H), 0.82 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.47 (C-13), 171.83 (C-1), 157.45 (-NH CO-), 148.92 (C-9), 145.03 (C-13), 120.54 (C-12), 118.99 (C-11), 115.88 (-CN), 112.76 (C-2), 66.88, 55.39, 52.61, 48.14, 44.83, 44.76,43.56, 42.85, 42.79, 42.25, 40.78, 36.57, 34.82, 31.64, 31.43, 30.90, 29.02,28.88, 28.41, 27.44, 27.21, 25.55, 21.68, 21.42, 19.69, 18.27. HRMS m / z calcdfor C 36 H 51 N3O3 + [M+H] + 574.4008, found 574.4011.

[0072] Example 20: 1-(2-cyano-3-oxo-18) β Preparation of oleanolic acid-1,9(11),12-trien-20-yl)-3-(4-hydroxymethylpiperidin-1-yl)urea

[0073] Following the preparation method of Example 18, the compound of Example 20 was prepared by replacing the 3-hydroxyazacyclobutamine in Example 18 with 4-piperidinemethanol. Yield: 24%. 1 H NMR (600 MHz, DMSO- d6 ) δ 8.58 (s, 1H,H-1), 6.18 (d, J = 5.8 Hz, 1H, H-11), 5.72 (d, J = 5.8 Hz, 1H, H-12), 5.54(s, 1H, -N H CO-), 4.45 (t, J = 5.3 Hz, 1H, -CH2O H ), 3.95 (d, J = 13.1 Hz, 2H,-N(C H2 CH2)2-), 3.22 (t, J = 5.8 Hz, 2H, -C H2 OH), 2.65–2.56 (m, 2H, -N(C H2CH2)2-), 2.14 (d, J = 9.0 Hz, 1H), 2.07–2.02 (m, 1H), 2.02–1.91 (m, 2H), 1.85–1.78 (m, 1H), 1.77–1.67 (m, 2H), 1.65 (t, J = 4.4 Hz, 2H), 1.59 (d, J =7.0 Hz, 2H), 1.51 (t, J = 13.2 Hz, 2H), 1.40 (s, 3H), 1.40 (s, 3H), 1.38 –1.37 (m, 1H), 1.18 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 1.04 –1.00 (m, 2H), 0.98 (d, J = 2.9 Hz, 2H), 0.93 (s, 3H), 0.82 (s, 3H). 13 C NMR (151 MHz, DMSO-) d 6 ) δ 198.46 (C-3), 171.82 (C-1), 157.49 (-NH C O-), 148.93 (C-9), 145.02 (C-13), 120.52 (C-12), 118.99 (C-11), 115.87 (-CN), 112.76 (C-2), 66.48, 55.38, 52.61, 48.14, 44.88, 44.76, 43.62, 42.85, 42.25, 41.26, 40.78,39.15, 36.57, 34.67, 31.63, 31.44, 31.42, 30.90, 29.08, 28.99, 28.93, 28.38,27.43, 22.98, 22.54, 21.67, 21.44, 19.68, 18.27. HRMS m / z calcd for C 37 H 53 N3O3 + [M+H] + 588.4175, found 588.4158.

[0074] Example 21: 1-(2-cyano-3-oxo-18) β Preparation of -oleanolane-1,9(11),12-trien-20-yl)-3-(4-cyanopiperidin-1-yl)urea

