A deubiquitinating enzyme targeted chimeric compound and its preparation method and application

Abstract

The present invention discloses a deubiquitinase targeting chimeric compound and a preparation method and application thereof. The structure of the deubiquitinase targeting chimeric compound is as shown in Formula I. The present invention modifies and transforms a FXR ligand and selects a suitable Linker connection site; the deubiquitinase ligand is connected to the FXR ligand through different flexible or rigid Linkers to synthesize FXR-targeting DUBTACs molecules with different spatial structures, and designs a reasonable synthesis route, so that the compound has great prospects in the application of FXR stabilizers.

Description

Technical Field

[0001] The present invention relates to a chimeric compound, in particular to a deubiquitinating enzyme targeted chimeric compound, and also to a preparation method and application of the chimeric compound. Background Art

[0002] Metabolic dysfunction-associated steatohepatitis (MASH) is a liver disease that is closely related to systemic metabolic dysfunction. It is characterized by abnormal accumulation of fat in the liver, accompanied by inflammation and hepatocyte damage. MASH originates from non-alcoholic fatty liver disease (NAFLD), but is more severe and is closely related to multiple metabolic syndrome components such as obesity, hypertension, diabetes, and dyslipidemia.

[0003] In the field of MASH treatment, metabolic regulators are attracting widespread attention as an emerging treatment strategy. This type of drug improves the patient's metabolic status through multiple mechanisms, thereby achieving the purpose of treating MASH. Among them, GLP-1 receptor agonists and SGLT2 inhibitors are two representative drug types, which have shown good efficacy and application prospects in the treatment of MASH.

[0004] In the field of MASH treatment, although some progress has been made in recent years, existing drugs still have obvious limitations in improving liver inflammation and fibrosis. This lack of efficacy is mainly reflected in the following aspects: Single-target drugs have limited effects: Most current MASH treatment drugs are designed for a single target, such as FXR agonists and GLP-1 receptor agonists. However, MASH is a complex multifactorial disease, and it is difficult for single-target drugs to fully cover all its pathological mechanisms. For example, although FXR agonists can improve liver inflammation and fibrosis to a certain extent, their effects are often not ideal and may have side effects. Long-term efficacy is unclear: Even if some drugs show certain effects in short-term clinical trials, such as GLP-1 receptor agonists, their long-term efficacy still needs to be further verified. Whether long-term use of these drugs can continuously improve liver inflammation and fibrosis, and whether it will bring new complications, these questions need to be answered by long-term follow-up studies. Lack of specific biomarkers: There is currently a lack of reliable biomarkers to accurately evaluate the treatment effect of MASH. This means that it is difficult for doctors and researchers to judge in a timely manner whether a drug is truly effective, and it is impossible to accurately track the progression of the disease. In this case, the evaluation of drug efficacy often relies on invasive liver biopsy, which is expensive and risky, limiting the development of large-scale clinical trials. Lack of individualized treatment options: There is a large heterogeneity among MASH patients, and different patients may require different treatment strategies. However, current drug treatments are often a "one-size-fits-all" approach that is difficult to meet the needs of different patients. This lack of personalized treatment options may result in some patients not being able to receive effective treatment or experiencing unnecessary side effects; new compounds are urgently needed to be developed and put into use. Summary of the invention

[0005] The purpose of the present invention is to provide a deubiquitinase targeting chimeric compound, and also to provide a preparation method of the chimeric compound and its application in an FXR stabilizer.

[0006] Technical solution: The present invention discloses a deubiquitinase targeting chimeric compound, the structure of which is shown in Formula I: ;

[0007] Wherein, R1 is hydrogen or cyano substituted on the carbon-carbon double bond;

[0008] R2 is hydrogen or cyclopropane substituted on the carbon-carbon double bond;

[0009] Targeting Ligand is a ligand of FXR protein, and its structure is as follows: ;

[0010] Linker is a connecting group that connects the ligand bound by OTUB1 and the ligand of FXR protein, and is one of saturated fatty chain, unsaturated fatty chain, polyethylene glycol chain, nitrogen-containing five-membered / six-membered heterocycle, and short polypeptide chain of amino acid condensation.

[0011] The Linker has the following structure: ;

[0012] Z0, Z1, and Z2 are each independently selected from , , In any group, m0, m1, m2, m3, m4, m5, and m6 are each independently selected from any integer from 0 to 6.

[0013] The Linker has the following structure: ;

[0014] Cx is selected from a 3-8 membered heterocyclic ring, a 3-8 membered cycloalkyl group, a 6-8 membered aryl group or a single bond, wherein the heterocyclic ring contains 1-4 heteroatoms, and the heteroatoms are selected from at least one of O, S and N;

[0015] Cy is selected from a 3-8 membered heterocyclic ring, a 3-8 membered cycloalkyl group, a 6-8 membered aryl group or a single bond, wherein the heterocyclic ring contains 1-4 heteroatoms, and the heteroatoms are selected from at least one of O, S and N;

[0016] Z is -CH2, -NH or -O; m is an integer between 1 and 6, and n is an integer between 0 and 6.

[0017] The compound also includes its pharmaceutically acceptable salt, hydrate or isomer.

[0018] Wherein, the compound is any of the following compounds: .

[0019] The method for preparing the above-mentioned deubiquitinase targeting chimeric compound comprises the following steps:

[0020] ;

[0021] (a) Compound 1 and 2 are reacted under alkaline conditions to obtain compound 3; (b) Compound 3 and 4 are coupled under nitrogen protection and alkaline conditions to obtain compound 5; (c) Compound 5 is reacted under hydrogen conditions to obtain compound 6; (d) Compound 6 and compound 7 are reacted under alkaline conditions to obtain compound 8; (e) Compound 8 is deprotected under acidic conditions to obtain compound 9; (f) Compound 9 and compound 10 are reacted through amide condensation to obtain the compound represented by formula I.

[0022] Wherein, in step (a), the base used in the alkaline condition is NaH, and the solvent is tetrahydrofuran; in step (b), the base used in the alkaline condition is K2CO3, and the solvent is 1,4-dioxane; in step (d), the base used in the alkaline condition is triethylamine, and the solvent is dichloromethane; in step (e), the acid used in the acidic condition is trifluoroacetic acid, and the solvent is dichloromethane; in step (f), the condensing agent used for amide condensation is HATU, the weak base is N,N-diisopropylethylamine, and the molar ratio of compound 7, compound 8, HATU, and N,N-diisopropylethylamine is 1.2:1:1.2:3.

[0023] Specifically, tert-butyl dicyclohexylphosphonoacetate reacts with 5-bromofuran-2-carboxaldehyde to obtain an (E)-3-(5-bromofuran-2-yl) acrylate intermediate; the intermediate undergoes a coupling reaction with 4-benzyloxycarbonyl-2-piperazinone to generate (E)-3-(5-(4-benzyloxycarbonyl-2-piperazinone-1-yl) furan-2-yl) acrylate; the intermediate is subjected to hydrogenation and acylation reactions to remove the tert-butyl protecting group to obtain an OTUB1 ligand; the FXR ligands OCA, CDCA, and LJN452 are subjected to an amide condensation reaction with a linker to deprotect the intermediate; the intermediate is subjected to an amide condensation reaction with OTUB1 to obtain the deubiquitinase targeting chimeric compound DUBTACs molecules D1-D33 of the present invention.

[0024] The above-mentioned deubiquitinase targeting chimeric compounds can also be used as FXR stabilizers.

[0025] Principle of the invention: The deubiquitinase-targeted chimeric compound of the present invention modifies and transforms the FXR ligand and selects a suitable "Linker" connection site by adjusting the key structural units of the FXR ligand and FXR; selects a suitable Linker, connects the deubiquitinase ligand and the FXR ligand through different flexible or rigid Linkers, designs and synthesizes FXR-targeted DUBTACs molecules with different spatial structures, and designs a reasonable synthesis route. The representative FXR agonists OCA, CDCA, and LJN-452 selected by the present invention all have carboxylic acid groups. By using this structure, the terminal carboxylic acid group is used as a Linker connection site to undergo an amide condensation reaction with a chain or cyclic Linker containing an amino group, and further connected with the OUTB1 ligand to construct a DUBTACs molecule, thereby obtaining a dual-target small molecule having both a FXR agonist pharmacophore and a deubiquitinase binding group.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the deubiquitinase targeted chimeric compound of the present invention has the characteristics of targeted stabilization of FXR, can significantly upregulate the FXR protein level in HepG2 cells, and shows good FXR stabilization activity, providing a research basis for the development of new drugs targeting FXR and even nuclear receptor drugs; while ensuring the effectiveness of the FXR stabilizer, it can be used in combination with related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the structural formula of the deubiquitinase targeting chimeric compound of the present invention;

[0028] Figure 2 Statistical significance of cell viability after treatment with compounds D1 to D6;

[0029] Figure 3 The statistical significance of cell viability after treatment with compounds from D7 to D12;

[0030] Figure 4 Statistical significance of cell viability after treatment with compounds from D13 to D18;

[0031] Figure 5 The statistical significance of cell viability after treatment with compounds from D19 to D24;

[0032] Figure 6 The statistical significance of cell viability after treatment with D25 to D30 compounds;

[0033] Figure 7 When D7 is administered to cells under physiological conditions, FXR protein interacts with the deubiquitinating enzyme OTUB1 protein;

[0034] Figure 8 The stabilizer D7 of the present invention regulates the level of FXR protein in cells under physiological conditions;

[0035] Fig. 9 The stabilizer D7 of the present invention regulates the FXR protein level in cells with bile acid imbalance;

[0036] Fig.10 The protective effect of the stabilizer D7 of the present invention on intrahepatic cholestasis caused by ANIT, (A) serum ALT level; (B) serum AST level; (C) serum ALP level; (D) serum TBA level; (E) liver H&E staining. DETAILED DESCRIPTION

[0037] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0038] Example 1: (4R)-N-(4-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)butyl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D1);

[0039] Chemical reaction steps for preparing compound D1: ;

[0040] (1) Intermediate 3: (E)-tert-butyl 3-(5-bromofuran-2-yl)acrylate: Dissolve tert-butyl diethylphosphonoacetate (9.71 g, 9.08 mL, 38.5 mmol) in THF (220 mL), cool to 0°C, and then add 5-bromofuran-2-acetaldehyde (6.13 g, 35 mmol) in proportion for 5 minutes. The reaction was stirred at 0°C for 20 minutes, and a colloidal solid precipitate appeared. Water was added and extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (0-15% EtOAc / Hex) to obtain the target compound (E)-tert-butyl 3-(5-bromofuran-2-yl)acrylate as a light yellow oil with a yield of 82%;

[0041] (2) Intermediate 5: tert-butyl (E)-3-(5-(4-benzyloxycarbonyl-2-piperazin-1-yl)furan-2-yl)acrylate: Intermediate 3 (1.62 g, 5.94 mmol) was dissolved in dioxane (30 mL), and 4-benzyloxycarbonyl-2-piperazinone (1.4 g, 5.94 mmol), K2CO3 (2.46 g, 17.8 mmol), N,N'-dimethylethylenediamine (0.167 mL, 1.49 mmol) and CuI (114 mg, 0.59 mmol) were added. The mixture was stirred under nitrogen reflux for 40 h, cooled to room temperature, 5 mL of saturated NH4Cl solution was added, stirred for 30 min, then diluted with ethyl acetate, filtered, water was added, the mixture was separated, and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (0-35% EtOAc / Hex) to obtain the target compound (E)-tert-butyl 3-(5-(4-benzyloxycarbonyl-2-piperazinone-1-yl)furan-2-yl)acrylate as an orange oil in a yield of 77%.

