Pentacyclic triterpenoid derivative as well as preparation method and medical application thereof

Abstract

The invention discloses a pentacyclic triterpenoid derivative as well as a preparation method and medical application thereof, and relates to the technical field of biological medicines. The structure of the pentacyclic triterpenoid derivative is shown as a general formula I: # imgabs0. The pentacyclic triterpenoid derivative provided by the invention can be used as an anti-inflammatory small molecule, shows a good inflammation inhibition effect on a cellular level, has a potential clinical application prospect, and can be used for treating self-inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly relates to a pentacyclic triterpenoid derivative, a preparation method thereof, and a medical use thereof. Background Art

[0002] Innate immunity is the first line of immune defense in mammals. When the body is invaded by pathogenic microorganisms, pattern recognition receptors (PRRs) on innate immune cells can quickly recognize pathogen-associated molecular patterns, trigger a cascade reaction, and amplify immune signals, thereby exerting an immune defense function. However, when the body experiences endogenous injuries such as nuclease metabolic disorders, endoplasmic reticulum stress, and mitochondrial dysfunction, PRRs will recognize host-derived related molecules, trigger a cytokine storm, and cause acute or chronic inflammation.

[0003] Autoinflammatory diseases (AIDs) are a group of diseases caused by gene mutations that lead to disorders of the body's innate immune system, thereby triggering systemic inflammatory responses and multi-system involvement. AIDs are diverse in type, complex in pathogenesis, and diverse in clinical manifestations. They are mainly characterized by repeated or persistent inflammatory reactions and lack the participation of the adaptive immune system. Common AIDs include familial Aicardi-Goutières syndrome, systemic juvenile idiopathic arthritis, psoriasis, etc. According to the disease pathological mechanism, AIDs can be classified into the following categories: inflammasome diseases, interferon signaling pathway diseases, NF-KappaB signaling pathway diseases, protein folding disorder diseases, other cytokine-related diseases, etc. Currently, the therapeutic drugs for AIDs include non-steroidal anti-inflammatory drugs, glucocorticoids, immunosuppressants, etc., among which small molecule drugs are relatively few. Small molecule drugs have significant advantages over other types of preparations due to their low molecular weight, easy synthesis and modification of the structure, rapid onset in the body, and convenient treatment. Therefore, the development of new and highly active small molecule drugs against AIDs has important clinical application value. Summary of the Invention

[0004] The purpose of the present invention is to provide a pentacyclic triterpenoid derivative, a preparation method thereof, and a use thereof as a new and highly active anti-inflammatory drug, aiming to provide new ideas and directions for the development of anti-inflammatory drugs.

[0005] To achieve the above object, the present invention mainly provides the following technical solutions:

[0006] In the first aspect of the present invention, there is provided a pentacyclic triterpenoid derivative or an isomer, salt, ester, or solvate thereof, characterized in that it has a general formula structure shown in Formula I:

[0007]

[0008] Among them, R1 is one of the following groups: azido group, amino group, acyl chloride, -COOR3, -CONHR4, -NHCOR5, -CONR6R7;

[0009] Among them, R2 is one of the following groups: hydrogen cyano group, methyl ester, ethyl ester, aldehyde group, carboxyl group, amino group, CONHR8, CONHR9R 10 。

[0010] The object of the present invention and the technical problems to be solved can be further realized by the following technical measures.

[0011] Further, R3 is one of the following groups: hydrogen methyl benzyl, trifluoroethyl benzotriazolyl;

[0012] R4 is one of the following groups: substituted C1-C6 alkyl or unsubstituted C1-C6 alkyl, saturated alicyclic hydrocarbon group, substituted phenyl or unsubstituted phenyl, substituted benzyl or unsubstituted benzyl, substituted 5-6 membered heteroaryl or unsubstituted 5-6 membered heteroaryl;

[0013] R5 is one of the following groups: substituted C1-C6 alkyl or unsubstituted C1-C6 alkyl, saturated alicyclic hydrocarbon group, substituted phenyl or unsubstituted phenyl, substituted benzyl or unsubstituted benzyl, substituted 5-6 membered heteroaryl or unsubstituted 5-6 membered heteroaryl;

[0014] R6 and R7 are disubstituted groups on the N atom, or, a substituted C3-C8 ring or an unsubstituted C3-C8 ring formed by R6 and R7 and the heteroatom N:

[0015]

[0016] Among them, R is one of the following groups: halogen trifluoromethyl methyl methoxy X is halogen, which is any one of F, Cl, Br;

[0017] R8 is one of the following groups: substituted C1-C6 alkyl or unsubstituted C1-C6 alkyl, saturated alicyclic hydrocarbon group, substituted phenyl or unsubstituted phenyl, substituted benzyl or unsubstituted benzyl, substituted 5-6 membered heteroaryl or unsubstituted 5-6 membered heteroaryl;

[0018] R9 and R 10 are disubstituted groups on the N atom, or, R9 and R 10A substituted or unsubstituted C3-C8 ring formed with a heteroatom N.

[0019] Furthermore, the saturated alicyclic hydrocarbon group is any one of the groups in; and / or the unsubstituted C1-C6 alkyl group is any one of the groups in; and / or the substituted C1-C6 alkyl group is any one of the groups in.

[0020] Preferably, the pentacyclic triterpenoid derivative is one of the following compounds:

[0021]

[0022]

[0023]

[0024] In a second aspect of the present invention, a method for preparing a triterpenoid derivative is provided, which comprises the following steps:

[0025]

[0026] Converting the initial raw material oleanolic acid into a general intermediate II through multiple steps, and then obtaining the triterpenoid derivative represented by the general formula I through multiple steps from the general intermediate II.

[0027] Preferably, the preparation method includes the following synthetic route:

[0028]

[0029] and / or

[0030]

[0031] wherein, II-1 is one of the intermediates represented by the general formula II;

[0032] I-1, I-2, I-3 and I-4 are partial pentacyclic triterpenoid derivatives represented by the general formula I;

[0033] wherein, the Pd / C is wet palladium carbon with a mass fraction of 5%-10%;

[0034] The solvents other than methanol, ethanol, ether and water are all ultra-dry solvents;

[0035] The separation of all compounds is achieved by a Flash automatic column chromatography machine or preparative thin layer plate chromatography technology.

[0036] In a third aspect of the present invention, there is provided the use of the above-mentioned pentacyclic triterpenoid derivatives or their isomers, salts, esters or solvates in the preparation of anti-inflammatory small molecule drugs.

[0037] In a fourth aspect of the present invention, there is provided the use of the compounds having an anti-inflammatory effect among the above-mentioned pentacyclic triterpenoid derivatives or their isomers, salts, esters or solvates in the preparation of anti-inflammatory drugs for treating autoimmune diseases. The compounds having an anti-inflammatory effect are preferably the above-mentioned Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20, Compound 21, Compound 22, Compound 23, Compound 24, Compound 25, Compound 26, Compound 27, Compound 28, Compound 29, Compound 31, Compound 32, Compound 34 and / or Compound 38, and the compounds having an anti-inflammatory effect are more preferably the above-mentioned Compound 19.

[0038] In a fifth aspect of the present invention, there is provided an anti-inflammatory small molecule drug composition, which comprises the above-mentioned pentacyclic triterpenoid derivatives or their isomers, salts, esters or solvates, and at least one pharmaceutically acceptable excipient.

[0039] In a sixth aspect of the present invention, there is provided an anti-inflammatory drug composition for treating autoimmune diseases, which comprises: the compounds having an anti-inflammatory effect among the above-mentioned pentacyclic triterpenoid derivatives or their isomers, salts, esters or solvates, and at least one pharmaceutically acceptable excipient; wherein, the compounds having an anti-inflammatory effect are preferably the above-mentioned Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20, Compound 21, Compound 22, Compound 23, Compound 24, Compound 25, Compound 26, Compound 27, Compound 28, Compound 29, Compound 31, Compound 32, Compound 34 and / or Compound 38, and the compounds having an anti-inflammatory effect are more preferably the above-mentioned Compound 19.