[0075] Following the preparation method of Example 18, the starting material 3-hydroxyazacyclobutamine in Example 18 was replaced with piperidine-4-carboxynitrile to prepare the compound of Example 21. Yield: 21%. 1 H NMR (600 MHz, DMSO- d6 ) δ 8.58 (s, 1H,H-1), 6.19 (d, J = 6.0 Hz, 1H, H-11), 5.73 (s, 1H, -N H CO-), 5.71 (d, J = 6.1Hz, 1H, H-12), 3.57–3.52 (m, 2H, -N(C H2 CH2)2-), 3.15 – 3.09 (m, 2H, -N(C H2 CH2)2-), 3.07–3.02 (m, 1H, -N(CH2CH2)2C H -), 2.15–2.11 (m, 1H), 2.04 (dd, J= 13.3, 3.2 Hz, 1H), 2.01–1.95 (m, 1H), 1.95–1.90 (m, 1H), 1.85–1.83 (m, 1H),1.82–1.78 (m, 2H), 1.76 –1.69 (m, 2H), 1.68–1.62 (m, 2H), 1.62–1.58 (m, 2H), 1.53 (t, J = 13.4 Hz, 1H), 1.40 (s, 3H), 1.28 (d, J = 3.8 Hz, 1H), 1.26 (d, J= 3.8 Hz, 1H), 1.24–1.22 (m, 1H), 1.18 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 1.05–1.01 (m, 1H), 0.93 (s, 3H), 0.93–0.87 (m, 1H), 0.85 (s,1H), 0.83 (s, 3H). 13 C NMR (151 MHz, DMSO- d6 ) δ 198.47 (C-3), 171.81 (C-1), 157.36 (-NH C O-), 148.86 (C-9), 145.09 (C-13), 122.69 (-N(CH2CH2)2CH CN), 120.56(C-12), 118.96 (C-11), 115.88 (-CN), 112.77 (C-2), 55.39, 52.77, 48.13,44.85, 44.76, 43.56, 43.27, 42.85, 42.25, 40.78, 36.57, 31.64, 31.29, 30.90,29.02, 28.81, 28.48, 28.40, 27.43, 27.20, 27.02, 25.96, 25.54, 21.68, 21.44,19.69, 18.26. HRMS m / z calcd for C 37 H 50 N4NaO2 + [M+Na] + 605.3832, found 605.3880.

[0076] Example 22: 1-(2-cyano-3-oxo-18) β Preparation of -oleanolane-1,9(11),12-trien-20-yl)-3-(6-(hydroxymethyl)-3-azabicyclo[3.1.0]hexane-3-yl)urea

[0077] Following the preparation method of Example 18, the starting material 3-hydroxyazacyclobutylamine in Example 18 was replaced with 3-azabicyclo[3.1.0]hex-6-ylmethanol to prepare the compound of Example 22. Yield: 26%. 1 H NMR (600 MHz, DMSO- d6 ) δ 8.58 (s, 1H, H-1), 6.18 (d, J = 5.9 Hz, 1H, H-11), 5.71 (d, J = 5.9 Hz,1H, H-12), 5.02 (s, 1H, -N H CO-), 4.49 (t, J = 5.5 Hz, 1H, -CH2O H ), 3.56 (d, J= 10.1 Hz, 1H), 3.47 (d, J = 10.2 Hz, 1H), 3.29 (t, J = 6.1 Hz, 2H, -C H2 OH), 3.26–3.18 (m, 2H, -N(C) H2CH)2-), 2.13 (dd, J = 13.3, 4.5 Hz, 1H), 2.04 (dd, J =13.2, 3.3 Hz, 1H), 2.00–1.92 (m, 1H), 1.89–1.84 (m, 1H), 1.81 (td, J = 13.8,3.0 Hz, 2H), 1.78–1.65 (m, 2H), 1.66–1.63 (m, 1H), 1.63–1.42 (m, 1H), 1.40 (s, 6H), 1.24 – 1.22 (m, 1H), 1.17 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07(s, 3H), 1.03 (q, J = 4.3, 3.3 Hz, 1H), 1.01 (s, 1H), 0.94 (d, J = 6.7 Hz,1H), 0.92 (s, 3H), 0.85 (s, 1H), 0.84 (s, 1H), 0.83 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.45 (C-3), 171.82 (C-1), 156.98 (-NH C O-), 148.79 (C-9), 145.06(C-13), 120.63 (C-12), 118.99 (C-11), 115.88 (-CN), 112.76 (C-2), 62.48,52.67, 48.40, 48.25, 48.14, 44.76, 43.79, 42.85, 42.25, 41.26, 40.76, 36.51,34.67, 31.62, 30.91, 29.01, 28.90, 28.39, 27.44, 25.54, 25.41, 25.26, 22.96,21.68, 21.44, 21.02 (d, J = 18.1 Hz), 19.71, 18.27. HRMS m / z calcd forC 37 H 51 N3NaO3 + [M+Na] + 608.3828, found 608.3825.