[0042] (3) Intermediate 6: tert-butyl 3-(5-(4-acryloyl-2-piperazin-1-yl)furan-2-yl)propanoate: Intermediate 5 (1.95 g, 4.59 mmol) was dissolved in ethyl acetate (25 mL) and Pd / C (200 mg, 10% wt. Pd) was added. The reaction was stirred vigorously at room temperature overnight under H2 protection. The reaction mixture was filtered through celite twice and the solvent was removed by rotary evaporation. The crude product was then redissolved in DCM (25 mL), cooled to 0°C, and TEA (1.28 mL, 9.18 mmol) was added dropwise. Then, a solution of acryloyl chloride (445 mL, 5.51 mmol) dissolved in DCM (5 mL) was added dropwise for 2 minutes. After stirring the reaction mixture for 20 minutes, water was added and extracted three times with DCM. The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (0-75% EtOAc / Hex) to obtain the target compound tert-butyl 3-(5-(4-acryloyl-2-piperazinone-1-yl)furan-2-yl)propanoate as a light yellow oil in a yield of 53%;

[0043] (4) Intermediate 7: 3-(5-(4-acryloyl-2-piperazinone-1-yl)furan-2-yl)propanoic acid: Dissolve intermediate 6 (140 mg, 0.4 mmol) in DCM (6 mL), add TFA (3 mL), stir at room temperature for 4 h, monitor the reaction progress by TLC until the starting material is consumed, remove the solvent by rotary evaporation, add DCM and rotary evaporate again. The orange-yellow oily compound 3-(5-(4-acryloyl-2-piperazinone-1-yl)furan-2-yl)propanoic acid is obtained and used in the next step without further purification;

[0044] (5) Intermediate 10: tert-butyl (4-((R)-4-((3R,5S,6R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamido)butyl)carbamate: Compound 8 (0.420 g, 1 mmol), compound 9 (N-tert-butyloxycarbonyl-1,4-butanediamine, 0.376 g, 2 mmol), N,N-diisopropylethylamine (0.646 g, 5 mmol) and 1-hydroxybenzotriazole (0.135 g, 1 mmol) were dissolved in 1 mL of dichloromethane, and EDCI (0.240 g, 1.23 mmol) was added. The reaction was stirred at room temperature for 2 days, and then water was added, the mixture was separated, and the aqueous layer was extracted three times with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (0-60% EtOAc / Hex) to obtain a white powdery compound intermediate 10 in a yield of 94%;

[0045] (6) Intermediate 11: (R)-N-(4-aminobutyl)-4-((3R, 5S, 6R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide: Intermediate 10 was dissolved in 1 mL of ethyl acetate, 1 mL of hydrochloric acid ethyl acetate solution was added, and the mixture was stirred at room temperature for 1 h. The solvent was removed by rotary evaporation, and the resulting mixture was dissolved in DCM and treated with saturated aqueous NaHCO3. The resulting mixture was extracted three times with DCM, the organic phases were combined, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation to obtain a white oily compound intermediate 11, which was used in the next step without further purification;

[0046] (7) Compound D1: (4R)-N-(4-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)butyl)-4-((3R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide: Intermediate 7 (0.292 g, 1 mmol) was dissolved in 1.5 mL DMF, N,N-diisopropylethylamine (1.034 g, 8 mmol) was added, and then intermediate 11 (0.462 g, 1 mmol) was added. Then HATU (0.475 g, 1.25 mmol) was added and stirred at room temperature for 16 h. Water was added, and the mixture was extracted three times with DCM, and the organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (0-5% MeOH / DCM) to obtain compound D1. Compound D1 is a yellow-brown powder with a yield of 30%; 1H NMR (300MHz, DMSO-d6) δ (ppm): δ7.68-7.54 (m, 2H), 6.01-5.88 (m, 2H), 5.56 (s, 1H), 4.22-4.06 (m, 2H), 3.85 (d, J = 6Hz, 1H), 3.75-3.58 (m, 3H), 3.30 (m, 1H), 2.97-2.8 (m, 5H), 2.60-2.42 (m, 2H), 2.17 (t, J = 6Hz, 1H), 0.40 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ (ppm): δ173.33, 35.79, 35.49, 34.01, 33.76, 33.11, 32.83,32.11, 30.89, 28.86, 28.33, 27.12, 27.05, 23.97, 23.56, 22.64, 20.87, 18.80,12.18; HRMS C44H68N4O7, m / z: [M]+ calcd 764.51, found.764.51.

[0047] Example 2: (4R)-N-(2-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)ethyl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D2);

[0048] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 7.91-7.76(m, 1H), 6.20-6.07(m, 1H), 5.75(s, 1H), 4.45-4.24(m, 2H), 4.06(d, J =3Hz, 1H), 3.93-3.65(m, 2H), 3.47(s,1H), 3.15-2.95(m, 5H), 2.78-2.61(m, 1H), 2.34(t, J =9Hz, 1H), 0.57(s, 3H); 13 CNMR (101 MHz, DMSO- d 6) δ (ppm): δ 172.76, 170.96, 150.07, 128.82, 106.91,100.45, 71.05, 68.84, 56.02, 50.59, 45.80, 42.47, 41.72, 38.87, 38.73, 35.99,35.67, 35.47, 34.01, 33.74, 33.12, 32.84, 32.13, 31.62, 30.89, 29.61, 28.33,26.56, 23.98, 23.57, 22.64, 20.88, 18.80, 12.19, 12.17; HRMS C 42 H 64 N4O7, m / z:[M] + calcd 736.48, found.736.48.

[0049] Example 2 was prepared according to the general method described in Example 1, except that an equal molar amount of mono-Boc-ethylenediamine was used in step (5) instead of N-tert-butyloxycarbonyl-1,4-butanediamine, and other operations were the same as in Example 1. The yield was 40%.

[0050] Example 3: (4R)-N-(5-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)pentyl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D3);

[0051] Example 3 was prepared according to the general method described in Example 1, except that an equal molar amount of N-(5-aminopentyl)carbamic acid tert-butyl ester was used in step (5) instead of N-tert-butyloxycarbonyl-1,4-butanediamine, and the other operations were the same as in Example 1. The yield was 41%. 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 8.00-7.82(m, 1H), 6.23-6.11(m,2H), 5.78(s, 1H), 4.44-4.35(m, 2H), 4.09(d, J =6Hz, 1H), 3.97-3.81(m, 3H),3.50(s, 1H), 3.17-3.01(m, 5H), 2.79(m, 2H), 2.39(t, J =6Hz, 1H), 0.61(s, 3H); 13 CNMR (75 MHz, DMSO- d 6) δ (ppm): δ 172.82, 171.01, 150.07, 128.83, 106.92,100.45, 71.04, 68.85, 68.80, 68.79, 65.16, 55.50, 51.42, 49.37, 47.03, 42.46,41.72, 38.85, 38.71, 35.67, 33.11, 32.13, 31.58, 29.29, 27.74, 24.22, 23.97,23.57, 22.64, 20.86, 18.81, 12.19; HRMS C 45 H 70 N4O7,m / z: [M] + calcd 778.52,found.778.52.

[0052] Example 4: (4R)-N-(2-(2-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)ethoxy)ethyl)-4-((3R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D4);

[0053] Example 4 was prepared according to the general method described in Example 1, except that an equal molar amount of [2-(2-aminoethoxy)ethyl]carbamic acid tert-butyl ester was used in step (5) instead of N-tert-butyloxycarbonyl-1,4-butanediamine, and the other operations were the same as in Example 1. The yield was 35%. 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 8.03-7.88(m, 2H), 6.23-6.11(m, 2H), 5.78(s, 1H), 4.44-4.28(m, 2H), 4.09(d, J =3Hz, 1H), 3.97-3.80(m,3H), 3.51(s, 1H), 3.23-3.18(m, 5H), 2.91(s, 2H), 2.83-2.71(m, 4H), 2.42(t, J =9Hz, 1H), 0.61(s, 3H); 13 C NMR (101 MHz, DMSO- d 6) δ (ppm): δ 173.19, 171.36,164.64, 162.79, 150.02, 128.80, 106.92, 100.45, 71.07, 69.42, 69.31, 68.85,56.02, 55.39, 50.59, 45.81, 42.47, 41.73, 38.84, 36.26, 35.99, 35.68, 35.51,34.01, 33.61, 33.12, 32.74, 32.02, 31.24, 30.90, 28.32, 23.88, 23.57, 22.64,20.88, 18.79, 12.18;HRMS C 44 H 68 N4O8, m / z: [M] + calcd 780.50, found.780.50.

[0054] Example 5: (4R)-N-(2-(2-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)ethoxy)ethoxy)ethyl)-4-((3R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D5);

[0055] Example 5 was prepared according to the general method described in Example 1, except that an equal molar amount of 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamic acid tert-butyl ester was used in step (5) instead of N-tert-butyloxycarbonyl-1,4-butanediamine, and the other operations were the same as in Example 1. The yield was 37%.

[0056] 1H NMR (300MHz, DMSO-d6) δ (ppm): δ 8.04-7.85(m, 1H), 6.24-6.11(m, 1H), 5.78(d, J =6Hz, 1H), 4.44-4.28(m, 2H), 4.08(d, J =3Hz, 1H), 3.97-3.80(m, 2H),3.51(s, 8H), 3.22-3.15(m, 6H), 2.80(t, J =6Hz, 1H), 2.41(t, J =6Hz, 1H), 0.61(s, 3H); 13C NMR (101 MHz, DMSO-d6) δ (ppm):δ 173.15, 171.34, 149.99, 128.83,106.92, 100.42, 71.06, 70.00, 69.65, 68.85, 55.99, 55.39, 50.59, 45.79,42.47, 41.72, 38.91, 35.67, 35.51, 33.59, 33.12, 32.71, 32.03, 28.33, 23.87,23.57, 22.64, 20.88, 18.80, 12.18; HRMS C46H72N4O9, m / z: [M]+ calcd 824.53,found.824.53.

[0057] Example 6: (4R)-N-(15-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)-13-oxo-3,6,9-trioxa-12-azapentadecyl)-4-((3R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D6);

[0058] Example 6 was prepared according to the general method described in Example 1, except that an equal molar amount of 13-amino-5,8,11-trioxa-2-azatridecanoic acid 1,1-dimethylethyl ester was used instead of N-tert-butyloxycarbonyl-1,4-butanediamine in step (5). The other operations were the same as in Example 1. The yield was 33%.