[0040] Compared with the prior art, the pentacyclic triterpenoid derivatives, preparation methods and pharmaceutical uses of the present invention have the following advantages and beneficial effects:

[0041] The present invention designs and synthesizes novel pentacyclic triterpenoid oleanolic acid derivatives, which have anti-inflammatory effects at the cellular level and can be used to prepare anti-inflammatory drugs for treating autoinflammatory diseases. Detailed implementation manners

[0042] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following will, in combination with preferred embodiments, describe in detail the specific implementation manners, structures, features and their effects of the application according to the present invention. The specific features, structures, or characteristics in the following embodiments can be combined in any suitable form, and the specific description content is only for illustrative purposes and should not limit the protection scope of the present invention.

[0043] Taking General Formula I-2, General Formula I-3 and General Formula I-4 as examples, the synthetic routes of the present application are introduced in detail as follows:

[0044]

[0045] 1) Preparation of benzyl 3β-hydroxyolean-12-ene-28-carboxylate:

[0046] Oleanolic acid (10 g, 21.9 mmol) and K2CO3 (6.05 g, 43.79 mmol) were placed in DMF (88 mL), and benzyl bromide (3.4 mL, 28.46 mmol) was added dropwise within 20 minutes at 50 - 55 °C. After the addition was completed, the reaction was continued for 2 - 4 hours. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with DMF (5 mL × 3). The filtrate was poured into ice water (300 mL), and a large amount of white solid precipitated. After standing until the solid particles became larger, suction filtration was carried out to collect the solid, which was washed thoroughly with water and dried to obtain a white solid (10.85 g, 91%). This compound is a known compound with a CAS number of 303114-51-4.

[0047] 2) Preparation of 3β-acetoxyolean-12-ene-28-carboxylate:

[0048] Benzyl 3β-hydroxyolean-12-ene-28-carboxylate (10.85 g, 19.84 mmol) was dissolved in 40 mL of pyridine, and acetic anhydride (18.6 mL, 198.41 mmol) was slowly added dropwise at 0 °C. After the addition was completed, DMAP (242 mg, 1.98 mmol) was added, and a solid precipitated. The reaction was continued overnight at room temperature. An appropriate amount of dichloromethane (100 mL) was added to dissolve the solution, and the solution was washed three times each with 5% HCl solution, saturated sodium bicarbonate solution, and saturated brine, and dried over anhydrous sodium sulfate. After removing the solvent under reduced pressure, a white solid (10.8 g, 92%) was obtained. This compound is a known compound with a CAS number of 357953-27-6.

[0049] 3) Preparation of 3β-acetoxy-12-oxooleanane-28-carboxylate:

[0050] Dissolve 3β-acetoxyolean-12-ene-28-carboxylate (10.8 g, 18.34 mmol) in an appropriate amount of dichloromethane (100 mL), add formic acid (18.68 mL), 30% H2O2 (7.57 mL, 73.36 mmol), and react at room temperature for 24 hours. Monitor the reaction progress by TLC. After the disappearance of the starting material spot, wash the reaction solution with saturated sodium bicarbonate solution until nearly neutral, wash it 3 times with saturated brine, dry it over anhydrous sodium sulfate, remove the solvent under reduced pressure to obtain a light yellow solid, and perform flash column chromatography to obtain a white solid (7.88 g, 71%). This compound is a known compound with a CAS number of 357953-28-7.

[0051] 4) Preparation of 3β-acetoxy-12-oxoolean-9(11)-ene-28-carboxylate:

[0052] Dissolve 3β-acetoxy-12-oxooleanane-28-carboxylate (5.22 g, 8.63 mmol) in acetic acid (43 mL), add a solution of 33% HBr (0.64 mL, 3.88 mmol) in acetic acid dropwise, heat to 50 °C, slowly add a solution of Br2 (0.44 mL, 8.63 mmol) dropwise. After the addition is complete, stir for a while, then add another solution of Br2 (0.22 mL, 4.31 mmol), and react overnight at room temperature. After the reaction is completed, pour the solution into ice water, collect the precipitated solid, wash it with saturated sodium bisulfite solution and water respectively, and dry it to obtain a crude white solid (4.05 g, 78%).

[0053] 5) Preparation of benzyl 3β-hydroxy-12-oxoolean-9(11)-ene-28-carboxylate:

[0054] Dissolve 3β-acetoxy-12-oxoolean-9(11)-ene-28-carboxylate (4.05 g, 6.72 mmol) and KOH (7.54 g, 134.36 mmol) in methanol (33 mL), heat under reflux for 1 hour, remove the solvent under reduced pressure, neutralize the remaining solid to nearly neutral with 3 mol / L HCl solution, extract the aqueous layer with dichloromethane (100 mL × 3), wash the organic layer 3 times each with saturated sodium bicarbonate solution and saturated brine, dry it over anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain a white solid (3.77 g, 100%).

[0055] 6) Preparation of 3,12-dioxoolean-9(11)-ene-28-carboxylate Ⅱ-1:

[0056] Dissolve benzyl 3β-hydroxy-12-oxoolean-9(11)-ene-28-carboxylate (3.77 g, 6.72 mmol) in acetone (67 mL). Slowly add Jones reagent (2.6 mL, 33.61 mmol) dropwise at 0 °C. After the addition is complete, react at room temperature for 30 minutes, and monitor the reaction progress by TLC. After the starting material spot disappears, remove the solvent under reduced pressure, add water to the residue, extract with dichloromethane (100 mL × 3). The organic layer is washed 3 times with saturated sodium bicarbonate solution and saturated brine respectively, dried over anhydrous sodium sulfate, and the solvent is removed under reduced pressure to obtain a yellow pasty crude product. The white solid II-1 (3.65 g, 97%) is obtained by rapid silica gel column chromatography.

[0057] 7) Preparation of compound VII:

[0058] To a stirred solution of compound II-1 (2.0 g, 3.58 mmol) in THF (300 mL) at -78 °C, add lithium bis(trimethylsilyl)amide (1.0 M in THF, 3.58 mL, 3.58 mmol) dropwise and stir for 1.5 hours. Add methyl cyanoformate (353 μL, 3.58 mmol) to this solution at -78 °C and stir the resulting mixture at the same temperature for 1 hour. After the reaction is complete, quench with saturated aqueous NH4Cl (20 mL) and extract the mixture with dichloromethane (3 × 30 mL). The combined organic layers are washed with brine (20 mL) and dried over anhydrous Na2SO4. Remove the solvent in vacuo and purify the residue by Flash automatic column chromatography (n-hexane / ethyl acetate 10:1 to 5:1) to obtain the white solid ester VII (2.15 g, 97%).

[0059] 8) Preparation of compound VIII:

[0060] Under a nitrogen atmosphere at 0 °C, add a solution of potassium bis(trimethylsilyl)amide (10.5 mL, 1 M in THF) to a solution of compound VII (2.15 g, 3.49 mmol) in anhydrous THF (3.0 mL). After the addition, stir the reaction mixture at 0 °C for an additional 10 minutes and add TMSCl (0.89 mL, 6.96 mmol) dropwise at the same temperature. Stir the resulting reaction mixture at 0 °C for 20 minutes, quench with H2O (40 mL), extract with ethyl acetate (40 mL × 3), wash with brine, and concentrate in vacuo to obtain a crude product for direct use in the next step. Then dissolve the crude product in anhydrous acetonitrile (20 mL) and add Pd(OAc)2 (1.0 g, 4.4 mmol). Then stir overnight in an oil bath at 60 °C and concentrate in vacuo. The residue is separated by Flash automatic column chromatography (n-hexane / ethyl acetate 2:1 to 1:1) to obtain compound VIII (yellow solid, 1.05 g, 49%).

[0061] 9) Preparation of Compound IX:

[0062] Dissolve the above Compound VIII (500 mg, 0.813 mmol) in 10 mL of tetrahydrofuran in a reaction flask, evacuate and replace with H2, add 5% or 10% wet palladium on carbon (50 mg), and react at room temperature for 6 hours until TLC analysis indicates the reaction is complete. Filter and concentrate by rotary evaporation, and separate the crude product by Flash automatic column chromatography (n-hexane / ethyl acetate 1:1) to obtain light yellow solid IX (300 mg, 70.3%).

[0063] 10) Preparation of Compound of General Formula I-2:

[0064] Prepare the compound of general formula I-2 by referring to the method of Examples 2-7.