[0078] Example 23: 1-(2-cyano-3-oxo-18) βPreparation of oleanolic acid-1,9(11),12-trien-20-yl)-3-(2,2-difluoropropyl)urea

[0079] Following the preparation method of Example 18, the compound of Example 23 was prepared by replacing the 3-hydroxyazacyclobutamine in Example 18 with 2,2-difluoropropylamine hydrochloride. Yield: 35%. 1 H (600 MHz, DMSO- d 6 ) δ 8.56 (s,1H, H-1), 6.19 (d, J = 5.9 Hz, 1H, H-11), 6.14 (t, J = 6.4 Hz, 1H, -NHCON H -),5.71 (s, 1H, -N H CONH-), 5.58 (d, J = 5.7 Hz, 1H, H-12), 3.49–3.36 (m, 2H, -NHC H2 CF2CH3), 2.18–2.15 (m, 1H), 2.00 – 1.95 (m, 1H), 1.85–1.74 (m, 2H), 1.68–1.62 (m, 1H), 1.60 – 1.51 (m, 2H), 1.47 – 1.44 (m, 2H), 1.40 (s, 1H), 1.33–1.24 (m, 3H), 1.20 (s, 3H), 1.16 (s, 3H), 1.15 (s, 3H), 1.07 (s, 1H), 1.05–1.03 (m, 3H), 0.94 (s, 3H), 0.90 – 0.86 (m, 1H), 0.84 (s, 3H). 13 C NMR (151MHz, DMSO- d 6 ) δ 197.97 (C-3), 171.24 (C-1), 156.77 (-NH CO-), 148.46 (C-9),144.81 (C-13), 123.64 (-CF2CH3), 119.82 (C-12), 118.48 (C-11), 115.37 (-CN),112.30 (C-2), 51.62, 47.59, 44.29, 44.26, 43.84, 43.63, 42.36, 41.77, 40.28,35.68, 31.22, 30.95, 30.37, 28.36, 28.24, 27.81, 26.92, 26.43, 25.07, 21.20,21.06, 19.28, 17.77, 13.96. HRMS m / z calcd for C 34 H 47 F2N3NaO2 + [M+Na] + 590.3534, found 590.3597.

[0080] Example 24: 1-(2-cyano-3-oxo-18) β Preparation of -oleanolane-1,9(11),12-trien-20-yl)-3-(3,3-difluorozacriane-1-yl)urea

[0081] Following the preparation method of Example 18, the compound of Example 24 was prepared by replacing the 3-hydroxyazacyclobutamine in Example 18 with 3,3-difluoropiperidine. Yield: 36%. 1 H NMR (600 MHz, DMSO- d6) δ 8.58 (s, 1H), 6.19 (d, J = 5.9 Hz, 1H), 6.00 (s, 1H), 5.69 (d, J = 5.8 Hz, 1H), 4.22 – 4.16 (m, 4H), 2.16 – 2.13 (m, 1H), 1.98 – 1.95 (m, 2H), 1.89 – 1.82 (m, 1H), 1.74– 1.72 (m, 2H), 1.66 – 1.63 (m, 2H), 1.57 – 1.52 (m, 1H), 1.40 (s, 3H), 1.31– 1.24 (m, 4H), 1.18 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 1.05– 1.02 (m, 1H), 0.93 (s, 3H), 0.86 – 0.88 (m, 2H), 0.84 (s, 3H). 13 C NMR (151MHz, DMSO- d 6 ) δ 197.99, 171.31, 159.03, 148.32, 144.69, 120.12, 118.48,116.39, 115.40, 112.29, 61.09, 60.91, 52.43, 47.65, 44.29, 44.12, 42.79,42.39, 41.77, 40.25, 35.91, 31.15, 30.96, 30.41, 28.40, 28.09, 27.91, 26.94,26.63, 25.07, 21.20, 21.00, 19.25, 13.96. HRMS m / z calcd for C 34 H 45 F2N3NaO2 + [M+Na] + 588.3372, found 588.3298.

[0082] Example 25: 1-(2-cyano-3-oxo-18) β Preparation of -oleanolic acid-1,9(11),12-trien-20-yl)-3-(4,4-difluoropiperidin-1-yl)urea