[0059] 1H NMR (300MHz, DMSO-d6) δ (ppm): δ 7.94-7.86(m, 1H), 6.24-6.11(m, 1H), 5.78(d, J =9Hz, 1H), 4.44-4.28(m, 2H), 4.09(d, J =3Hz, 1H), 3.97-3.82(m, 2H),3.52(s, 12H), 3.22-3.12(m, 6H), 2.80(t, J =6Hz, 1H), 2.41(t, J =6Hz, 1H),0.61(s, 3H); 13C NMR (75 MHz, DMSO-d6) δ (ppm):δ 173.17, 171.37, 150.05,128.84, 106.96, 100.47, 71.10, 70.23, 70.08, 69.67, 68.89, 56.05, 50.63,45.84, 42.51, 41.77, 38.96, 35.71, HRMS C48H76N4O10, m / z: [M]+calcd 868.56, found.868.56.

[0060] Example 7: (4R)-N-(3-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)propyl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D7);

[0061] Example 7 was prepared according to the general method described in Example 1, except that an equal molar amount of N-tert-butyloxycarbonyl-1,3-propylenediamine was used instead of N-tert-butyloxycarbonyl-1,4-butylenediamine in step (5). The other operations were the same as in Example 1. The yield was 32%. 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 7.93-7.80(m, 1H), 6.23-6.10(m, 1H), 5.78(s, 1H), 4.44-4.27(m, 1H), 4.11(d, J =3Hz, 1H), 3.97-3.80(m, 2H), 3.51(s, 4H),3.13-2, 98(m, 5H), 2.80(t, J =6Hz, 1H), 2.38(t, J =9Hz, 1H), 0.61(s, 3H); 13 C NMR (101 MHz, DMSO- d 6) δ (ppm): δ 172.98, 171.14, 169.54, 150.02, 149.15, 139.91, 134.96, 128.82, 128.29, 120.00, 106.94, 100.47, 71.07, 68.84, 55.99, 55.38,50.58, 45.79, 42.46, 42.06, 41.73, 36.84, 36.75, 36.69, 35.98, 35.66, 35.50,34.00, 33.77, 33.11, 32.84, 32.10, 30.89, 29.73, 28.32, 23.95, 23.56, 23.07,22.64, 20.87, 18.80, 12.18, 12.16; HRMS C 43 H 66 N4O7, m / z: [M] + calcd 750.49,found. 750.49.

[0062] Example 8: (4R)-N-(6-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)hexyl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D8);

[0063] Example 8 was prepared according to the general method described in Example 1, except that an equal molar amount of N-(6-aminohexyl)carbamic acid tert-butyl ester was used in step (5) instead of N-tert-butyloxycarbonyl-1,4-butanediamine, and the other operations were the same as in Example 1. The yield was 34%. 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 7.90-7.73(m, 2H), 6.22-6.09(m,2H), 5.76(s, 1H), 4.43-4.27(m, 2H), 4.07(d, J =6Hz, 1H), 3.98-3.77(m, 3H),3.49(s, 1H), 3.17-2.96(m, 6H), 2.78(t, J =9Hz, 1H), 2.37(t, J =6Hz, 1H), 0.60(s, 3H); 13 C NMR (75 MHz, DMSO- d 6) δ (ppm): δ 172.79, 170.97, 169.35, 150.06,128.83, 106.90, 71.06, 68.84, 56.02, 55.38, 50.59, 45.79, 42.46, 41.72,38.74, 35.98, 35.67, 35.46, 34.00, 33.74, 33.12, 32.84, 32.13, 30.89, 29.60,28.34, 26.64, 26.56, 23.98, 23.56, 23.07, 22.64, 20.88, 18.79, 12.17; HRMSC 46 H 72 N4O7, m / z: [M] + calcd 792.54, found. 792.54.

[0064] Example 9: (4R)-N-(18-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)-16-oxo-3,6,9,12-tetraoxa-15-azaoctadecyl)-4-((3R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D9);

[0065] Example 9 was prepared according to the general method described in Example 1, except that an equal molar amount of 16-amino-5,8,11,14-tetraoxa-2-azahexadecanoic acid 1,1-dimethylethyl ester was used instead of N-tert-butyloxycarbonyl-1,4-butanediamine in step (5). The other operations were the same as in Example 1. The yield was 34%. 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 8.08-7.88(m, 1H), 6.24-6.11(m, 1H), 5.78(s, 1H), 4.44-4.28(m, 2H), 4.11(d, J =6Hz, 1H),3.99-3.79(m, 3H), 3.52(s, 20H), 3.24-3.12(m, 5H), 2.80(t, J =6Hz, 1H), 2.41(t, J =6Hz, 1H), 0.61(s, 3H); 13 C NMR (101 MHz, DMSO- d 6) δ (ppm): δ 173.13, 171.34,164.63, 150.00, 128.83, 128.23, 106.93, 100.43, 71.06, 70.25, 70.19, 70.04,69.64, 69.61, 69.57, 68.84, 56.00, 50.59, 46.86, 45.80, 42.47, 41.72, 39.02,38.92, 35.98, 35.67, 35.51, 34.01, 33.60, 33.12, 32.72, 32.03, 30.89, 28.32,23.87, 23.56, 23.01, 22.64, 20.87, 18.80, 12.17; HRMS C 50 H 80 N4O 11 , m / z: [M] +calcd 912.58, found. 912.58.

[0066] Example 10: (4R)-N-(2-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)ethyl)-4-((3R, 7R, 8R, 9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D10);

[0067] Chemical reaction steps for preparing compound D10: ;

[0068] Example 10 was prepared according to the general method described in Example 2, except that an equal molar amount of compound 12 (CDCA) was used instead of compound 8 (OCA) in step (5), and the other operations were the same as in Example 1. The yield was 36%. 1 H NMR (300 MHz, DMSO- d 6) δ (ppm): δ 7.98-7.84(m, 2H), 6.23-6.11(m, 2H), 5.78(s, 1H), 4.44-4.16(m, 2H), 3.97(d, J =6Hz, 1H), 3.87-3.76(m, 3H), 3.64(s, 1H), 3.20-3.08(m, 5H),2.80(t, J =9Hz, 1H), 2.38(t, J =9Hz, 1H), 0.61(s, 3H); 13 C NMR (101 MHz, DMSO- d 6)δ (ppm): δ 173.23, 171.38, 164.64, 150.02, 145.48, 128.83, 128.28, 128.06,106.94, 100.47, 70.79, 66.63, 56.01, 55.38, 50.48, 49.30, 47.57, 46.87,42.37, 41.87, 38.73, 35.77, 35.53, 35.29, 35.20, 33.79, 32.84, 32.74, 31.97,31.01, 28.27, 23.87, 23.62, 23.18, 20.72, 18.80, 12.11; HRMS C 40 H 60 N4O7, m / z:[M]+ calcd 708.45, found. 708.45.

[0069] Example 11: (4R)-N-(3-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)propyl)-4-((3R, 7R, 8R, 9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D11);

[0070] Example 11 was prepared according to the general method described in Example 10, except that an equal molar amount of the compound N-tert-butyloxycarbonyl-1,3-propylenediamine was used instead of mono-Boc-ethylenediamine in step (5), and the other operations were the same as in Example 10. The yield was 36%. 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 7.94-7.77(m, 2H), 6.23-6.10(m, 2H), 5.77(d, J =9Hz, 1H), 4.27-4.23(m, 1H), 3.96-3.78(m, 3H), 3.65-3.42(m, 7H), 3.08-3.01(m, 2H), 2.80(t, J =6Hz, 1H), 2.39(t, J =6Hz, 1H), 0.60(s, 3H); 13 C NMR (75MHz, DMSO- d 6) δ (ppm): δ 173.05, 171.21,150.08, 128.83, 106.97, 100.53,70.84, 66.67, 56.53, 56.06, 50.52, 49.09, 42.41, 41.92, 36.88, 36.74, 35.81,35.54, 35.32, 35.23, 32.92, 32.78, 32.14, 31.05, 29.76, 28.30, 23.98, 23.65,23.19, 20.75, 19.03, 18.81, 12.12; HRMS C 41 H 62 N4O7, m / z: [M] + calcd 722.46,found. 722.46.

[0071] Example 12: (4R)-N-(4-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)butyl)-4-((3R, 7R, 8R, 9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D12);

[0072] Example 12 was prepared according to the general method described in Example 10, except that an equal molar amount of the compound N-tert-butyloxycarbonyl-1,4-butanediamine was used instead of mono-Boc-ethylenediamine in step (5), and the other operations were the same as in Example 10. The yield was 36%. 1 H NMR (300MHz, DMSO- d 6)δ (ppm):δ 7.91-7.73(m, 2H), 6.24-6.10(m, 2H), 5.79(d, J =9Hz, 2H), 4.44-4.28(m, 1H), 4.13(s, 1H), 3.97-3.80(m, 3H), 3.64(s, 1H), 3.20-3.17(m, 1H), 3.08-2.99(m, 4H), 2.83-2.66(m, 2H), 2.38(t, J =9Hz, 1H),0.61(s, 3H); 13 C NMR (75 MHz, DMSO- d 6) δ (ppm): δ 172.83, 171.01, 150.05,128.83, 106.92, 100.48, 70.80, 66.63, 56.02, 55.38, 50.49, 42.37, 41.87,38.69, 38.54, 35.77, 35.51, 35.29, 35.20, 33.76, 32.86, 32.74, 32.11, 31.01,28.29, 27.12, 27.05, 23.98, 23.63, 23.18, 20.72, 18.79, 12.10; HRMS C 42 H 64 N4O7,m / z: [M] + calcd 736.48, found. 736.48.

[0073] Example 13: Preparation of (4R)-N-(6-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)hexyl)-4-((3R, 7R, 8R, 9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D13);

[0074] Example 13 was prepared according to the general method described in Example 10, except that an equal molar amount of the compound N-(6-aminohexyl)carbamic acid tert-butyl ester was used in step (5) instead of mono-Boc-ethylenediamine, and the other operations were the same as in Example 10. The yield was 36%. 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 7.95-7.77(m, 2H), 6.25-6.11(m, 1H), 5.79(d, J =9Hz, 1H), 4.45-4.30(m, 1H), 4.09-3.82(m, 3H), 3.20(s, 5H), 3.08-2.99(m, 4H), 2.84-2.67(m, 2H), 2.45-2.40(m, 1H), 0.62(s, 3H); 13 C NMR (101 MHz, DMSO- d 6) δ (ppm): δ 172.85, 171.03, 158.74, 158.42, 150.08, 128.81, 119.00,116.03, 106.90, 100.48, 70.80, 66.63, 60.22, 56.05, 50.48, 49.04, 42.37,41.88, 38.87, 38.74, 37.07, 35.76, 35.48, 35.28, 35.19, 34.69, 33.74, 32.88,32.75, 32.13, 31.00, 29.57, 29.53, 28.27, 28.12, 26.54, 23.97, 23.61, 23.16,21.20, 20.71, 18.76, 14.52, 12.07; HRMS C 44 H 68 N4O7, m / z: [M] + calcd 764.51,found. 764.51.