[0065] 11) Preparation of Compound I-3:

[0066] Dissolve Compound I-2 in 1,2-dichloroethane (0.1 mmol / L), and add trimethyltin hydroxide (4 equivalents). React in an oil bath at 80 °C until TLC analysis indicates the reaction is complete. Adjust the pH to 2-3 with 3N hydrochloric acid, extract with ethyl acetate 3 times, wash with brine once, dry over anhydrous sodium sulfate, filter, and remove the solvent under vacuum. Separate the crude product by preparative thin layer chromatography on a TLC plate with a thickness of 1 mm (n-hexane / ethyl acetate) to obtain light yellow solid product I-3.

[0067] 12) Preparation of Compound I-4:

[0068] Prepare the compound of general formula I-4 by referring to the method of Examples 26 and 27.

[0069] Example 1

[0070] Preparation of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (Compound 1)

[0071]

[0072] The preparation of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (Compound 1) starts from commercially available oleanolic acid as the initial raw material, and after 11 steps of reaction, Compound 1 (trade name: bardoxolone, abbreviated as CDDO, CAS No.: 218600-44-3) is finally obtained, with a total yield of about 10%. 11H NMR (400 MHz, CDCl3) δ 8.04 (s, 1H), 5.98 (s, 1H), 3.04–2.93 (m, 2H), 1.96–1.49 (m, 13H), 1.46 (s, 3H), 1.32 (s, 3H), 1.14 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H) ppm.

[0073] Example 2

[0074] Preparation of (4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropicene-4a(2H)-carboxylate 2,2,2-trifluoroethyl ester

[0075]

[0076] To a solution of compound 1 (50 mg, 0.102 mmol) and 2,2,2-trifluoroethanol (11 μL, 0.152 mmol) in dry dichloromethane (1.1 mL) was added 4-dimethylaminopyridine (12.4 mg, 0.102 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (35 mg, 0.183 mmol) and N,N-diisopropylethylamine (53 μL, 0.305 mmol). The reaction was stirred at room temperature until TLC analysis indicated completion. Separation by preparative thin layer chromatography on a 1 mm thick plate (hexane / ethyl acetate 1:1) gave compound 3 (30 mg, 51%) as a white solid. 1 1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 5.97 (s, 1H), 4.65–4.56 (m, 1H), 4.53–4.41 (m, 1H), 3.06 (d, J = 13.0 Hz, 1H), 2.94 (d, J = 4.7 Hz, 1H), 1.30 (s, 3H), 1.25 (s, 3H), 1.16 (s, 3H), 1.01 (s, 3H), 0.99 (s, 3H), 0.90 (s, 3H) ppm; 13CNMR(101MHz,CDCl3)δ198.49,196.65,176.14,168.59,165.80,124.21,114.75,114.49,60.32,59.96,49.62,47.85,47.57,45.87,45.15,42.65,42.20,41.15,35.74,34.46,33.27,32.79,31.80,31.62,30.70,29.84,28.02,27.15,26.81,24.67,23.09,22.74,21.69,21.66,18.35。

[0077] Example 3

[0078] Preparation of (4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropyrene-4a(2H)-carboxylate 1H-benzotriazol-1-yl

[0079]

[0080] To a dry N,N-dimethylformamide (1.0 mL) solution of compound 1 (50 mg, 0.102 mmol) was added 1H-benzotriazol-1-yloxytrispyrrolidinophosphonium hexafluorophosphate PyBop (79 mg, 0.153 mmol) and N,N-diisopropylethylamine (53 μL, 0.305 mmol). The reaction was stirred at room temperature until TLC analysis indicated completion. Purification by preparative TLC (1 mm thickness, hexane / ethyl acetate 3:1) gave compound 4 (40 mg, 71%) as a white solid. 1 HNMR(400MHz,CDCl3)δ8.06(d,J=8.4Hz,1H),8.04(s,1H),7.57–7.51(m,1H),7.46–7.40(m,1H),7.38–7.33(m,1H),5.99(s,1H),3.24–3.16(m,2H),2.32–2.14(m,2H),2.05–1.98(m,3H),1.84–1.58(m,7H),1.49(s,3H),1.44(s,3H),1.43–1.29(m,3H),1.26(d,J=2.2Hz,3H),1.17(s,3H),1.08(s,3H),1.07(s,3H),0.97(s,3H)ppm;13 13C NMR (101 MHz, CDCl3) δ 197.99, 196.60, 174.13, 168.91, 165.63, 143.67, 128.91, 128.76, 124.97, 123.95, 120.71, 114.69, 114.48, 108.00, 49.61, 47.90, 47.87, 46.12, 45.14, 42.71, 42.30, 35.43, 34.31, 33.18, 32.98, 32.01, 31.78, 30.69, 28.36, 27.03, 26.68, 24.88, 23.12, 22.88, 21.71, 21.67, 18.30.

[0081] Example 4

[0082] Preparation of N-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropicen-4a(2H)-yl)-2,2-difluoropropanamide

[0083]

[0084] Compound 1 (100 mg, 0.203 mmol) and triethylamine (85 μL, 0.610 mmol) were dissolved in dry toluene (2.0 mL). Diphenylphosphoryl azide DPPA (66 μL, 0.305 mmol) was added at 0 °C. After 5 minutes, the reaction was transferred to room temperature until TLC analysis indicated completion of the reaction. The formyl azide (theoretical yield 105 mg) was separated by Flash automatic column chromatography (n-hexane / ethyl acetate 1:0 to 20:1) as a white solid.

[0085] The above formyl azide (105 mg, 0.203 mmol) was dissolved in benzene (1.2 mL) and heated to 80 °C for 2 hours. The reaction mixture was directly evaporated to dryness for the next reaction without purification of the crude product.

[0086] The crude product (theoretical yield 99.3 mg) was dissolved in acetonitrile (1.0 mL). Concentrated hydrochloric acid (12 N, 0.45 mL) was slowly added dropwise under an ice bath, and the reaction was transferred to room temperature for 1 hour. It was cooled again to 0 °C, and a 10% NaOH (2.5 mL) solution was slowly added and stirred. Then saturated NaHCO3 solution was added, and the mixture was extracted with ethyl acetate, washed with water, washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to obtain the amino triterpene compound intermediate (94 mg), which was used directly for the next reaction without purification.

[0087] The above intermediate (94 mg, 0.203 mmol) and 2,2-difluoropropanoic acid (26.8 mg, 0.243 mmol) were dissolved in dichloromethane (2.0 mL). N,N'-dicyclohexylcarbodiimide DCC (63 mg, 0.305 mmol) and 4-dimethylaminopyridine (9.5 mg, 0.077 mmol) were added and stirred overnight until TLC analysis indicated that the reaction was complete. The final product 5 (85 mg, total yield of 4 steps 75.4%) was obtained as a white solid by separation using an automatic Flash column chromatography machine (n-hexane / ethyl acetate 1:0 to 5:1).

[0088] Example 5

[0089] Preparation of (4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-N-pentyl-1,3,4,5,6,6a,6b,7,8a,9,10,12a,14,14a,14b-hexadecahydropicene-4a(2H)-carboxamide

[0090]

[0091] Compound 1 (50 mg, 0.102 mmol), 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (79.4 mg, 0.153 mmol) and N,N-diisopropylethylamine (53 μL, 0.305 mmol) were dissolved in anhydrous N,N-dimethylformamide (1.0 mL). After stirring at room temperature for 15 minutes, n-pentylamine (14 μL, 0.122 mmol) was added and the reaction was carried out at room temperature for 4 hours. After TLC detection showed that the reaction was complete, it was diluted with ethyl acetate, washed 3 times with water, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The crude product was separated by preparative thin layer chromatography on a plate with a thickness of 1 mm (n-hexane / ethyl acetate 3:1) to obtain compound 7 (white solid, 38 mg, 67%). 11H NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 5.98 (s, 1H), 5.88 (t, J = 6.0 Hz, 1H), 3.29–3.19 (m, 2H), 3.05 (d, J = 4.5 Hz, 1H), 2.86 (d, J = 13.2 Hz, 1H), 1.95 (td, J = 13.6, 3.6 Hz, 1H), 1.84–1.41 (m, 18H), 1.32 (s, 3H), 1.30–1.25 (m, 5H), 1.24 (s, 3H), 1.15 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H), 0.86 (t, J = 7.1 Hz, 3H) ppm; 13 13C NMR (101 MHz, CDCl3) δ 199.22, 196.70, 177.01, 168.90, 165.99, 124.08, 114.64, 114.53, 49.65, 47.80, 46.55, 46.02, 45.12, 42.68, 42.24, 39.74, 36.24, 34.71, 34.20, 33.39, 32.10, 31.78, 30.74, 29.65, 29.28, 27.87, 27.09, 26.72, 24.99, 23.23, 23.10, 22.45, 21.86, 21.66, 18.35, 14.12.