[0083] Following the preparation method of Example 18, the compound of Example 25 was prepared by replacing the 3-hydroxyazacyclobutamine in Example 18 with 4,4-difluoropiperidine. Yield: 29%. 1 H NMR (600 MHz, DMSO- d 6 ) δ 8.58 (s, 1H,H-1), 6.19 (d, J = 5.9 Hz, 1H, H-11), 5.92 (s, 1H, -N H CONH-), 5.69 (d, J =5.8 Hz, 1H, H-12), 3.42 (t, J = 5.7 Hz, 4H, -N(CH2C H2 )2CF2, 2.13 – 2.10 (m,1H), 2.05 – 2.02 (m, 1H), 1.99 – 1.96 (m, 1H), 1.93 – 1.86 (m, 4H), 1.84 –1.79 (m, 1H), 1.74 – 1.71 (m, 2H), 1.67 – 1.63 (m, 2H), 1.56 – 1.51 (m, 1H), 1.40 (s, 3H), 1.30 – 1.24 (m, 4H), 1.19 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 1.04 – 1.02 (m, 1H), 0.93 (s, 3H), 0.87 – 0.84 (m, 2H), 0.82 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 197.98 (C-3), 171.31 (C-1), 156.63 (-NH CO-), 148.36 (C-9), 144.66 (C-13), 122.95 (-CF2), 120.04 (C-12), 118.46 (C-11), 115.40 (-CN), 112.29 (C-2), 52.40, 47.65, 44.46, 44.28, 42.94, 42.36,41.77, 40.30, 36.10, 33.60, 33.45, 33.31, 31.16, 30.95, 30.86, 30.40, 28.52,28.24, 27.90, 26.94, 26.70, 25.06, 21.19, 20.96, 19.20, 17.77. HRMS m / z calcdfor C 36 H 49 F2N3NaO2 + [M+Na] + 616.3691, found 616.3728.

[0084] Example 26: 1-(2-cyano-3-oxo-18) β Preparation of -oleanolane-1,9(11),12-trien-20-yl)-3-(6,6-difluoro-3-azabicyclo[3.1.0]hexane-3-yl)urea

[0085] Following the preparation method of Example 18, the starting material 3-hydroxyazacyclobutane in Example 18 was replaced with 6,6-difluoro-3-azabicyclo[3.1.0]hexane to prepare the compound of Example 26. Yield: 27%. 1 H NMR (600 MHz, DMSO- d6 ) δ 8.58 (s, 1H, H-1), 6.18 (d, J = 5.9 Hz, 1H, H-11), 5.69 (d, J = 5.8 Hz,1H, H-12), 5.21 (s, 1H, -N H CON-), 3.76 (d, J = 10.9 Hz, 1H, -N(C H 2)2-), 3.70(d, J = 10.9 Hz, 1H, -N(C H 2)2-), 3.51 (t, J = 11.0 Hz, 2H, -N(C H2)2-), 2.13(dd, J = 12.7, 4.0 Hz, 1H), 2.01 (dd, J = 10.6, 2.8 Hz, 1H), 1.96 (dd, J =13.6, 4.4 Hz, 1H), 1.91 – 1.86 (m, 1H), 1.85 – 1.77 (m, 1H), 1.75–1.69 (m,2H), 1.66–1.63 (m, 2H), 1.51 (t, J = 13.5 Hz, 1H), 1.45 – 1.41 (m, 1H), 1.40 (s, 1H), 1.38 –1.36 (m,1H), 1.28 – 1.24 (m,1H), 1.23 (s, 1H), 1.18 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 1.04 – 1.00 (m, 1H), 0.92 (s, 3H), 0.90 – 0.86 (m, 1H), 0.83 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.47 (C-3),171.83 (C-1), 155.90 (-NH C O-), 148.83 (C-9), 145.07 (C-13), 120.57 (C-12), 118.98 (C-11), 115.88 (-CN), 112.76 (C-2), 55.39, 52.82, 48.13, 45.77, 45.73,44.76, 44.70, 43.55, 42.85, 42.25, 40.76, 40.53, 36.45, 31.60, 31.31, 30.90,28.98, 28.77, 28.40, 27.43, 27.20, 26.06, 25.54, 21.68, 21.45, 19.69, 18.26.HRMS m / z calcd for C 36 H 47 F2N3NaO2 + [M+Na] + 614.3534, found 614.3567.