[0075] Example 14: (4R)-N-(5-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)pentyl)-4-((3R, 7R, 8R, 9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D14);

[0076] Example 14 was prepared according to the general method described in Example 10, except that an equal molar amount of the compound N-(5-aminopentyl)carbamic acid tert-butyl ester was used in step (5) instead of mono-Boc-ethylenediamine, and the other operations were the same as in Example 10. The yield was 36%. 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 7.93-7.76(m, 2H), 6.25-6.13(m, 2H), 5.81(d, J =6Hz, 1H), 4.45-4.31(m, 1H), 4.15-3.82(m, 3H), 3.67(s, 2H), 3.27-3.18(m, 2H), 3.10-2.95(m, 4H), 2.85-2.68(m, 2H), 2.41(t, J =9Hz, 1H), 0.64(s, 3H); 13 C NMR (75 MHz, DMSO- d 6) δ (ppm): δ 172.87, 171.05, 150.12, 128.82, 106.94,100.51, 70.84, 66.67, 56.07, 50.52, 42.42, 41.93, 38.79, 35.82, 35.53, 35.33,35.23, 33.81, 32.91, 32.80, 32.16, 31.05, 29.35, 28.31, 24.26, 24.01, 23.66,23.21, 20.76, 18.82, 12.13; HRMS C 43 H 66 N4O7, m / z: [M] + calcd 750.49, found.750.49.

[0077] Example 15: (4R)-N-(2-(1-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionyl)piperidin-4-yl)ethyl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D15);

[0078] Example 15 was prepared according to the general method described in Example 1, except that an equal molar amount of 2-(N-Boc-4-piperidinyl)ethylamine was used in step (5) instead of N-tert-butyloxycarbonyl-1,4-butanediamine, and the other operations were the same as in Example 1. The yield was 36%. 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 7.77(t, J =6Hz, 1H), 6.89-6.76(m, 1H),6.22(d, J =3Hz, 1H), 6.17-6.12(m, 1H), 5.76(t, J =6Hz, 2H), 4.43-4.27(m, 4H),4.07(d, J =3Hz, 1H), 3.98-3.80(m, 5H), 3.50(s, 1H), 3.17-3.02(m, 3H), 2.80-2.75(q, 2H), 2.67-2.55(m, 4H), 0.60(s, 3H); 13 C NMR (101 MHz, DMSO- d 6) δ (ppm): δ 172.81,169.36, 158.61, 158.29, 150.23, 128.83, 128.05, 119.11, 116.13,107.09, 100.55, 71.06, 68.84, 56.02, 55.37, 50.60, 46.84, 45.79, 45.49,42.46, 41.85, 41.71, 36.20, 35.98, 35.66, 35.47, 34.73, 34.00, 33.35, 33.12,32.84, 32.62, 32.11, 31.94, 30.85, 28.34, 23.71, 23.56, 22.63, 20.87, 18.77,12.17; HRMS C 47 H 72N4O7, m / z: [M] + calcd 804.54, found. 804.54.

[0079] Example 16: (4R)-N-(2-(1-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionyl)piperidin-4-yl)ethyl)-4-((3R, 7R, 8R, 9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D16);

[0080] Example 16 was prepared according to the general method described in Example 10, except that an equal molar amount of the compound 1-tert-butyloxycarbonyl-4-aminomethylpiperidine was used instead of mono-Boc-ethylenediamine in step (5), and the other operations were the same as in Example 23. The yield was 36%.

[0081] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 7.79-7.75(m, 1H), 6.24-6.14(m, 2H), 5.77(s, 2H), 4.48-4.28(m, 2H), 4.13(s, 1H), 3.97-3.82(m, 4H), 3.64(s, 1H),3.25-2.75(m, 6H), 2.65-2.56(m, 2H), 0.61(s, 3H); 13 C NMR (75 MHz, DMSO- d 6) δ(ppm): δ 172.84, 169.40, 150.29, 128.84, 107.13, 100.59, 70.84, 66.66, 56.08,55.41, 50.54, 42.42, 41.91, 36.26, 35.53, 35.23, 34.80, 33.41, 32.80, 31.05,28.34, 23.66, 23.22, 20.77, 18.81, 12.15; HRMS C 45 H 68 N4O7, m / z: [M] + calcd776.51, found. 776.51.

[0082] Example 17: (4R)-N-(2-(2-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)ethoxy)ethyl)-4-((3R, 7R, 8R, 9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D17);

[0083] Example 17 was prepared according to the general method described in Example 10, except that an equal molar amount of the compound [2-(2-aminoethoxy)ethyl]carbamic acid tert-butyl ester was used in step (5) instead of mono-Boc-ethylenediamine, and the other operations were the same as in Example 23. The yield was 36%. 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 8.00-7.81(m, 2H), 6.24-6.10(m, 2H), 5.78(d, J =6Hz, 3H), 4.48-4.28(m, 2H), 4.12(s, 1H), 3.97-3.80(m, 4H), 3.64(s, 1H), 3.25-3.06(m, 6H), 2.83-2.66(m, 2H), 2.42(t, J =6Hz, 1H), 0.60(s,3H); 13 C NMR (101 MHz, DMSO- d 6) δ (ppm): δ 173.18, 171.35, 164.62, 158.76,150.00, 145.46, 128.82, 128.05, 120.94, 106.91, 100.43, 82.94, 70.79, 69.41,69.32, 66.62, 56.02, 55.38, 50.48, 47.56, 46.87, 42.37, 41.88, 38.95, 38.83,35.77, 35.53, 35.29, 35.20, 33.61, 32.75, 32.03,31.01, 28.84, 28.28, 23.87,23.62, 23.18, 20.72, 18.77, 12.10; HRMS C 42 H 64 N4O8, m / z: [M] + calcd 752.47,found. 752.47.

[0084] Example 18: (4R)-N-(2-(2-(2-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)ethoxy)ethoxy)ethyl)-4-((3R, 7R, 8R, 9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D18);

[0085] Example 18 was prepared according to the general method described in Example 10, except that an equal molar amount of the compound [tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate was used instead of mono-Boc-ethylenediamine in step (5), and the other operations were the same as in Example 23. The yield was 36%.

[0086] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 8.03-7.81(m, 2H), 6.24-6.11(m, 2H), 5.79(d, J =9Hz, 2H), 4.44-4.28(m, 2H), 4.13(s, 1H), 3.97-3.80(m, 4H), 3.64(s,1H), 3.51(s, 6H), 3.25-3.06(m, 5H)2.83-2.66(m, 2H), 2.41(t, J =9Hz, 1H), 0.61(s, 3H); 13 C NMR (101 MHz, DMSO- d 6) δ (ppm): δ 173.16, 171.35, 164.62, 149.99,145.45, 128.83, 128.06, 106.93, 100.42, 70.79, 70.00, 69.65, 69.57, 66.63,56.01, 55.38, 50.48, 49.31, 47.55, 46.87, 42.44, 42.37, 41.87, 39.02, 38.91,35.77, 35.53, 35.29, 35.20, 33.60, 32.75, 32.03, 31.01, 29.46, 28.28, 23.87,23.62, 23.18, 20.72, 18.78, 12.10; HRMS C 44 H 68 N4O9, m / z: [M] +calcd 796.50,found. 796.50.

[0087] Example 19: 4-acryloyl-1-(5-(3-(4-((4R)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)piperazin-1-yl)-3-oxopropyl)furan-2-yl)piperazin-2-one (D19);

[0088] Example 19 was prepared according to the general method described in Example 1, except that an equal molar amount of N-BOC-piperazine was used in place of N-tert-butyloxycarbonyl-1,4-butanediamine in step (5), and the other operations were the same as in Example 1. The yield was 35%.

[0089] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 6.90-6.74(m, 1H), 6.22-6.12(m, 2H), 5.79-5.75(m, 1H), 4.50-4.27(m, 3H), 4.07(d, J =3Hz, 1H), 3.97-3.76(m, 3H), 3.50-3.41(m, 5H), 3.17-3.05(m, 2H), 2.82-2.66(m, 3H), 2.37-2.18(m, 2H), 0.61(s, 3H); 13 C NMR (101 MHz, DMSO- d 6) δ (ppm): δ 171.76, 170.05, 128.85, 128.06,107.19, 71.07, 68.86, 55.98, 55.38, 50.58, 45.78, 42.50, 41.72, 35.98, 35.67,35.58, 34.01, 33.12, 31.76, 30.89, 29.48, 28.32, 23.57, 22.64, 20.87, 18.90,14.43, 12.19; 44 H 66 N4O7, m / z: [M] + calcd 762.49, found. 762.49.

[0090] Example 20: 4-acryloyl-1-(5-(3-(4-((4R)-4-((3R, 7R, 8R, 9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)piperazin-1-yl)-3-oxopropyl)furan-2-yl)piperazin-2-one (D20);

[0091] Example 20 was prepared according to the general method described in Example 10, except that an equal molar amount of the compound N-BOC-piperazine was used in step (5) instead of mono-Boc-ethylenediamine, and the other operations were the same as in Example 23. The yield was 36%.

[0092] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 6.90-6.73(m, 1H), 6.21-6.14(m, 2H), 5.77(t, J =3Hz, 2H), 4.42-4.27(m, 3H), 4.13(d, J =3Hz, 1H), 3.97-3.75(m, 4H), 3.63(s, 2H), 3.19-3.17(m, 2H), 2.82-2.64(m, 3H), 2.38-2.31(m, 1H), 2.26-2.15(m, 2H), 0.61(s, 3H); 13 C NMR (101 MHz, DMSO- d 6) δ (ppm): δ 171.79, 170.05,158.52, 150.84, 139.50, 128.83, 127.96, 119.56, 119.16, 107.19, 70.80, 66.64,55.98, 55.37, 50.47,46.48, 44.09, 42.41, 41.87, 35.76, 35.57, 35.28, 35.20,32.75, 31.01, 28.27, 23.66, 23.18, 22.77, 20.72, 18.89, 12.13; HRMS C 42 H 62 N4O7,m / z: [M] + calcd 734.46, found. 734.46.

[0093] Example 21: (4R)-N-(1-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionyl)piperidin-4-yl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D21);

[0094] Example 21 was prepared according to the general method described in Example 1, except that an equal molar amount of 1-BOC-4-aminopiperidine was used instead of N-tert-butyloxycarbonyl-1,4-butanediamine in step (5). The other operations were the same as in Example 1. The yield was 35%.