[0092] Example 6

[0093] (4aS, 6aR, 6bS, 8aR, 12aS, 14aR, 14bS)-11-Cyano-N-(3-(dimethylamino)propyl)-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropicene-4a(2H)-carboxamide Preparation

[0094]

[0095] Compound 1 (50 mg, 0.102 mmol) was dissolved in anhydrous dichloromethane (2.0 mL). Oxalyl chloride (86 μL, 1.02 mmol) was slowly added dropwise with stirring at room temperature, and the mixture was stirred for 1 hour. After the reaction was complete as detected by TLC, the solvent was removed under reduced pressure to obtain a white solid. The white solid was dissolved in benzene (1.0 mL), and 3-dimethylaminopropylamine (26 μL, 0.203 mmol) and a drop of pyridine solution were slowly added dropwise with stirring, and the reaction was heated to reflux overnight. After the reaction was complete as detected by TLC, the solvent was removed by distillation under reduced pressure. The residue was diluted with dichloromethane (10 mL), and the organic phase was washed successively with saturated sodium bicarbonate solution (10 mL × 3), water (10 mL × 3), and saturated brine (10 mL × 1), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The obtained crude product was separated by preparative thin-layer chromatography on a TLC plate with a thickness of 1 mm (n-hexane / ethyl acetate 1:1) to obtain Compound 8 (white solid, 52 mg, 88.8%). 1 H NMR (600 MHz, CDCl3) δ 8.03 (s, 1H), 7.84 (t, J = 5.1 Hz, 1H), 5.93 (s, 1H), 3.39–3.27 (m, 2H), 3.04 (d, J = 4.5 Hz, 1H), 2.84 (d, J = 13.1 Hz, 1H), 2.47 (td, J = 6.3, 1.9 Hz, 2H), 2.26 (s, 6H), 1.90 (t, J = 13.6, 1H), 1.81–1.48 (m, 12H), 1.45 (s, 3H), 1.31 (s, 3H), 1.22 (s, 3H), 1.13 (s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.87 (s, 3H) ppm; 13 C NMR (151 MHz, CDCl3) δ 199.05, 196.66, 177.44, 168.54, 165.86, 124.02, 114.61, 114.49, 58.75, 49.54, 47.80, 46.46, 45.95, 45.09, 45.00, 42.62, 42.27, 39.93, 36.19, 34.79, 34.10, 33.38, 31.89, 31.75, 30.71, 27.95, 27.03, 26.64, 25.14, 24.94, 23.08, 22.80, 21.83, 21.64, 18.35。

[0096] Example 7

[0097] Preparation of Ethyl (R)-2-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropyrene-4-carboxamido)pentanoate

[0098]

[0099] Compound 1 (50 mg, 0.102 mmol) was dissolved in anhydrous dichloromethane (2.0 mL), and oxalyl chloride (86 μL, 1.02 mmol) was slowly added dropwise with stirring at room temperature for 1 hour. After the reaction was monitored by TLC and completed, the solvent was removed under reduced pressure to obtain a white solid. The white solid was dissolved in anhydrous dichloromethane (1.0 mL), and L-norvaline ethyl ester hydrochloride (37 mg, 0.203 mmol) and triethylamine (28 μL, 0.203 mmol) were added with stirring. The reaction was carried out overnight at room temperature. After the reaction was monitored by TLC and completed, it was separated by preparative thin-layer chromatography on a TLC plate with a thickness of 1 mm (n-hexane / ethyl acetate 2:1) to obtain Compound 9 (white solid, 53 mg, 84%). 1 HNMR(400MHz,CDCl3)δ8.06(s,1H),6.24(d,J=7.8Hz,1H),5.98(s,1H),4.59–4.54(m,1H),4.14(q,J=7.1Hz,2H),3.15(d,J=4.5Hz,1H),2.93(d,J=13.2Hz,1H),2.03–1.92(m,1H),1.84–1.49(m,12H),1.47(s,3H),1.41–1.17(m,16H),1.14(s,3H),1.00(s,3H),0.97(s,3H),0.92(t,J=7.3Hz,3H),0.88(s,3H)ppm; 13 C NMR(101MHz,CDCl3)δ199.04,196.73,177.17,172.97,168.63,166.12,124.13,114.59,114.54,61.42,51.95,49.53,47.77,46.64,45.99,45.09,42.64,42.25,36.14,34.68,34.43,34.16,33.34,31.85,31.81,30.71,27.85,27.11,26.78,24.93,23.21,21.81,21.64,19.05,18.34,14.27,13.78,13.69。

[0100] Example 8

[0101] (4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-Cyano-N-cyclopentyl-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropicene-4a(2H)-carboxamide Preparation

[0102]

[0103] Referring to the method of Example 5, Compound 10 (white solid, 39.5 mg, 70%) was prepared by reacting Compound 1 (50 mg, 0.102 mmol) with cyclopentylamine (12 μL, 0.122 mmol). 1 H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 5.96 (s, 1H), 5.66 (d, J = 7.3 Hz, 1H), 4.25–4.15 (m, 1H), 3.05 (d, J = 4.5 Hz, 1H), 2.85 (d, J = 13.0 Hz, 1H), 2.01 - 1.90 (m, 3H), 1.85–1.41 (m, 19H), 1.37–1.21 (m, 12H), 1.15 (s, 4H), 0.99 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H) ppm; 13 C NMR (101 MHz, CDCl3) δ 199.15, 196.69, 176.65, 168.71, 165.93, 124.09, 114.64, 114.51, 51.34, 49.55, 47.80, 46.31, 46.03, 45.11, 42.65, 42.21, 36.18, 34.69, 34.13, 33.39, 33.26, 33.14, 32.01, 31.75, 30.72, 27.84, 27.08, 26.68, 24.90, 23.86, 23.25, 23.13, 21.84, 21.66, 18.33.

[0104] Example 9

[0105] Preparation of (4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-N-(furan-2-ylmethyl)-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,3,4,5,6,6a,6b,7,8a,9,9,10,12a,14,14a,14b-hexadecahydropicene-4a(2H)-carboxamide

[0106]

[0107] Compound 11 (white solid, 51 mg, 80%) was prepared by reacting compound 1 (55 mg, 0.112 mmol) with 2-furanmethanamine (12 μL, 0.134 mmol) according to the method of Example 5. 1 H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.29–7.26 (m, 1H), 6.31–6.22 (m, 2H), 6.20 (d, J = 3.2 Hz, 1H), 5.96 (s, 1H), 4.43 (d, J = 5.5 Hz, 2H), 3.02 (d, J = 4.5 Hz, 1H), 2.90 (d, J = 13.3 Hz, 1H), 1.97 (td, J = 13.7, 3.8 Hz, 1H), 1.85–1.47 (m, 10H), 1.45 (s, 3H), 1.36–1.24 (m, 3H), 1.23 (s, 3H), 1.18 (s, 3H), 1.14 (s, 3H), 0.98 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H) ppm; 13 C NMR (101 MHz, CDCl3) δ 199.16, 196.70, 176.97, 168.85, 166.01, 151.86, 142.06, 124.01, 114.60, 114.53, 110.55, 107.57, 49.49, 47.78, 46.64, 45.91, 45.10, 42.65, 42.21, 36.54, 36.14, 34.67, 34.04, 33.37, 31.94, 31.74, 30.71, 27.78, 27.07, 26.67, 24.72, 23.20, 23.14, 21.81, 21.65, 18.33.