[0086] Example 27: 1-(2-cyano-3-oxo-18) βPreparation of -oleanolane-1,9(11),12-trien-20-yl)-3-(1,1-dioxide thiomorpholin-4-yl)urea

[0087] Following the preparation method of Example 18, the starting material 3-hydroxyazacyclobutamine in Example 18 was replaced with thiomorpholine-1,1-dioxide to prepare the compound of Example 27. Yield: 22%. 1 H NMR (600 MHz, DMSO- d6 ) δ8.57 (s, 1H, H-1), 6.19 (d, J = 5.9 Hz, 1H, H-11), 6.08 (s, 1H, -N H CON-),5.69 (d, J = 5.9 Hz, 1H, H-12), 3.76 (t, J = 5.3 Hz, 4H, -N(C H2)2 -), 3.06 (t,J = 5.2 Hz, 4H, -(C H2 ) 2 SO2), 2.10 (dd, J = 13.2, 4.4 Hz, 1H), 2.05–2.00 (m,1H), 1.99–1.97 (m, 1H), 1.96–1.94 (m, 1H), 1.85–1.79 (m, 1H), 1.76–1.69 (m,2H), 1.65 (q, J = 6.1 Hz, 2H), 1.55 (t, J = 13.5 Hz, 1H), 1.41 (s, 3H), 1.32–1.27 (m, 1H), 1.24 (d, J = 6.8 Hz, 1H), 1.19 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.07 (s, 3H), 1.06 – 1.01 (m, 2H), 0.93 (s, 3H), 0.92–0.88 (m, 1H), 0.85 (s, 1H), 0.83 (s, 3H). 13 C NMR (151 MHz, DMSO- d 6 ) δ 198.45 (C-3), 171.77 (C-30), 156.49 (-NH CO-), 148.77 (C-9), 145.20 (C-13), 120.57 (C-12), 118.93 (C-11), 115.88 (-CN), 112.78 (C-2), 55.38, 53.18, 51.72, 48.13, 45.02, 44.76, 43.71,43.23, 42.26, 40.77, 36.61, 34.67, 31.67, 31.38, 30.89, 29.00, 28.63, 28.38,27.42, 27.17, 25.53, 22.96, 21.67, 21.45, 19.69, 18.25. HRMS m / z calcd forC 35 H 49 N3NaO4S + [M+Na] + 630.3342, found 630.3367.

[0088] Test Example 1: Pharmacological Study of the Compounds of the Invention: 1. Evaluation of target degradation activity; To evaluate the degradation ability of the target compounds on HDAC3 protein, Western blot analysis was performed. Human THP-1 cells were treated with 2.5 μM and 5 μM concentrations of compounds 1-27 (examples) for 24 hours, and changes in HDAC3 protein levels were detected. The results showed that some of the example compounds could effectively degrade HDAC3 protein at the stated concentrations, with several compounds exhibiting significant degradation capabilities.

[0089] The specific experimental procedure includes: (1) Cell culture: THP-1 cells were cultured in RPMI medium and verified by STR genotyping. The medium was supplemented with 10% (V / V) fetal bovine serum and 100 U / mL penicillin-100 μg / mL streptomycin. All cells used in the experiment were cultured in an incubator at 37℃ and 5% CO2 saturated humidity.

[0090] (2) Western Blot experiment: After THP-1 cells were co-incubated with the specified concentration of the compound from the example for 24 hours, the cell pellet was collected by centrifugation. Cells were lysed on ice for 30 minutes using RIPA lysis buffer, and then the supernatant was collected by centrifugation at 12,000 rpm for 5 minutes at 4°C. Protein concentration was determined using a BCA protein quantification kit, and the corresponding loading amount was calculated based on the determination results. The protein sample was mixed with 5× loading buffer at a ratio of 4:1 and heated in a metal bath at 95°C for 5 minutes to complete protein denaturation. Protein separation was performed using a discontinuous SDS-polyacrylamide gel electrophoresis system, and then the protein was transferred to a 0.45 μm PVDF membrane by wet transfer. After the transfer, the PVDF membrane was first washed with TBST buffer, and then blocked with TBST buffer containing 5% skim milk powder at room temperature for 1.5 hours. Then, specific primary antibody was added and incubated overnight at 4°C. The next day, after thorough washing with TBST, HRP-labeled secondary antibody of the corresponding species was added and incubated at room temperature for 1.5 hours. After TBST cleaning, the membrane surface was covered with an ECL chemiluminescence reagent kit (equal volumes of solution A and solution B were mixed), and finally the signal was acquired using a fully automated chemiluminescence imaging system.