[0095] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 8.71(s, 1H), 7.79(d, J =3Hz, 1H),6.89-6.76(m, 1H), 6.22-6.13(m, 2H), 5.78-5.75(m, 1H), 4.47-4.18(m, 2H), 3.98-3.68(m, 5H), 3.67-3.57(m, 3H), 3.18-3.04(m, 4H), 2.81-2.61(m, 3H), 0.60(s,3H); 13 C NMR (75 MHz, DMSO- d 6) δ (ppm): δ 172.30, 169.50, 150.22, 128.83,107.09, 100.57, 68.82, 55.95, 53.89, 50.59, 45.96, 45.79, 42.46, HRMS C 45 H 68 N4O7, m / z: [M] + calcd 776.51, found. 776.51.

[0096] Example 22: (4R)-N-((1-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionyl)piperidin-4-yl)methyl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D22);

[0097] Example 22 was prepared according to the general method described in Example 1, except that an equal molar amount of 1-tert-butyloxycarbonyl-4-aminomethylpiperidine was used instead of N-tert-butyloxycarbonyl-1,4-butanediamine in step (5). The other operations were the same as in Example 1. The yield was 31%;

[0098] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 8.55(s, 1H), 7.85(t, J =6Hz, 1H),6.89-6.76(m, 1H), 6.22-6.13(m, 2H), 5.77(d, J =6Hz, 1H), 4.46-4.27(m, 3H),4.07(s, 1H), 3.98-3.75(m, 4H), 3.65-3.59(m, 2H), 3.17-3.09(m, 3H), 2.92(t, J =12Hz, 2H), 2.78(t, J =9Hz, 1H), 2.61(t, J =6Hz, 1H), 0.59(s, 3H); 13 C NMR (75MHz, DMSO- d 6) δ (ppm): δ 173.01, 169.38, 150.19, 128.83, 107.08, 100.48,71.06, 68.82, 56.00, 53.91, 50.61, 46.84, 45.79, 44.17, 42.46, 42.19, 41.72,36.40, 35.98, 35.66, 35.45, 33.12, 32.79, 32.14, 30.87, 29.83, 28.37, 23.70,23.56, 22.64, 20.88, 18.77, 18.46, 17.16, 12.82, 12.18, 12.14; HRMS C 46 H 70N4O7,m / z: [M] + calcd 790.52, found. 790.52.

[0099] Example 23: (4R)-N-(1-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propanoyl)azetidin-3-yl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D23);

[0100] Example 23 was prepared according to the general method described in Example 1, except that an equal molar amount of 1-tert-butyloxycarbonyl-3-aminocyclobutylamine was used instead of N-tert-butyloxycarbonyl-1,4-butanediamine in step (5). The other operations were the same as in Example 1. The yield was 36%;

[0101] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 8.45 (d, J =6Hz, 1H), 6.90-6.77(m,1H), 6.23-6.12(m, 2H), 5.77(t, J =6Hz, 2H), 4.43-4.27(m, 4H), 4.09-4.02(m, 2H), 4.00-3.75(m, 5H), 3.68-3.63(dd, J 1=9Hz, J 2=6Hz, 1H), 3.50(s, 1H), 3.14(s,1H), 2.76(t, J =9Hz, 2H), 2.45-2.33(m, 2H), 0.60(s, 3H); 13 C NMR (75 MHz, DMSO- d6) δ (ppm): δ 172.78, 169.33, 158.21, 150.22, 128.84, 119.63, 107.09,100.57,100.52, 71.06, 68.82, 56.01, 53.91, 50.60, 45.79, 42.46, 42.20, 41.71, 36.21,35.66, 35.48, 33.35, 33.12, 32.83, 30.84, 28.79, 23.57, 22.65, 20.88, 18.77,18.46, 17.16, 12.82, 12.18; HRMS C 43 H 64 N4O7, m / z: [M] + calcd 748.48, found.748.48.

[0102] Example 24: 4-acryloyl-1-(5-(3-(4-((4-((4R)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)piperazin-1-yl)methyl)piperidin-1-yl)-3-oxopropyl)furan-2-yl)piperazin-2-one (D24); Example 24 was prepared according to the general method described in Example 1, except that an equal molar amount of 4-(piperazin-1-ylmethyl)piperidine-1-carboxylic acid tert-butyl ester was used in step (5) instead of N-tert-butyloxycarbonyl-1,4-butanediamine, and other operations were the same as in Example 1. The yield was 36%;

[0103] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 6.90-6.77(m, 1H), 6.23-6.17(m, 2H), 5.78(d, J =6Hz, 2H), 4.43-4.27(m, 4H), 4.07(d, J =6Hz, 1H), 3.96-3.50(m, 9H),3.14(s, 1H), 3.01-2.76(m, 4H), 2.64-2.59(m, 2H), 2.32-2.20(m, 6H)0.61(s, 3H); 13 C NMR (75 MHz, DMSO- d6) δ (ppm): δ 171.42, 169.38, 150.19, 128.85, 107.13,100.51, 71.06, 68.85, 55.96, 55.40, 50.58, 46.88, 45.78, 42.50, 41.73, 35.67,35.59, 33.11, 30.88, 28.34, 23.72, 23.58, 22.65, 20.89, 18.89, 12.19; HRMSC 50 H 77 N5O7, m / z: [M] + calcd 859.58, found. 859.58.

[0104] Example 25: 4-Acryloyl-1-(5-(3-(1-((4R)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)hexahydropyrrolo[3,4-b]pyrrol-5(1H)-yl)-3-oxopropyl)furan-2-yl)piperazin-2-one (D25); prepared according to the general method described in Example 1, except that an equal molar amount of 5-BOC-hexahydropyrrolo[3,4-b]pyrrole was used in step (5) instead of N-tert-butyloxycarbonyl-1,4-butanediamine, and other operations were the same as in Example 1. The yield was 36%;

[0105] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 6.86-6.74(m, 1H), 6.22-6.12(m, 2H), 5.75(d, J =6Hz, 2H), 4.48-4.16(m, 3H), 4.02-3.60(m, 6H), 3.59-3.46(m, 4H), 3.23-3.06(m, 2H), 2.94-2.87(m, 2H), 2.79-2.64(m, 4H), 2.55-2.52(m, 1H), 2.24-2.12(m, 2H), 0.58(s, 3H); 13 C NMR (75 MHz, DMSO- d6) δ (ppm): δ 169.69, 162.79,128.85, 107.05, 71.07, 68.86, 56.07, 55.38, 50.57, 46.87, 45.77, 42.48,41.71, 36.26, 35.99, 35.66, 35.51, 34.01, 33.10, 31.23, 30.88, 28.35, 23.56,23.23, 22.64, 20.87, 18.89, 12.18; 46 H 68 N4O7, m / z: [M] + calcd 788.51,found. 788.51.

[0106] Example 26: (4R)-N-(3-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)cyclobutyl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D26);

[0107] Example 26 was prepared according to the general method described in Example 1, except that an equal molar amount of N-BOC-1,3-cyclobutanediamine was used instead of N-tert-butyloxycarbonyl-1,4-butanediamine in step (5). The other operations were the same as in Example 1. The yield was 36%;

[0108] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 8.28-7.98 (m, 2H), 6.95-6.76 (m, 1H), 6.23-6.08 (m, 2H), 5.77 (d, J =6Hz, 2H), 4.42-4.15(m, 3H), 4.05-3.74(m, 5H), 3.50-3.41(m, 2H), 3.19-3.06(m, 1H), 2.81-2.69(m, 2H), 2.48-2.28(m, 2H), 0.60(s, 3H); 13 C NMR (75 MHz, DMSO- d6) δ (ppm): δ 172.30, 170.72, 170.47, 150.05,128.85, 106.91, 100.50, 71.06, 68.83, 55.94, 55.39, 50.59, 45.79, 42.46,41.72, 41.30, 38.57, 37.26, 35.67, 35.51, 34.01, 33.11, 32.00, 30.90, 28.33,23.56, 22.65, 20.88, 18.83, 12.18; HRMS C 44 H 66 N4O7, m / z: [M] + calcd 762.49,found. 762.49.

[0109] Example 27: 4-acryloyl-1-(5-(3-(4-((1-((4R)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)piperidin-4-yl)methyl)piperazin-1-yl)-3-oxopropyl)furan-2-yl)piperazin-2-one (D27); Example 27 was prepared according to the general method described in Example 1, except that an equal molar amount of 4-(piperidin-4-ylmethyl)piperazine-1-carboxylic acid tert-butyl ester was used in step (5) instead of N-tert-butyloxycarbonyl-1,4-butanediamine, and other operations were the same as in Example 1. The yield was 36%;

[0110] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 7.38-7.26 (m, 1H), 6.89-6.70 (m, 1H), 6.22-6.13 (m, 2H), 5.78 ((d, J =6Hz, 1H), 4.43-4.27(m, 4H), 4.05-3.96(m, 3H), 3.86-3.76(m, 4H), 3.50-3.47(m, 3H), 3.14-2.90(m, 3H), 2.82-2.74(m, 2H), 2.65-2.60(m, 1H), 2.34-2.13(m, 8H), 0.61(s, 3H); 13 C NMR (75 MHz, DMSO- d6) δ (ppm): δ171.15, 170.33, 164.96, 162.16, 150.58, 149.30, 128.80, 107.14, 100.55,71.69, 70.29, 68.88, 63.99, 55.99, 50.58, 47.03, 46.80, 45.92, 45.79, 45.21,44.52, 42.51, 41.73, 35.67, 33.13, 30.84, 30.61, 29.56, 28.81, 23.58, 22.63,18.89, 14.96, 12.18; HRMS C 50 H 77 N5O7, m / z: [M] + calcd 859.58, found. 859.58.

[0111] Example 28: (4R)-N-(1-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionyl)pyrrolidin-3-yl)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D28);

[0112] Example 28 was prepared according to the general method described in Example 1, except that an equal molar amount of tert-butyl 3-aminopyrrolidine-1-carboxylate was used instead of N-tert-butyloxycarbonyl-1,4-butanediamine in step (5). The other operations were the same as in Example 1. The yield was 36%;

[0113] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 8.15-8.02(m, 1H), 6.91-6.49(m, 1H), 6.22-6.12(m, 2H), 5.76(t, J =6Hz, 1H), 4.47-4.13(m, 3H), 4.07-3.48(m, 8H), 3.22-3.08(m, 3H), 2.82-2.56(m, 2H), 0.59(s, 3H); 13 C NMR (75 MHz, DMSO- d6) δ(ppm): δ 173.08, 128.84, 107.08, 100.57, 71.06, 68.83, 55.92, 55.40, 53.93,50.59, 47.89, 45.79, 42.46, 42.21, 41.72, 35.98, 35.66, 35.50, 34.00, 33.11,32.61, 31.97, 30.89, 29.50, 28.34, 23.56, 23.17, 22.64, 20.87, 18.82, 18.48,17.17, 12.85, 12.18; HRMS C 44 H 66 N4O7, m / z: [M] + calcd 762.49, found. 762.49.