[0108] Example 10

[0109] Preparation of N-(1-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropyrene-4a-carbonyl)-1H-pyrazol-3-yl)acetamide

[0110]

[0111] Compound 13 (white solid, 58 mg, 95%) was prepared by reacting compound 1 (50 mg, 0.102 mmol) with 2-furfurylamine (25.5 mg, 0.203 mmol) according to the method of Example 6. 1 H NMR (600 MHz, CDCl3) δ 8.66–8.62 (m, 2H), 8.17 (s, 1H), 6.95 (s, 1H), 6.10 (s, 1H), 4.13–4.09 (m, 1H), 2.81 (s, 1H), 2.17 (s, 3H), 1.45 (s, 3H), 1.25 (s, 3H), 1.24 (s, 3H), 1.19 (s, 3H), 1.06 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H) ppm; 13 C NMR (151 MHz, CDCl3) δ 198.82, 197.12, 175.22, 169.08, 168.38, 150.50, 131.26, 124.82, 114.79, 114.45, 102.65, 50.20, 48.29, 45.96, 45.27, 42.88, 42.38, 36.42, 34.50, 33.61, 31.56, 31.41, 31.03, 28.05, 27.56, 27.38, 24.45, 24.22, 23.94, 22.70, 22.31, 21.73, 18.45.

[0112] Example 11

[0113] (4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-N-methoxy-N,2,2,6a,6b,9,9,12a-octamethyl-10,14-dioxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropyrene-4a(2H)-carboxamide Preparation

[0114]

[0115] Referring to the method of Example 7, Compound 14 (white solid, 39 mg, 72%) was prepared by reacting Compound 1 (50 mg, 0.102 mmol) with N,O-dimethylhydroxylamine hydrochloride (20 mg, 0.203 mmol). 1 H NMR (500 MHz, CDCl3) δ 8.03 (s, 1H), 5.93 (s, 1H), 3.67 (s, 3H), 3.23–3.18 (m, 1H), 3.15 (s, 3H), 3.02 (d, J = 4.2 Hz, 1H), 2.02–1.49 (m, 13H), 1.46 (s, 3H), 1.31 (s, 3H), 1.23 (s, 3H), 1.15 (s, 3H), 1.01 (s, 3H), 0.98 (s, 3H), 0.88 (s, 3H) ppm; 13 C NMR (125 MHz, CDCl3) δ 199.34, 196.74, 178.60, 168.70, 165.97, 124.06, 114.62, 114.52, 60.58, 50.20, 48.10, 47.80, 45.94, 45.11, 42.63, 42.20, 36.42, 34.57, 34.45, 33.42, 31.70, 31.15, 30.76, 29.17, 28.30, 27.09, 26.70, 24.89, 23.27, 22.39, 22.24, 21.66, 18.34.

[0116] Example 12

[0117] (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-8a-(Indole-1-carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-3,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropyrene-2-carbonitrile Preparation

[0118]

[0119] Referring to the method of Example 7, Compound 15 (light yellow solid, 38.5 mg, 64%) was prepared by reacting Compound 1 (50 mg, 0.102 mmol) with indoline (23 μL, 0.203 mmol). 11H NMR (400 MHz, CDCl3) δ 8.06 (d, J = 10.0 Hz, 1H), 8.05 (s, 1H), 7.21 (d, J = 7.4 Hz, 1H), 7.19–7.14 (m, 1H), 7.01 (td, J = 7.4, 1.0 Hz, 1H), 5.95 (s, 1H), 4.35–4.26 (m, 1H), 4.23–4.15 (m, 1H), 3.36–3.23 (m, 2H), 3.21–3.09 (m, 2H), 2.13–2.03 (m, 1H), 1.98–1.88 (m, 2H), 1.82–1.64 (m, 8H), 1.45 (s, 3H), 1.31 (s, 3H), 1.23 (s, 3H), 1.13 (s, 3H), 1.05 (s, 3H), 1.04 (s, 3H), 0.93 (s, 3H) ppm; 13 13C NMR (101 MHz, CDCl3) δ 199.11, 196.70, 175.76, 168.03, 165.88, 144.78, 131.10, 127.38, 124.54, 124.00, 123.91, 118.56, 114.52, 114.50, 49.99, 49.68, 49.01, 47.83, 45.99, 45.08, 42.61, 42.46, 36.16, 34.38, 33.37, 31.69, 30.64, 30.24, 29.86, 28.33, 26.97, 26.58, 24.62, 23.85, 22.13, 21.65, 18.28.

[0120] Example 13

[0121] Preparation of (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-4,4,6a,6b,11,11,14b-heptamethyl-8a-((S)-2-methylindole-1-carbonyl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-2-carbonitrile and (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-4,4,6a,6b,11,11,14b-heptamethyl-8a-((R)-2-methylindole-1-carbonyl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-2-carbonitrile

[0122]

[0123] According to the method of Example 7, Compound 16 (light yellow solid, 21 mg, 34%) and Compound 17 (light yellow solid, 31.5 mg, 51%) were prepared by reacting Compound 1 (50 mg, 0.102 mmol) with 2-methylindoline (27 mg, 0.203 mmol).

[0124] 1H NMR data of Compound 16: 1 H NMR(600MHz,CDCl3)δ8.02(s,1H),7.85(d,J=8.0Hz,1H),7.24(d,J=7.4Hz,1H),7.18(t,J=7.8Hz,1H),7.04(t,J=7.4Hz,1H),5.94(s,1H),5.06–5.01(m,1H),3.33(dd,J=14.9,7.3Hz,1H),3.18–3.16(m,2H),2.60(d,J=14.8Hz,1H),1.45(s,3H),1.33(s,3H),1.25(s,3H),1.20(d,J=6.3Hz,3H),1.15(s,3H),1.09(s,3H),1.06(s,3H),0.95(s,3H)ppm; 13 C NMR(151MHz,CDCl3)δ199.02,196.68,176.15,168.92,165.75,143.45,131.32,127.05,125.16,124.19,123.96,120.35,114.70,114.53,57.41,50.10,49.05,47.85,46.23,45.14,42.65,42.21,37.84,36.49,34.45,33.62,31.64,31.39,31.31,30.70,28.36,27.08,26.59,24.92,24.20,23.92,22.66,21.69,21.16,18.34。

[0125] 1H NMR data of Compound 17: 11H NMR (600 MHz, CDCl3) δ 8.07 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 7.4 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.04 (t, J = 7.4 Hz, 1H), 5.96 (s, 1H), 4.95–4.90 (m, 1H), 3.54 (d, J = 4.7 Hz, 1H), 3.30–3.22 (m, 2H), 2.56 (d, J = 14.8 Hz, 1H), 2.15–2.08 (m, 1H), 1.49 (s, 3H), 1.40 (s, 3H), 1.24 (s, 3H), 1.23 (d, J = 5.9 Hz, 3H), 1.15 (s, 3H), 1.06 (s, 3H), 0.99 (s, 3H), 0.91 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 199.10, 196.72, 175.40, 167.39, 165.97, 143.16, 131.57, 126.98, 125.14, 124.30, 124.28, 120.75, 114.55, 114.52, 57.24, 49.40, 48.86, 47.94, 45.97, 45.12, 42.63, 42.53, 37.67, 36.13, 34.68, 33.43, 33.11, 31.82, 31.09, 30.56, 29.15, 27.02, 26.69, 24.52, 23.80, 21.68, 21.65, 21.61, 20.71, 18.35.

[0126] Example 14

[0127] (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-4,4,6a,6b,11,11,14b-Heptamethyl-8a-(4-methylindoline-1-carbonyl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropyrene-2-carbonitrile Preparation

[0128]

[0129] Referring to the method of Example 7, compound 18 (light yellow solid, 49 mg, 79.4%) was prepared by reacting compound 1 (50 mg, 0.102 mmol) with 4-methylindoline (24 μL, 0.203 mmol). 11H NMR (600 MHz, CDCl3) δ 8.05 (s, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 6.85 (d, J = 7.5 Hz, 1H), 5.95 (s, 1H), 4.34–4.30 (m, 1H), 4.25–4.17 (m, 1H), 3.32 (d, J = 13.0 Hz, 1H), 3.26 (s, 1H), 3.09–3.03 (m, 2H), 2.25 (s, 3H), 2.11–2.03 (m, 1H), 1.99–1.88 (m, 2H), 1.81–1.65 (m, 7H), 1.48 (d, J = 12.6 Hz, 1H), 1.45 (s, 3H), 1.42–1.33 (m, 2H), 1.30 (s, 3H), 1.28–1.25 (m, 1H), 1.23 (s, 3H), 1.20 (s, 1H), 1.14 (s, 3H), 1.05 (s, 3H), 1.04 (s, 3H), 0.93 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 199.08, 196.67, 175.70, 165.85, 144.55, 133.88, 129.81, 127.54, 124.87, 124.00, 116.04, 114.53, 114.50, 49.87, 49.69, 49.07, 47.88, 46.01, 45.09, 42.62, 42.49, 36.20, 34.41, 33.38, 31.71, 30.65, 30.20, 28.61, 28.36, 26.97, 26.56, 24.63, 23.86, 22.13, 21.64, 18.79, 18.29.