[0091] Table 1: Efficiency of compound 1-27 in degrading HDAC3 protein at 2.5 μM and 5 μM

[0092] 2. Study on the mechanism of action of the compound in Example 8; Studies have shown that HDAC3 plays a crucial role in the innate immune response by regulating mitochondrial metabolism and promoting NLRP3 inflammasome activation. Particularly in macrophages, HDAC3-mediated mitochondrial remodeling promotes NLRP3 inflammasome-dependent IL-1β maturation and secretion. To investigate whether the compounds in the examples exert their anti-inflammatory effects by degrading HDAC3 and thus inhibiting NLRP3 inflammasome activation, we conducted a mechanistic study in THP-1-derived macrophages. Figure 1 As shown in Figure A, KEGG combined enrichment analysis of transcriptomic and proteomic data from the drug-treated group and the model group revealed that the anti-inflammatory effect of the compound in Example 8 was closely related to the NOD-like receptor signaling pathway. Further experimental results showed that ( Figure 1 B) LPS / ATP stimulation significantly promoted the increase of extracellular maturation levels of IL-1β and caspase-1, while Example 8 induced HDAC3 degradation in a dose-dependent manner and simultaneously inhibited the release of IL-1β and caspase-1. These results indicate that Example 8, by specifically degrading HDAC3, blocked the activation process of the NLRP3 inflammasome, providing a precise mechanistic explanation for its anti-inflammatory effect and laying a theoretical foundation for targeted therapy of related inflammatory diseases.

[0093] The specific experimental procedure includes: (1) Quantitative proteomics analysis; After removing the sample cells from the -80℃ freezer, an appropriate amount of lysis buffer (8M urea, 1 mM PMSF, 2 mM EDTA) was added, and the cells were sonicated on ice for 5 minutes. The supernatant was then collected by centrifugation at 15000 g for 10 minutes at 4℃. Protein concentration was determined using a BCA kit. 100 μg of protein solution was taken, and the volume was adjusted to 200 μL with 8 M urea. DTT (final concentration 5 mM) was added, and the solution was reduced at 37℃ for 45 minutes. Subsequently, alkylation was performed at room temperature in the dark with iodoacetamide (final concentration 11 mM) for 15 minutes. 800 μL of 25 mM ammonium bicarbonate solution and 2 μL of trypsin were added, and the solution was incubated overnight at 37℃. The digested peptides were adjusted to pH 2-3 with 20% TFA and then desalted using C18 resin. Finally, the peptide concentration was determined using a quantitative peptide assay kit.

[0094] (2) Liquid chromatography-mass spectrometry detection and mRNA sequencing analysis; Peptide samples were separated using an ultra-high performance liquid chromatography (UHPLC) system. Data acquisition-independent mass spectrometry analysis was performed on a high-resolution mass spectrometer. The obtained raw mass spectrometry data were analyzed using a library-free method with DIA-NN software. Simultaneously, total RNA was extracted using the phenol-guanidine isothiocyanate method and dissolved in DEPC water. RNA concentration was precisely quantified using a fluorometer, and RNA integrity values ​​(RIN values) were detected using a high-throughput biological fragment analyzer to ensure sample quality met library construction standards. Qualified samples were used to construct mRNA libraries. After quality control, the libraries were mixed according to their effective concentration and target sequencing data volume, and sequencing was performed to generate 150 bp paired-end reads. This sequencing technology is based on the principle of simultaneous synthesis sequencing: four fluorescently labeled dNTPs, DNA polymerase, and adaptor primers were added to the sequencing flow cell for amplification. During the extension of the complementary strand within each sequencing cluster, the addition of a fluorescently labeled dNTP releases a corresponding fluorescence signal; the sequencer captures these signals, and computer software converts them into a sequencing peak diagram, thereby obtaining the sequence information of the target fragment.

[0095] 3. Evaluation of the in vivo anti-inflammatory activity of the compound in preferred embodiment 8; To verify the in vivo anti-inflammatory efficacy of the compound in Example 8, this invention first used an LPS-induced endotoxin shock model to evaluate the systemic inflammatory response: Treatment with Example 8 (15 mg / kg, ip) resulted in a 75% survival rate in mice after 96 hours, significantly improving lethal inflammatory damage. Figure 3Furthermore, in a DSS-induced acute colitis model, treatment with Example 8 (10 mg / kg, ip) significantly inhibited disease progression, with the colon length recovering from 4.46 cm in the model group to 5.76 cm in the treated group. Pathological examination also showed restoration of mucosal integrity and an increase in the number of goblet cells. Figure 2 , Figure 4 ). With the lead compound COOTO-Me: This serves as the control group.