[0114] Example 29: 4-acryloyl-1-(5-(3-(4-((4R)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)-1,4-diazepan-1-yl)-3-oxopropyl)furan-2-yl)piperazin-2-one (D29);

[0115] Example 29 was prepared according to the general method described in Example 1, except that an equal molar amount of 1,4-diazepane-1-carboxylic acid tert-butyl ester was used instead of N-tert-butyloxycarbonyl-1,4-butanediamine in step (5). Other operations were the same as in Example 1. The yield was 36%;

[0116] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 6.87-6.69(m, 1H), 6.16-6.04(m, 2H),5.71(t, J =6Hz, 1H), 4.44-4.22(m, 2H), 4.04-3.70(m, 4H), 3.61-3.52(m, 3H), 3.45-3.43(m, 5H), 3.12-3.00(m, 2H), 2.77-2.69(m, 1H), 2.62-2.52(m, 1H), 2.25-2.09(m, 2H), 0.55(s, 3H); 13 C NMR (75 MHz, DMSO- d6) δ (ppm): δ 170.66, 164.43,150.14, 128.84,100.45, 71.06, 68.85, 56.11, 55.40, 53.89, 50.57, 46.85,45.79, 42.49, 42.17, 41.72, 35.99, 35.67, 35.60, 34.01, 33.10, 30.90, 28.32,23.57, 22.65, 20.87, 18.88, 18.46, 17.16, 12.81, 12.18; HRMS C 45 H 68 N4O7, m / z:[M] + calcd 776.51, found. 776.51.

[0117] Example 30: 4-acryloyl-1-(5-(3-(8-((4R)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)-2,8-diazaspiro[4.5]decane-2-yl)-3-oxopropyl)furan-2-yl)piperazin-2-one (D30); Example 30 was prepared according to the general method described in Example 1, except that an equal molar amount of 2-BOC-2,8-diaza-spiro[4.5]decane was used instead of N-tert-butyloxycarbonyl-1,4-butanediamine in step (5), and the other operations were the same as in Example 1. The yield was 36%;

[0118] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 6.90-6.77(m, 1H), 6.22-6.11(m, 2H),5.75(t, J =9Hz, 1H), 4.48-4.27(m, 2H), 4.05-3.75(m, 4H), 3.64-3.57(m, 2H), 3.50-3.49(m, 4H), 3.31(s, 2H), 3.20-3.06(m, 3H), 2.81-2.67(m, 2H), 2.57-2.50(m, 1H), 2.35-2.16(m, 2H), 0.61(s, 3H); 13 C NMR (75 MHz, DMSO- d6) δ (ppm): δ171.17, 150.24, 128.84, 107.07, 100.56, 71.06, 68.85, 55.98, 55.40, 53.92,50.58, 45.79, 42.50, 42.20, 41.72, 41.48, 35.99, 35.67, 34.96, 33.11, 31.64,29.96, 28.36, 23.57, 22.65, 20.88, 18.90, 18.48, 17.17, 12.84, 12.19; HRMSC 48 H 72 N4O7, m / z: [M] + calcd 816.54, found. 816.54.

[0119] Example 31: 4-acryloyl-1-(5-(3-(6-((4R)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)-2,6-diazaspiro[3.3]heptane-2-yl)-3-oxopropyl)furan-2-yl)piperazin-2-one (D31); Example 31 was prepared according to the general method described in Example 1, except that an equal molar amount of 2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester was used in step (5) instead of N-tert-butyloxycarbonyl-1,4-butanediamine, and other operations were the same as in Example 1. The yield was 36%;

[0120] 1 H NMR (300MHz, DMSO- d 6) δ (ppm): δ 6.88-6.77(m, 1H), 6.22-6.17(m, 2H), 6.12-6.09(m, 1H), 5.79-5.74(m, 1H), 4.43(s, 1H), 4.33-4.22(m, 3H), 4.07-3.95(m, 4H), 3.89-3.74(m, 5H), 3.63-3.49(m, 3H), 3.18-3.10(m, 1H), 2.83-2.70(m,2H), 2.44(t, J =6Hz, 1H), 2.32(t, J =3Hz, 1H), 0.59(s, 3H); 13 C NMR (101 MHz, DMSO- d6) δ (ppm): δ 173.19, 172.94, 172.18, 170.81, 164.60, 149.85, 145.54,128.83, 128.27, 128.04, 107.16, 106.98, 100.71, 100.37, 71.06, 68.86, 60.23,59.89, 55.93, 55.39, 55.22, 53.03, 50.57, 49.11, 46.87, 45.78, 42.47, 41.72,35.98, 35.67, 35.52, 34.01, 33.73,33.10,31.80,30.89,28.30,27.99,24.01,23.56,22.64,21.23,20.87,18.79,14.55,12.18; HRMS C 45 H 66 N4O7, m / z: [M] + calcd 774.49, found. 774.49.

[0121] Example 32 (4R)-N-(3-(3-(5-(4-((E)-2-cyano-3-cyclopropylacryloyl)-2-oxopiperazin-1-yl)furan-2-yl)propionamido)-4-((3R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide (D32);

[0122] Chemical reaction steps for preparing compound D32: ;

[0123] Step 8, Intermediate 17 (Z)-tert-butyl 3-(5-(4-(2-cyano-3-cyclopropylacryloyl)-2-oxopiperazin-1-yl)furan-2-yl)propanoate

[0124] Intermediate 5 (1.95 g, 4.59 mmol) was dissolved in ethyl acetate (25 mL) and Pd / C (200 mg, 10% wt. Pd) was added. The reaction was stirred vigorously at room temperature overnight under H2 protection. The reaction mixture was filtered through diatomaceous earth twice and the solvent was removed by rotary evaporation. The crude product was then redissolved in DCM (25 mL), cooled to 0°C, TEA (1.28 mL, 9.18 mmol) was added dropwise, and then a solution of compound 16 (2.278 g, 5.51 mmol) dissolved in DCM (5 mL) was added dropwise for 2 minutes. After stirring the reaction mixture for 20 minutes, water was added and extracted with DCM three times. The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (0-75% EtOAc / Hex) to obtain the target compound (Z)-3-(5-(4-(2-cyano-3-cyclopropylacryloyl)-2-oxopiperazin-1-yl)furan-2-yl)propanoic acid tert-butyl ester as a light yellow oil in a yield of 53%.

[0125] Step 9, Intermediate 18: (Z)-3-(5-(4-(2-cyano-3-cyclopropylacryloyl)-2-oxopiperazin-1-yl)furan-2-yl)propanoic acid

[0126] Intermediate 17 (140 mg, 0.4 mmol) was dissolved in DCM (6 mL), TFA (3 mL) was added, and the mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC until the starting material was consumed. The solvent was removed by rotary evaporation, and DCM was added and rotary evaporated again. The orange-yellow oily compound (Z)-3-(5-(4-(2-cyano-3-cyclopropylacryloyl)-2-oxopiperazin-1-yl)furan-2-yl)propanoic acid was obtained and used in the next step without further purification.

[0127] Step 10, intermediate 20: tert-butyl (3-((R)-4-((3R,5S,6R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamido)propyl)carbamate

[0128] Compound 8 (0.420 g, 1 mmol), compound 19 (N-tert-butyloxycarbonyl-1,3-propylenediamine, 0,348 g, 2 mmol), N,N-diisopropylethylamine (0.646 g, 5 mmol) and 1-hydroxybenzotriazole (0.135 g, 1 mmol) were dissolved in 1 mL of dichloromethane, and then EDCI (0.240 g, 1.23 mmol) was added. The reaction was stirred at room temperature for 2 days, and then water was added, the mixture was separated, and the aqueous layer was extracted three times with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (0-60% EtOAc / Hex) to obtain a white powdery compound intermediate 20 with a yield of 94%;

[0129] Step 11, Intermediate 21: (R)-N-(3-aminopropyl)-4-((3R, 5S, 6R, 7R, 8S, 9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide

[0130] Intermediate 20 was dissolved in 1 mL of ethyl acetate, and 1 mL of hydrochloric acid ethyl acetate solution was added and stirred at room temperature for 1 h. The solvent was removed by rotary evaporation, and the resulting mixture was dissolved in DCM and treated with saturated aqueous NaHCO3. The resulting mixture was extracted three times with DCM, the organic phases were combined, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation to obtain intermediate 21 as a white oil, which was used in the next step without further purification;

[0131] Step 12, Compound D32: (4R)-N-(3-(3-(5-(4-((E)-2-cyano-3-cyclopropylacryloyl)-2-oxopiperazin-1-yl)furan-2-yl)propionamido)-4-((3R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanamide

[0132] Dissolve intermediate 18 (0.357 g, 1 mmol) in 1.5 mL DMF, add N,N-diisopropylethylamine (1.034 g, 8 mmol), and then add intermediate 21 (0.476 g, 1 mmol). Then add HATU (0.475 g, 1.25 mmol) and stir at room temperature for 16 h. Add water, extract with DCM three times, combine the organic phases, wash with saturated brine, dry with anhydrous Na2SO4, and remove the solvent by rotary evaporation. The crude product is purified by silica gel column chromatography (0-5% MeOH / DCM) to obtain compound D32 as a yellow-brown powder with a yield of 30%.

[0133] Example 33: N-(3-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)propyl)-2-(3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-azabicyclo[3.2.1]octan-8-yl)-4-fluorobenzo[d]thiazole-6-carboxamide (D33);

[0134] Chemical reaction steps for preparing compound D33: ;

[0135] Step 12, Intermediate 23: (E)-2-(Trifluoromethoxy)benzaldehyde oxime; To an aqueous solution of sodium hydroxide (0.952 g, 23.8 mmol) (16.5 mL) was added an aqueous solution of NH2OH•HCl (1.598 g, 23 mmol) at 0°C. The resulting solution was stirred at 0°C for 10 minutes. A solution of compound 22 (2-(trifluoromethoxy)benzaldehyde, (3.805 g, 20 mmol, 1.00 equiv)) in ethanol (16.5 mL) was added and the resulting solution was stirred at room temperature for another hour. The reaction was diluted with water (200 mL) and extracted with ethyl acetate (2 × 200 mL). The organic layers were combined, washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain intermediate 23 (E)-2-(trifluoromethoxy)benzaldehyde oxime), which was directly carried to the next step to obtain 3.486 g of a yellow oil with a yield of 85%;

[0136] Step 13, Intermediate 24: N-Hydroxy-2-(trifluoromethoxy)benzoimidyl chloride; NCS (2.542 g, 19.04 mmol) was slowly added to a solution of (Intermediate 23 (3.486 g, 17 mmol,) in DMF (10 mL) while maintaining the internal temperature below 25°C. The reaction mixture was stirred at room temperature for 1 hour. The resulting solution was diluted with water (200 mL) and extracted with ethyl acetate (2×200 mL). The organic layers were combined, washed with brine (5×100 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to give 4.063 g of Intermediate 24 (N-hydroxy-2-(trifluoromethoxy)benzoimidyl chloride) as a light yellow residue. This material was used directly without further purification in a yield of 99%;