[0130] Example 15

[0131] Preparation of (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-4,4,6a,6b,11,11,14b-heptamethyl-8a-(5-methylindoline-1-carbonyl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropyrene-2-carbonitrile

[0132]

[0133] According to the method of Example 7, Compound 19 (light yellow solid, 53.5 mg, 86.7%) was prepared by reacting Compound 1 (50 mg, 0.102 mmol) with 5-methylindoline (26 μL, 0.203 mmol). 1 H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.03 (s, 1H), 6.97 (d, J = 8.4 Hz, 1H), 5.94 (s, 1H), 4.34–4.24 (m, 1H), 4.23–4.13 (m, 1H), 3.37–3.21 (m, 2H), 3.21–3.03 (m, 2H), 2.30 (s, 3H), 1.45 (s, 3H), 1.30 (s, 3H), 1.23 (s, 3H), 1.14 (s, 3H), 1.04 (s, 6H), 0.92 (s, 3H) ppm; 13 C NMR (101 MHz, CDCl3) δ 199.15, 196.72, 175.40, 165.92, 142.42, 133.60, 131.19, 127.82, 125.17, 123.97, 118.23, 114.51, 77.36, 50.06, 49.68, 48.88, 47.80, 45.97, 45.07, 42.59, 42.44, 36.14, 34.37, 33.40, 31.67, 30.65, 30.21, 29.81, 28.29, 26.98, 26.59, 24.62, 23.84, 22.16, 21.65, 21.04, 18.27。

[0134] Example 16

[0135] (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-4,4,6a,6b,11,11,14b-Heptamethyl-8a-(6-methylindoline-1-carbonyl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropyrene-2-carbonitrile Preparation

[0136]

[0137] According to the method of Example 7, Compound 20 (light yellow solid, 47 mg, 76%) was prepared by reacting Compound 1 (50 mg, 0.102 mmol) with 6-methylindoline (26 μL, 0.203 mmol). 11H NMR (600 MHz, CDCl3) δ 8.05 (s, 1H), 7.94 (d, J = 1.5 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H), 6.84 (dd, J = 7.6, 1.5 Hz, 1H), 5.95 (s, 1H), 4.32–4.28 (m, 1H), 4.22–4.15 (m, 1H), 3.35–3.30 (m, 1H), 3.26 (s, 1H), 3.17–3.08 (m, 2H), 2.31 (s, 3H), 1.46 (s, 3H), 1.32 (s, 3H), 1.24 (s, 3H), 1.14 (s, 3H), 1.05 (s, 3H), 1.04 (s, 3H), 0.93 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 199.04, 196.69, 175.73, 168.02, 165.88, 144.95, 137.30, 128.13, 124.63, 124.10, 124.02, 119.29, 114.54, 114.49, 50.27, 49.71, 49.09, 47.86, 46.00, 45.09, 42.62, 42.50, 36.20, 34.40, 33.36, 31.72, 30.65, 30.29, 29.47, 28.38, 26.99, 26.61, 24.63, 23.84, 22.12, 21.76, 21.64, 18.30.

[0138] Example 17

[0139] (4aR, 6aS, 6bR, 8aS, 12aS, 12bR, 14bS)-4,4,6a,6b,11,11,14b-Heptamethyl-8a-(7-methylindoline-1-carbonyl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropyrene-2-carbonitrile Preparation

[0140]

[0141] Referring to the method of Example 7, Compound 21 (light yellow solid, 35 mg, 56.7%) was prepared by reacting Compound 1 (50 mg, 0.102 mmol) with 7-methylindoline (26 μL, 0.203 mmol). 11H NMR (600 MHz, CDCl3) δ 8.05 (s, 1H), 7.10–7.06 (m, 1H), 7.04–7.00 (m, 2H), 6.00 (s, 1H), 4.42–4.35 (m, 1H), 4.16–4.10 (m, 1H), 3.48–3.41 (m, 1H), 3.12–3.08 (m, 1H), 3.02 (d, J = 4.6 Hz, 1H), 2.99–2.94 (m, 1H), 2.20 (s, 3H), 1.49 (s, 3H), 1.40 (s, 3H), 1.25 (s, 3H), 1.16 (s, 3H), 1.07 (s, 3H), 1.04 (s, 3H), 0.92 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 198.88, 196.69, 175.57, 168.53, 166.09, 143.68, 133.96, 129.78, 129.69, 125.32, 124.57, 121.77, 114.68, 114.47, 51.60, 50.13, 48.60, 47.89, 46.10, 45.15, 42.63, 42.35, 36.77, 34.46, 32.99, 32.94, 31.97, 31.88, 31.12, 30.73, 28.42, 27.17, 27.03, 25.85, 24.27, 23.39, 21.82, 21.70, 21.63, 18.33.

[0142] Example 18

[0143] (4aR, 6aS, 6bR, 8aS, 12aS, 12bR, 14bS)-8a-(5-Fluoroindoline-1-carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-2-carbonitrile Preparation

[0144]

[0145] Referring to the method of Example 7, compound 22 (light yellow solid, 53.5 mg, 86%) was prepared by reacting compound 1 (50 mg, 0.102 mmol) with 5-fluoroindoline (26 μL, 0.203 mmol). 11H NMR (600 MHz, CDCl3) δ 8.04 (s, 1H), 8.03 (dd, J = 8.9, 4.8 Hz, 1H), 6.89 (dd, J = 8.1, 2.7 Hz, 1H), 6.83 (td, J = 8.9, 2.8 Hz, 1H), 5.95 (s, 1H), 4.34–4.30 (m, 1H), 4.21 (q, J = 9.2 Hz, 1H), 3.32–3.27 (m, 1H), 3.24 (d, J = 4.6 Hz, 1H), 3.20–3.08 (m, 2H), 2.07 (td, J = 14.2, 3.4 Hz, 1H), 1.96–1.87 (m, 2H), 1.79–1.64 (m, 7H), 1.52–1.45 (m, 1H), 1.42–1.32 (m, 2H), 1.32–1.27 (m, 4H), 1.25–1.20 (m, 5H), 1.13 (s, 3H), 1.04 (s, 3H), 1.03 (s, 3H), 0.92 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 198.97, 196.63, 175.53, 167.99, 165.78, 160.22, 158.61, 140.88, 133.09, 133.03, 124.04, 119.38, 119.33, 114.55, 114.48, 113.66, 113.52, 111.72, 111.57, 50.17, 49.68, 48.97, 47.91, 46.01, 45.09, 42.63, 42.49, 36.20, 34.39, 33.33, 32.40, 31.73, 30.62, 30.28, 29.78, 28.39, 26.96, 26.54, 24.56, 23.85, 22.07, 21.96, 21.63, 18.29.