[0096] The specific experimental procedure includes: The animals used in the experiment were male C57Bl / 6 mice (SPF grade, weight 20±2g, 8 weeks old) purchased from Liaoning Changsheng Biotechnology Co., Ltd. (laboratory animal production license). The mice were housed in a barrier environment at the SPF-grade animal experimental center of Shenyang Pharmaceutical University with a temperature of 22±1℃, humidity of 50±10%, and a 12-hour light / dark cycle.

[0097] (1) Acute shock model; After 7 days of acclimatization, mice were randomly divided into groups of 10 each. The mice were administered LPS saline solution via intraperitoneal injection (7.5 / 15 mg / kg). Two hours later, LPS saline solution was injected intraperitoneally at a dose of 20 mg / kg. Survival status and clinical symptoms (such as piloerection, respiratory distress, and decreased activity) were observed every 12 hours after LPS injection. The survival data for each group were plotted as Kaplan-Meier curves for analysis.

[0098] (2) Acute colitis model; After 7 days of environmental acclimatization, mice were randomly assigned to groups (n=7) and acute colitis was induced by 2.5% (w / v) sodium dextran sulfate (DSS). Mice had free access to drinking water from days 1 to 3; from days 4 to 10, the water was replaced with 2.5% DSS solution; and from days 11 to 13, drinking water was restored. The compound (5 / 10 mg / kg) or solvent control group was administered intraperitoneally for 13 consecutive days. Daily assessment indicators included weight change, fecal characteristics score, and benzidine occult blood test. On day 13, mice were sacrificed, colon length was measured, and representative colonic segments were fixed in 4% paraformaldehyde for hematoxylin-eosin (H&E) histopathological analysis.

Claims

1. The 18-membered diene-containing segment represented by general formula I β - Glycyrrhetinic acid derivatives or their optical isomers or pharmaceutically acceptable salts: in, R1 is -C(O)NR2R3, -NHC(O)R4 or -NHC(O)NR5R6; R2 and R3 may be the same or different, and each is independently selected from hydrogen, substituted or unsubstituted C3-C5 alkyl, substituted or unsubstituted C3-C5 cycloalkane group, or R2 and R3 with the N atom attached to them to form a substituted or unsubstituted C4-C6 saturated heterocyclic group, substituted or unsubstituted C5 bridged ring group; the C4-C6 saturated heterocyclic group includes piperidinyl, aziridine butyl, thiomorpholinyl; the C5 bridged ring group is 3-azirbicyclo[3.1.0]hexyl; the C3-C5 alkyl includes propyl, butyl, pentyl; the substituent is one or more, selected from hydroxyl, halogen atom, hydroxymethyl, cyano; R4 is selected from monosubstituted or disubstituted C3-C6 cycloalkane groups, disubstituted C3-C6 alkyl groups, and monosubstituted or disubstituted five- or six-membered heterocyclic groups; the C3-C6 cycloalkane group includes cyclobutyl, cyclopentyl, and cyclohexyl; the C3-C6 alkyl group includes propyl, butyl, and pentyl; the five- or six-membered heterocyclic group includes pyrrolidinyl, piperidinyl, and 2-azaspiro[3.3]heptyl; the substituent is selected from hydroxyl, hydroxymethyl, cyano, halogen atom, and methyl; R5 and R6 may be the same or different, each independently selected from hydrogen, substituted or unsubstituted C3-C5 alkyl groups, or R5 and R6 forming substituted or unsubstituted C3-C6 saturated heterocyclic groups or substituted or unsubstituted C5 bridged cyclic groups with the N atom attached to them; the C3-C6 saturated heterocyclic groups include azirrobutyl, piperidinyl, thiomorpholinyl, and 3-azirbicyclo[3.1.0]hexyl; the C3-C5 alkyl groups include propyl, butyl, and pentyl; the substituents are one or more, selected from hydroxyl, hydroxymethyl, cyano, halogen atoms, and 1,1-dioxides.