[0137] Step 14, Intermediate 26: 5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole-4-carboxylic acid methyl ester; Potassium carbonate (1.506 g, 10.9 mmol) was suspended in THF (12 mL), and the mixture was cooled to -10 °C and treated dropwise with a solution of compound 25 (methyl 3-cyclopropyl-3-oxopropanoate (1.358 g, 10.6 mmol)) in 6 ml of THF over 5 minutes. A solution of intermediate 24 (2.390 g, 10 mmol) in THF (6 mL) was carefully added to the reaction, ensuring that the internal temperature did not exceed -5 °C during the addition; after complete addition, the reaction was warmed to 35 °C; after completion of the reaction, the mixture was diluted with water (200 mL) and extracted with ethyl acetate (2 × 300 mL). The organic layer was washed with brine (2 × 200 mL), dried over anhydrous sodium sulfate, concentrated under vacuum, and then purified by silica gel column chromatography using ethyl acetate / petroleum ether (1:20–1:15) as eluent to afford intermediate 20a (methyl 5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole-4-carboxylate) as a white solid, 1.722 g, yield 55%;

[0138] Step 15, Intermediate 27: (5-Cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)-methanol; A 250 mL round-bottom flask was purged and maintained with an inert atmosphere of nitrogen. A suspension of LiAlH4 (0.218 g, 5.74 mmol) in THF (4.5 mL) was added, followed by the dropwise addition of a solution of Intermediate 26 (0.626 g, 2 mmol) in tetrahydrofuran (9 mL) at -10 °C. The resulting solution was stirred at -10 °C for 30 min. The reaction was then quenched by the addition of ethyl acetate (3 mL), followed by water (3 mL), and finally 15% aqueous NaOH (3 mL). The resulting mixture was filtered through celite, and the filter cake was washed with ethyl acetate (200 mL). The filtrate was washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. As a result, 0.569 g of intermediate 27 (5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methanol was obtained as a yellow oil with a yield of 95%;

[0139] Step 16, Intermediate 28: 4-bromomethyl-5-cyclopropyl-3-(2-(trifluoromethoxy)-phenyl)isoxazole; In a 100 mL round bottom flask was placed Intermediate 27 (0.2 g, 0.66 mmol), followed by triphenylphosphine (0.262 g, 1 mmol) and dichloromethane (2 mL). The mixture was stirred until completely dissolved and then slowly added dropwise to a solution of carbon tetrabromide (0.331 g, 1 mmol) in dichloromethane (1 ml). The mixture was stirred for 1 hour and then the solvent was evaporated in vacuo. The residue was purified by silica gel chromatography with a gradient of 0-50% ethyl acetate / hexanes to give the product as a clear oil, 0.162 g, in 68% yield, and was used immediately in the next step (due to stability);

[0140] Step 17, Intermediate 29: tert-butyl 3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-azabicyclo[3.2.1]octane-8-carboxylate; In a dry 250 mL flask, N-Boc nortropine (0.113 g, 0.5 mmol) and 18-crown-6 (0.132 g, 0.5 mmol) were added, followed by anhydrous THF (3 mL). Potassium tert-butoxide (0.112 g, 1 mmol) was added portionwise, and the mixture was stirred vigorously under nitrogen for 1 hour. Intermediate 28 (0.162 g, 0.45 mmol) was dissolved in anhydrous THF (1 mL) and added dropwise. The reaction mixture was stirred overnight under positive nitrogen pressure. The solvent was removed in vacuo, and the mixture was diluted with water (100 mL) and ethyl acetate (100 mL). The organic layer was separated, dried (MgSO 4 ), evaporated in vacuo, and purified by silica gel chromatography with a gradient of 0–100% ethyl acetate / hexanes to afford a yellow oil, 0.203 g, in 89% yield;

[0141] Step 18, Intermediate 30: 4-((8-azabicyclo[3.2.1]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole; Intermediate 29 (0.203 g) was dissolved in 20% TFA in dichloromethane (30 mL). The solution was stirred at room temperature for 1 hour and the solvent was evaporated. The residue was dissolved in ethyl acetate (125 mL) and washed with saturated sodium bicarbonate solution (100 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated in vacuo. 0.156 g (99% yield);

[0142] Step 19, Intermediate 32: 2-(3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-azabicyclo[3.2.1]octan-8-yl)-4-fluorobenzo[d]thiazole-6-carboxylic acid methyl ester; In a 25 mL round-bottom flask equipped with a stirring bar, the following solutions were added in sequence: Intermediate 30 (0.156 g, 0.4 mmol), 1.1 mL DMA, cesium carbonate (0.326 g, 1 mmol) and compound 31 (2-chloro-4-methoxybenzo[d]thiazole-6-carboxylic acid methyl ester, (0.25 g, 0 .87 mmol)). After the resulting slurry was stirred at room temperature for 10 minutes, the mixture was heated to 60 ° C for 1 hour. The reaction slurry was cooled to room temperature, then diluted with ethyl acetate (200 mL) and washed with water (3×30 mL). The organic solvent extract was concentrated under vacuum and purified by silica gel chromatography (40 g silica gel column) using a 15 min gradient of 10–60% ethyl acetate / hexanes. The desired fractions were concentrated under vacuum and the resulting residue crystallized upon standing to give 0.23 g of the desired intermediate methyl ester 32 in 93% yield;

[0143] Step 20: Intermediate 33: 2-(3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-azabicyclo[3.2.1]octan-8-yl)-4-fluorobenzo[d]thiazole-6-carboxylic acid

[0144] In a 25 mL round-bottom flask equipped with a stirring bar, the following solutions were added in sequence: intermediate 32 (0.186 g, 0.3 mmol), THF (2 mL), MeOH (1.0 mL), and 3 N KOH aqueous solution (1 mL, 3 mmol). The resulting homogeneous solution was stirred at 70 °C for 1 hour and then cooled to room temperature. The reaction was quenched with AcOH (0.2 mL glacial acetic acid, 3 mmol) until pH = 6, as shown on pH strip paper (Whatman Class CF Cat. No. 2613991). The resulting solution was diluted with water (300 mL) and extracted with ethyl acetate (2 × 500 mL). The organic layers were combined, washed with brine (5 × 300 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to give intermediate 33, which was used directly without further purification in 99% yield;

[0145] Step 21: Intermediate 34: tert-butyl (3-(2-(3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-azabicyclo[3.2.1]octan-8-yl)-4-fluorobenzo[d]thiazole-6-carboxamido)propyl)carbamate; Intermediate 33 (0.181 g, 0.3 mmol), compound 9 (N-Boc-1,3-propanediamine (0.104 g, 0.6 mmol)), DIPEA (300 μL, 1.5 mmol) and HOBt (0.04 g, 0.3 mmol) were dissolved in DCM (10 mL), and EDCI (0.072 g, 0.375 mmol) was then added. The reaction was stirred at room temperature for 1 day, then water was added, the mixture was separated, and the aqueous layer was extracted twice with DCM. The combined organic extracts were washed with brine, dried over Na2SO4, concentrated, and the resulting crude was purified by silica gel chromatography (0-60% EtOAc / Hex) to afford intermediate 34 as a clear oil at 0.190 g in 83% yield;

[0146] Step 22, Intermediate 35: N-(3-aminopropyl)-2-(3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-azabicyclo[3.2.1]octan-8-yl)-4-fluorobenzo[d]thiazole-6-carboxamide; Intermediate 34 (0.189 g, 0.25 mmol) was dissolved in EA (3 mL), HCL·EA (5 mL) was added, and the solution was stirred at room temperature for 2 hours until the starting material was consumed according to TLC monitoring. The residue was evaporated, DCM was added, and evaporated again. Intermediate 35 was obtained and directly reacted in the next step without purification;

[0147] Step 19, Compound D33: N-(3-(3-(5-(4-acryloyl-2-oxopiperazin-1-yl)furan-2-yl)propionamido)propyl)-2-(3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-azabicyclo[3.2.1]octan-8-yl)-4-fluorobenzo[d]thiazole-6-carboxamide; Intermediate 7 (0.131 g, 0.45 mmol) was dissolved in DMF (6 ml), HATU (0.143 g, 0.375 mmol) and DIPEA (400 μL, 2 mmol) were added, and the reaction was carried out at room temperature for half an hour. Intermediate 34 was added and reacted overnight. Water was added and the resulting suspension was extracted three times with DCM. The combined organic extracts were washed with brine, dried over Na 2SO 4 and concentrated. The crude residue was purified by silica gel chromatography (0-5% MeOH / DCM) to afford D33, 0.112 g, 48% yield; 1H NMR (300 MHz, CDCl3) δ 7.91 (d, J = 1.6 Hz, 1H), 7.56 – 7.49 (m,3H), 7.39 (tt, J = 6.7, 1.3 Hz, 2H), 6.39 (dd, J = 16.7, 2.2 Hz, 2H), 6.18 (d, J = 3.2 Hz, 1H), 6.07 (d, J = 3.3 Hz, 1H), 5.92 – 5.72 (m, 1H), 4.33 (s,2H), 4.12 (q, J = 7.2 Hz, 2H), 3.83 (d, J = 5.8 Hz, 2H), 3.53 (s, 1H), 3.31(dq, J = 12.1, 5.9 Hz, 4H), 2.96 (t, J = 7.1 Hz, 2H), 2.55 (t, J = 7.1 Hz,2H), 2.13 – 2.10 (m, 1H), 2.04 (d, J = 1.3 Hz, 2H), 1.93 (d, J = 2.5 Hz, 4H), 1.75 (d, J = 14.9 Hz, 3H), 1.48 – 1.41 (m, 2H), 1.27 – 1.20 (m, 4H).

[0148] Embodiment 34

[0149] Verification of the application of the deubiquitinase targeting chimeric compound FXR-DUBTACs of the present invention:

[0150] The CCK-8 (Cell Counting Kit-8) method was used to determine the in vitro safety of the compound. The CCK-8 method is a commonly used method for detecting cell proliferation and cytotoxicity. WST-8 (2-(2-indoxyl)-3-[4-iodophenyl]-5-(4-nitrobenzyl)-2H-tetrazolium hydrochloride) is used as a substrate. WST-8 is reduced to generate orange-yellow water-soluble menadione amine salt under the influence of cell metabolic activity. The number of cells can be indirectly reflected by measuring the light absorption value (OD value).

[0151] First, human liver cancer HepG2 cells were plated in 96-well plates and cultured for 24 hours. Then, the cells were treated with 0, 1, 5, 10, and 50 μM FXR-DUBTACs for 24 hours and then CCK-8 was added for 30 min-2 h. The absorbance at 450 nm was measured using an ELISA reader, and the data were saved and processed using Graphpad Prism 8.0. The experimental results are shown in Table 1 and Figures 2~6 As shown;

[0152] Table 1. Cell experiment results of compounds D1~D12

[0153]

[0154] Based on the results of Western blot experiments, the compounds of the present invention with OCA as the FXR ligand can significantly upregulate the FXR protein level in HepG2 cells at a concentration of 1μM or 10μM, preliminarily showing good FXR stabilization activity and having the characteristics of targeted stabilization of FXR. In addition, the stabilization effect of DUBTACs (N series compounds) with CDCA as the FXR ligand on FXR protein was analyzed, and the results showed that it did not significantly increase the expression level of FXR protein in HepG2 cells. Therefore, the deubiquitinase targeted chimeric compound of the present invention has the characteristics of targeted stabilization of FXR, providing a research basis for the development of new drugs for FXR and even nuclear receptor drug targets; it can be used in combination with related drugs while ensuring the effectiveness of the FXR stabilizer.