[0146] Example 19

[0147] Preparation of (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-8a-(5-trifluoromethylindoline-1-carbonyl)-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropyrene-2-carbonitrile

[0148]

[0149] Refer to the method of Example 7. Compound 23 (light yellow solid, 46.3 mg, 69%) was prepared by reacting compound 1 (50 mg, 0.102 mmol) with 5-trifluoromethyl indoline (38 mg, 0.203 mmol). 1 H NMR (600 MHz, CDCl3) δ 8.16 (d, J = 8.5 Hz, 1H), 8.05 (s, 1H), 7.45–7.39 (m, 2H), 5.96 (s, 1H), 4.39–4.33 (m, 1H), 4.27 (q, J = 9.2 Hz, 1H), 3.33–3.19 (m, 4H), 1.45 (s, 3H), 1.29 (s, 3H), 1.23 (s, 3H), 1.05 (s, 3H), 1.04 (s, 3H), 0.93 (s, 3H) ppm; 13 C NMR (151 MHz, CDCl3) δ 198.89, 196.64, 176.50, 168.11, 165.74, 147.82, 131.69, 125.09, 124.06, 121.58, 118.18, 114.61, 114.49, 50.29, 49.68, 49.34, 47.90, 46.01, 45.11, 42.65, 42.47, 36.16, 34.34, 33.32, 31.74, 30.64, 30.26, 29.53, 28.39, 26.98, 26.59, 24.58, 23.84, 22.12, 21.65, 18.29.

[0150] Example 20

[0151] (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-8a-(5-Methoxyindoline-1-carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropyrene-2-carbonitrile Preparation

[0152]

[0153] Refer to the method of Example 7. Compound 24 (light yellow solid, 48.5 mg, 76.6%) was prepared by reacting compound 1 (50 mg, 0.102 mmol) with 5-methoxyindoline (30.4 mg, 0.203 mmol). 11H NMR (600 MHz, CDCl3) δ 8.04 (s, 1H), 8.01 (d, J = 8.9 Hz, 1H), 6.78 (s, 1H), 6.70 (dd, J = 9.0, 2.5 Hz, 1H), 5.95 (s, 1H), 4.31–4.28 (m, 1H), 4.22–4.17 (m, 1H), 3.78 (s, 3H), 3.31 (d, J = 13.4 Hz, 1H), 3.29–3.22 (m, 1H), 3.19–3.08 (m, 2H), 1.45 (s, 3H), 1.31 (s, 3H), 1.24 (s, 3H), 1.15 (s, 3H), 1.05 (s, 6H), 0.94 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 199.10, 196.67, 174.99, 167.96, 165.86, 156.47, 138.35, 132.59, 123.98, 119.16, 114.49, 112.05, 110.53, 55.74, 50.05, 49.67, 48.78, 47.86, 45.98, 45.06, 42.60, 42.47, 36.18, 34.39, 33.36, 31.69, 30.62, 30.25, 29.99, 28.34, 26.94, 26.53, 24.58, 23.83, 22.08, 21.89, 21.62, 18.27.

[0154] Example 21

[0155] (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-8a-(6-Fluoroindoline-1-carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-2-carbonitrile Preparation

[0156]

[0157] Referring to the method of Example 7, compound 25 (light yellow solid, 17.6 mg, 28.3%) was prepared by reacting compound 1 (50 mg, 0.102 mmol) with 6-fluoroindoline (24 μL, 0.203 mmol). 11H NMR (600 MHz, CDCl3) δ 8.04 (s, 1H), 7.85 (dd, J = 11.0, 2.2 Hz, 1H), 7.12–7.09 (m, 1H), 6.71 (td, J = 8.5, 2.4 Hz, 1H), 5.96 (s, 1H), 4.36–4.32 (m, 1H), 4.29–4.23 (m, 1H), 3.29 (d, J = 13.2 Hz, 1H), 3.22–3.17 (m, 1H), 3.15–3.12 (m, 2H), 1.46 (s, 3H), 1.31 (s, 3H), 1.24 (s, 3H), 1.14 (s, 3H), 1.05 (s, 3H), 1.04 (s, 3H), 0.93 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 198.97, 196.67, 176.07, 168.12, 165.79, 163.12, 161.52, 146.15, 146.07, 126.24, 124.74, 124.68, 124.05, 114.61, 114.50, 110.48, 110.33, 106.78, 106.59, 50.89, 49.70, 49.23, 47.92, 46.03, 45.13, 42.66, 42.48, 36.20, 34.38, 33.36, 31.76, 30.66, 30.25, 29.16, 28.41, 27.01, 26.61, 24.62, 23.88, 22.16, 21.67, 18.32.

[0158] Example 22

[0159] (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-8a-(6-chloroindoline-1-carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-2-carbonitrile Preparation

[0160]

[0161] Referring to the method of Example 7, compound 26 (light yellow solid, 40.8 mg, 64%) was prepared by reacting compound 1 (50 mg, 0.102 mmol) with 6-chloroindoline (24 μL, 0.203 mmol). 11H NMR (600 MHz, CDCl3) δ 8.14 (d, J = 2.0 Hz, 1H), 8.04 (s, 1H), 7.10 (d, J = 8.0 Hz, 1H), 7.00 (dd, J = 7.9, 1.9 Hz, 1H), 5.96 (s, 1H), 4.37–4.21 (m, 2H), 3.30 (d, J = 13.2 Hz, 1H), 3.23–3.11 (m, 3H), 1.47 (s, 3H), 1.32 (s, 3H), 1.24 (s, 3H), 1.15 (s, 3H), 1.05 (s, 3H), 1.05 (s, 3H), 0.94 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 198.93, 196.66, 176.13, 165.77, 145.98, 133.16, 129.42, 125.08, 124.09, 123.94, 118.96, 114.68, 50.52, 49.75, 49.30, 47.97, 46.07, 45.16, 42.69, 42.49, 36.25, 34.41, 33.39, 31.80, 30.71, 30.31, 29.85, 29.40, 28.45, 27.06, 26.69, 24.67, 23.92, 22.22, 21.70, 18.36.

[0162] Example 23

[0163] Preparation of (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-4,4,6a,6b,11,11,14b-heptamethyl-8a-(6-methyl-1,2,3,4-tetrahydroquinoline-1-carbonyl)-3,13-dioxo-3,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-2-carbonitrile

[0164]

[0165] Referring to the method of Example 7, Compound 27 (white solid, 17 mg, 27%) was prepared by reacting Compound 1 (50 mg, 0.102 mmol) with 1,2,3,4-tetrahydro-6-methylquinoline (30 mg, 0.203 mmol). 11H NMR (600 MHz, CDCl3) δ 8.05 (s, 1H), 7.28 (d, J = 8.2 Hz, 1H), 6.94 (s, 1H), 6.92 (d, J = 7.6 Hz, 1H), 5.96 (s, 1H), 4.09–4.05 (m, 1H), 3.68 (s, 1H), 3.32 (d, J = 13.6, 1H), 3.28 (d, J = 4.5 Hz, 1H), 2.75 (t, J = 7.0 Hz, 2H), 2.28 (s, 3H), 1.49 (s, 3H), 1.45 (s, 3H), 1.25 (s, 3H), 1.16 (s, 3H), 1.04 (s, 3H), 0.98 (s, 3H), 0.90 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 199.09, 196.73, 176.93, 167.97, 166.03, 138.25, 134.72, 129.61, 126.44, 125.69, 124.39, 114.64, 114.51, 49.98, 48.84, 47.94, 46.21, 46.09, 45.15, 42.63, 42.35, 36.54, 34.62, 33.41, 33.24, 31.88, 31.08, 30.56, 28.65, 27.12, 26.86, 26.27, 25.20, 24.12, 23.92, 21.98, 21.68, 20.98, 18.38.

[0166] Example 24

[0167] (4aR,6aR,6bR,8aS,12aS,12bR,14bR)-8a-(1H-Imidazole-1-carbonyl)-4,4,6a,6b,11,11,14b-heptamethyl-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a,14b-eicosahydrophenanthrene-2-carbonitrile Preparation

[0168]

[0169] Referring to the methods of Example 1 and Example 7, compound 28 (white solid, 135 mg, 82%) was prepared by reacting compound I-5 (150 mg, 0.304 mmol) with imidazole (21 mg, 0.304 mmol). 11H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.67–7.56 (m, 2H), 7.09 (d, J = 1.6 Hz, 1H), 3.00–2.90 (m, 2H), 2.45 (dd, J = 16.3, 4.8 Hz, 1H), 2.40–2.20 (m, 2H), 2.09 (d, J = 13.3 Hz, 1H), 2.02–1.92 (m, 3H), 1.87 (d, J = 14.4 Hz, 1H), 1.68–1.58 (m, 3H), 1.52–1.24 (m, 8H), 1.20 (s, 3H), 1.15 (s, 3H), 1.13 (s, 3H), 1.03 (s, 5H), 1.00 (s, 3H), 0.97 (s, 3H) ppm; 13 13C NMR (101 MHz, CDCl3) δ 208.20, 197.60, 175.44, 167.76, 137.41, 130.34, 117.67, 114.76, 114.69, 52.44, 51.20, 50.13, 45.09, 43.41, 42.52, 42.48, 40.53, 38.31, 36.11, 34.37, 33.18, 32.11, 31.08, 30.47, 27.83, 27.66, 23.80, 23.71, 21.55, 20.61, 18.86, 17.76, 16.62.