2. The 18-membered diene-containing fragment according to claim 1 β - A glycyrrhetinic acid derivative or its optical isomer or a pharmaceutically acceptable salt thereof, characterized in that... Selected from any one of the following compounds, their optical isomers, or their pharmaceutically acceptable salts: N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-triene-30-acyl)-3-hydroxyazacyclobutane; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-30-acyl)-4-hydroxypiperidine; N -(2-cyano-3-oxo-18) β -Oleanane-1,9(11),12-trien-30-acyl)-6-hydroxymethyl-3-azabicyclo[3.1.0]hexylamine; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-triene-30-acyl)-piperidine-4-carboxynitrile; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-30-acyl)-2,2-difluoropropylamine; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-triene-30-acyl)-3,3-difluorozahexacyclic butylamine; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-triene-30-acyl)-3,3-difluorocyclobutylamine; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-triene-30-acyl)-4,4-difluoropiperidine; N -(2-cyano-3-oxo-18) β -Oleanane-1,9(11),12-trien-30-acyl)-6,6-difluoro-3-azabicyclo[3.1.0]hexylamine; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-hydroxycyclobutane-1-carboxamide; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-4-hydroxycyclohexane-1-carboxamide; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-4-hydroxymethylcyclohexane-1-carboxamide; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-4-cyanocyclohexane-1-carboxamide; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-2,2-difluoropropionamide; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3,3-difluorocyclobutane-1-carboxamide; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-4,4-difluorocyclohexane-1-carboxamide; N -(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-1-methylpyrrolidine-2-carboxamide; 1-(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-(3-hydroxyazacyclobutane-1-yl)urea; 1-(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-(4-hydroxypiperidin-1-yl)urea; 1-(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-(4-hydroxymethylpiperidin-1-yl)urea; 1-(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-(4-cyanopiperidin-1-yl)urea; 1-(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-(6-(hydroxymethyl)-3-azabicyclo[3.1.0]hexane-3-yl)urea; 1-(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-(2,2-difluoropropyl)urea; 1-(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-(3,3-difluorozahexacyclic butane-1-yl)urea; 1-(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-(4,4-difluoropiperidin-1-yl)urea; 1-(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-(6,6-difluoro-3-azabicyclo[3.1.0]hexane-3-yl)urea; 1-(2-cyano-3-oxo-18) β -Oleanene-1,9(11),12-trien-20-yl)-3-(1,1-dioxide-thiomorpholin-4-yl)urea.

3. A pharmaceutical composition, characterized in that, 18 containing the diene-containing structural fragment of claim 1 β - Glycyrrhetinic acid derivatives or their optical isomers or their pharmaceutically acceptable salts and pharmaceutically acceptable excipients.

4. An 18-membered terephthalic acid containing a diene structural fragment as described in claim 1. β A method for preparing glycyrrhetinic acid derivatives or their optical isomers or pharmaceutically acceptable salts thereof, characterized in that... The following synthetic routes are included: Synthesis Route 1: Reagents and reaction conditions: a) NaBH4, NaOH, THF / H2O, reflux; b) HCl, THF, reflux; c) o-iodobenzoic acid, DMSO, 85℃; d) iodine, pyridine, THF, reflux; e) CuCN, NMP, 130℃, nitrogen protection; f) Substituted amines, DIPEA, HATU, anhydrous DMF, room temperature; Synthesis Route 2: Reagents and reaction conditions: a) Diphenyl azidophosphate, TEA, MeCN, reflux; b) Concentrated hydrochloric acid, MeCN, room temperature; c) Substituted acids, HOBt / EDCI or HATU, DIPEA, anhydrous DMF, room temperature; Synthesis Route 3: Reagents and reaction conditions: a) DPPA, TEA, MeCN, reflux; b) Substituted amines, HOBt / EDCI or HATU, DIPEA, room temperature; Wherein, R2, R3, R5, and R6 are the corresponding groups at the corresponding positions of the compounds shown in Formula I.

5. The 18-membered diene-containing fragment as described in claim 1 or 2 β - The use of glycyrrhetinic acid derivatives or their optical isomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition of claim 3, in the preparation of medicaments for the treatment and / or prevention of inflammatory diseases.

6. The application according to claim 5, characterized in that, The inflammatory diseases mentioned include rheumatoid arthritis, psoriasis, inflammatory bowel disease, systemic lupus erythematosus, gout / hyperuricemia, asthma, atopic dermatitis, Alzheimer's disease, multiple sclerosis, or hepatitis.

7. The 18-membered diene-containing fragment as described in claim 1 or 2 β - Use of glycyrrhetinic acid derivatives or their optical isomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition of claim 3, in the preparation of medicaments for diseases associated with HDAC3 inhibition or NLRP3 inhibition.

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