[0155] Embodiment 35

[0156] Verify the effect of D7 on the interaction between FXR protein and OTUB1 protein in normal hepatocytes:

[0157] 1. Experimental materials: HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. DMEM medium was purchased from GIBCO (Grand Island, New York, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, Utah, USA). Trypsin was purchased from Amersco (Solon, Ohio, USA). Cell culture consumables were purchased from Costar (USA). Protein A / G magnetic beads were purchased from ThermoFisher (USA);

[0158] 2. Experimental methods:

[0159] 2.1 Cell culture and drug treatment

[0160] The human liver cell line HepG2 was cultured in DMEM complete medium containing 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were taken and plated at 1.5×10 6 Each well was inoculated in a 6-well plate. D7 was given 24 hours after adherence, and the administration time was 8 hours;

[0161] 2.2 Co-immunoprecipitation (Co-IP)

[0162] 1) Wash the cells twice with pre-cooled PBS, add an appropriate amount of cell lysis buffer NP40 for the last time, and transfer the lysis buffer NP40 to a 1.5 ml EP tube. Lyse on ice, vortex every 5 minutes for 1 hour, until the cells are completely lysed. Centrifuge at 4℃, 13000 g for 10 minutes, and transfer the supernatant to a new EP tube. Measure protein concentration by BCA assay;

[0163] 2) Take 25 μl of Protein A / G magnetic beads and wash the beads 3 times by inverting them upside down several times with wash buffer / TBST. Add FXR antibody and IgG to the magnetic beads and shake them at 4 °C overnight. After washing 3 times, add protein lysate with uniform concentration to the magnetic beads and shake them at 4 °C for 8 hours to prepare magnetic beads-antigen-antibody complexes. After washing 3 times, add 1× Loading Buffer (100 μl) to the beads, boil them in boiling water for 57 minutes, and centrifuge them at 12000 rpm for 5 minutes. Perform Western blot and gel imaging analysis;

[0164] 2.3 Western blot

[0165] 1) After collecting the cells, place them on ice, add an appropriate amount of RIPA (containing protease inhibitors) cell lysis buffer, place the EP tube on ice, vortex once every 5 minutes, and sonicate after 30 minutes. The parameters are set as follows: ultrasonic time 15 seconds, ultrasonic intensity 20%, ultrasonic frequency: supersonic 5 seconds, stop 5 seconds. Centrifuge at 13000 g for 10 minutes, and pipette the supernatant into a new 1.5 mL EP tube. Use the BCA method to determine the protein content, add 4× loading buffer, mix well, and boil for 10 minutes before loading;

[0166] 2) Prepare 10% and 6% gels, and separate protein samples by electrophoresis. Shake the samples thoroughly, and the recommended protein loading amount is 60-80μg. The electrophoresis voltage is 150-200 V, and the electrophoresis can be stopped when the bromophenol blue indicator reaches the bottom edge. Use semi-dry transfer conditions for protein transfer, and place the transfer filter paper-activated PVDF membrane-gel-transfer filter paper in sequence to form a "sandwich" structure. Expel bubbles. Semi-dry transfer condition settings: constant voltage 25V, time 30 min;

[0167] 3) After the transfer, quickly wash the PVDF membrane in TBS / T solution for 5 minutes, and then transfer to 5% milk for blocking. The blocking conditions are: 37°C shaker, 1 hour. Prepare the antibody with the primary antibody diluent according to the recommended ratio in the manual, and incubate at 4°C overnight. Wash the primary antibody the next day. Prepare the antibody with the secondary antibody diluent according to the recommended ratio in the manual, and incubate the secondary antibody according to the corresponding species of the primary antibody. Shake at 37°C for 1 hour, wash the membrane, and wash 5 times with TBST, 5 minutes / time;

[0168] 4) Exposure was performed using the ChemiDocTM XRS+ gel imaging system (Bio-Rad), and densitometric analysis and semi-quantitative comparison were performed using Image Lab TMsoftware;

[0169] 3. Experimental results: Figure 7 As shown, under physiological conditions, when D7 is administered, the interaction between FXR protein and the deubiquitinating enzyme OTUB1 is enhanced, and the level of FXR protein increases.

[0170] Embodiment 36

[0171] Verify the regulation of D7 on FXR protein levels in cells under physiological and pathological conditions:

[0172] 1. Experimental materials: Compound D7 was synthesized in the laboratory and has the following structural formula: ;

[0173] The remaining reagents are the same as those in the previous embodiment;

[0174] 2. Experimental methods

[0175] 2.1 Cell culture and drug treatment: Human hepatocyte HepG2 cells were cultured in DMEM complete medium containing 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were taken and 1.5×10 6 The cells were inoculated into 6-well plates. After 24 hours, different concentrations of compound D7 were administered: 0.1 μM, 1 μM, and 10 μM. The cells were collected after 8 hours. The cells in the logarithmic growth phase were taken and plated at 1.5×10 6 Cells were inoculated in 6-well plates. Cholestasis model was established by DCA (100 μM) + TNFα (50 ng / μL). Compound D7 (5 μM) was administered for 4 h before the modeling agent was administered, and cells were collected 12 h after modeling.

[0176] 2.2 Western blot: same as the above embodiment;

[0177] 3. Experimental results

[0178] 3.1 Stabilizer D7 regulates FXR protein levels in cells under physiological conditions: Figure 8 As shown, in normal cells, administration of compound D7 can significantly increase the expression level of FXR protein, and this effect is dose-dependent;

[0179] 3.2 Stabilizer D7 regulates FXR protein levels in cells under pathological conditions: Fig. 9 As shown, after hepatocytes were incubated with bile acid, the level of FXR protein decreased; and compound D7 had a significant reversal effect on this; the results confirmed that compound D7 is a FXR protein stabilizer.

[0180] Embodiment 37

[0181] Protective effect of stabilizer D7 on ANIT-induced intrahepatic cholestasis in mice

[0182] 1. Experimental materials: Experimental mice (C57BL / 6) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; α-analothiocyanate (ANIT) was purchased from Sigma (USA), mineral oil was purchased from Sigma-Aldrich, and dimethyl sulfoxide (DMSO) was purchased from Sigma (USA). Other biological materials / reagents can be obtained from commercial sources;

[0183] The rest of the experimental materials are the same as those in the previous embodiment;

[0184] 2. Experimental methods

[0185] 2.1 Effect of stabilizer D7 on ANIT-induced cholestasis in mice

[0186] The breeding environment is: SPF animal laboratory, the temperature is maintained at 25±2℃, the relative humidity is maintained at 50±10%, the daylight and night cycles are simulated for 12 hours each, normal food intake and water intake are guaranteed, and adaptive breeding is carried out for one week. The body weight is maintained at 20-22g. This experiment is divided into blank control group, model group, D7 low-dose administration group (1mg / kg) and D7 high-dose administration group (2.5mg / kg); C57BL / 6 mice in the D7 low-dose administration group and the D7 high-dose administration group were pre-treated with intraperitoneal injection of 1mg / kg / day or 2.5mg / kg / dayD7, and the other groups were given the same dose of solvent control. ANIT modeling was performed 3 days later, and the blank control group was given the same dose of mineral oil. The mice were euthanized 48 hours later. The blood and liver of the mice were collected;

[0187] 2.2 Serum transaminase test: After the whole blood was allowed to stand at room temperature for 2 h, it was centrifuged at 8000 rpm for 10 min, twice. The supernatant was aspirated to obtain serum, which was used to test the blood biochemical indexes. The tests of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bile acids (TBA) were performed according to the instructions of the kit.

[0188] 2.3 Liver pathology analysis: This part of H&E staining was commissioned by Wuhan Saiweier Biotechnology Co., Ltd.

[0189] 3 Experimental results

[0190] 3.1 Effects of stabilizer D7 on serum biochemical indices: Fig.10 As shown in A-D, ANIT can cause a significant increase in the serum ALT, AST, ALP and TBA levels of mice, while the stabilizer D7 can significantly reduce the increase in serum ALT, AST, ALP and TBA levels caused by ANIT injection;

[0191] 3.2 The improvement effect of stabilizer D7 on liver pathology: According to the results of H&E staining of liver pathology analysis ( Fig.10As shown in E in the figure, the liver lobule structure of mice in the control group was intact, and the hepatocytes were arranged radially around the central vein, without fibrosis and inflammatory cells; the hepatocytes were large, with round nuclei in the center. After the injection of ANIT, the hepatocytes showed varying degrees of swelling, some hepatocytes showed ballooning, some hepatocytes had necrotic foci, some hepatocytes had edema and degeneration, inflammatory cells infiltrated, some intrahepatic hepatic lobule structures were destroyed, fibrous tissue in the portal area proliferated abnormally, collagen fibers proliferated significantly, and showed band-like hyperplasia. The disorder of the liver lobule structure in the D7-treated group was significantly improved, and the infiltration of inflammatory cells was reduced. This shows that D7 has a significant liver protective effect.

[0192] Embodiment 38

[0193] 50 mg of stabilizer D7 is mixed with 50 mg of microcrystalline cellulose and 60 mg of starch, and an appropriate amount of 20% ethanol is used as a wetting agent to form a soft material, which is granulated by conventional methods, and talc and magnesium stearate are added and mixed, and tableted to form tablets;

[0194] Stabilizer D7 50 mg was mixed with starch 50 mg, dextrin 10 mg, and sucrose 10 mg, and 30% ethanol was used as a wetting agent to make a soft material, and wet granulated to make granules;

[0195] Stabilizer D7 50 mg was mixed with starch 50 mg, dextrin 10 mg, and sucrose 10 mg, and 30% ethanol was used as a wetting agent to make a soft material, which was wet granulated and then filled into capsules to make capsules;

[0196] Stabilizer D7 50 mg, mixed with starch 40 mg and talc 10 mg, was made into powder using conventional technology;

[0197] 50 mg of stabilizer D7 is mixed with 15 mg of powdered sugar and 8 mL of water using conventional technology to prepare a syrup, and then 70 mg of starch is added to prepare a pellet using conventional technology;

[0198] Stabilizer D7 50 mg was prepared into oral solution with povidone, sucrose and water by conventional technology.

Claims

1. A deubiquitinase targeting chimeric compound, characterized in that: The compound structure is shown in Formula I: .

2. Use of the deubiquitinase targeting chimeric compound according to claim 1 in the preparation of FXR stabilizer drugs.

Images