[0170] Example 25

[0171] (4aS,6aR,6bS,8aR,12aS,14aR,14bS)-N-Methoxy-N,2,2,6a,6b,9,9,12a-octamethyl-10,14-dioxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropicene-4a(2H)-carboxamide Preparation

[0172]

[0173] Compound II-2 (2.5 g, 5.18 mmol) was dissolved in anhydrous N,N-dimethylformamide (52 mL). Lithium iodide (13.86 g, 103.58 mmol) was added with stirring, and the mixture was stirred at 155 °C overnight in an oil bath. After the reaction was completed as detected by TLC, most of the solvent was removed by distillation under reduced pressure. The residue was diluted with ethyl acetate and washed with water three times. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain a light yellow solid (1.48 g, 61%). According to the method of Example 7, the light yellow solid (1.25 g, 2.67 mmol) was reacted with N,O-dimethylhydroxylamine hydrochloride (520 mg, 5.33 mmol) to obtain Compound I-6 (white solid, 1.2 g, 88%).

[0174] Under a nitrogen atmosphere at 0 °C, potassium bis(trimethylsilyl)amide (0.38 mL, 1 M in THF) solution was added to a solution of Compound I-6 (100 mg, 0.20 mmol) in anhydrous THF (1.1 mL). After the addition, the reaction mixture was stirred at 0 °C for an additional 10 minutes, and TMSCl (50 μL, 0.39 mmol) was added dropwise at the same temperature. The resulting reaction mixture was stirred at 0 °C for 20 minutes, quenched with H2O (10 mL), extracted with ethyl acetate (10 mL × 3), washed with brine, and concentrated in vacuo to obtain a crude product which was directly subjected to the next step. Then the crude product was dissolved in anhydrous acetonitrile (1.1 mL), and Pd(OAc)2 (88.3 mg, 0.39 mmol) was added. Then the mixture was stirred overnight in an oil bath at 60 °C and concentrated in vacuo. The residue was separated by Flash automatic column chromatography (hexane / ethyl acetate 3:1 to 1:1) to obtain Compound VIII (yellow solid, 45.2 mg, 45.4%). 1 H NMR (600 MHz, CDCl3) δ 7.32 (d, J = 10.4 Hz, 1H), 5.98 (s, 1H), 5.91 (d, J = 10.4 Hz, 1H), 3.69 (s, 3H), 3.24 (dt, J = 13.7, 4.3 Hz, 1H), 3.17 (s, 3H), 2.99 (d, J = 4.4 Hz, 1H), 1.40 (s, 3H), 1.31 (s, 3H), 1.19 (s, 3H), 1.11 (s, 3H), 1.02 (s, 3H), 1.00 (s, 3H), 0.88 (s, 3H) ppm; 1313C NMR (151 MHz, CDCl3) δ 203.66, 200.08, 178.82, 171.85, 154.97, 125.99, 123.67, 60.60, 50.25, 48.46, 48.26, 45.88, 44.82, 42.17, 41.91, 34.64, 34.54, 33.49, 32.18, 31.12, 30.84, 29.29, 28.39, 27.23, 27.07, 24.89, 23.34, 22.59, 22.33, 21.79, 18.54。

[0175] Example 26

[0176] Preparation of Compounds 31, 32 and 33

[0177]

[0178] To a stirred solution of Compound II-1 (2.0 g, 3.58 mmol) in THF (300 mL) at -78 °C was added dropwise lithium bis(trimethylsilyl)amide (1.0 M in THF, 3.58 mL, 3.58 mmol), and the mixture was stirred for 1.5 h. Methyl cyanoformate (353 μL, 3.58 mmol) was added to the solution at -78 °C, and the resulting mixture was stirred at the same temperature for 1 h. After completion of the reaction, the reaction was quenched by adding saturated aqueous NH4Cl solution (20 mL), and the mixture was extracted with dichloromethane (3 × 30 mL). The combined organic layers were washed with brine (20 mL) and dried over anhydrous Na2SO4. The solvent was removed under vacuum, and the residue was purified by Flash automatic column chromatography (n-hexane / ethyl acetate 10:1 to 5:1) to give the ester VII as a white solid (2.15 g, 97%).

[0179] Under a nitrogen atmosphere at 0 °C, potassium bis(trimethylsilyl)amide (10.5 mL, 1 M in THF) solution was added to a solution of Compound VII (2.15 g, 3.49 mmol) in anhydrous THF (3.0 mL). After the addition, the reaction mixture was stirred at 0 °C for an additional 10 min, and TMSCl (0.89 mL, 6.96 mmol) was added dropwise at the same temperature. The resulting reaction mixture was stirred at 0 °C for 20 min, quenched with H2O (40 mL), extracted with ethyl acetate (40 mL × 3), washed with brine, and concentrated in vacuo to give the crude product which was directly subjected to the next step. The crude product was then dissolved in anhydrous acetonitrile (20 mL), and Pd(OAc)2 (1.0 g, 4.4 mmol) was added. The mixture was then stirred overnight in an oil bath at 60 °C and concentrated in vacuo. The residue was separated by Flash automatic column chromatography (n-hexane / ethyl acetate 2:1 to 1:1) to give Compound VIII as a yellow solid (1.05 g, 49%).

[0180] Dissolve the above compound VIII (500 mg, 0.813 mmol) in 10 mL of tetrahydrofuran in a reaction flask, evacuate and replace with H2, add 5% or 10% wet palladium-carbon (50 mg), and react at room temperature for 6 hours until TLC analysis indicates the completion of the reaction. Filter and concentrate by rotary evaporation. The crude product is separated by an automatic Flash column chromatography machine (n-hexane / ethyl acetate 1:1) to obtain a light yellow solid IX (300 mg, 70.3%).

[0181] Referring to the method of Example 7, compound 31 (light yellow solid, 115 mg, 31.4%) was prepared by reacting compound IX (300 mg, 0.572 mmol) with 5-methylindoline (152 mg, 1.14 mmol). 1 H NMR (600 MHz, CDCl3) δ 8.06 (s, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.02 (s, 1H), 6.97 (dd, J = 8.4, 1.8 Hz, 1H), 6.08 (s, 1H), 4.31–4.16 (m, 2H), 3.80 (s, 3H), 3.33 (d, J = 13.2 Hz, 1H), 3.25 (s, 1H), 3.16–3.06 (m, 2H), 2.30 (s, 3H), 1.35 (s, 3H), 1.29 (s, 3H), 1.17 (s, 3H), 1.16 (s, 3H), 1.05 (s, 6H), 0.93 (s, 3H) ppm; 13 C NMR (151 MHz, CDCl3) δ 199.59, 199.52, 175.53, 165.02, 160.60, 142.52, 133.59, 131.18, 129.79, 127.88, 125.16, 125.03, 118.33, 52.46, 50.11, 49.88, 49.02, 48.29, 46.00, 45.90, 42.50, 41.92, 36.35, 34.45, 33.45, 31.59, 30.71, 30.34, 29.88, 28.42, 27.99, 27.12, 24.49, 23.97, 22.15, 21.34, 21.05, 18.72。

[0182] Compound 31 (115 mg, 0.180 mmol) was dissolved in 1,2-dichloroethane (2 mL), and trimethyltin hydroxide (139 mg, 0.720 mmol) was added. The reaction was carried out in an oil bath at 80 °C until TLC analysis indicated the completion of the reaction. The pH was adjusted to 2 - 3 with 3N hydrochloric acid, and the mixture was extracted with ethyl acetate three times, washed with brine once, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum. The crude product was separated by preparative thin-layer chromatography on a TLC plate with a thickness of 1 mm (n-hexane / ethyl acetate 1:1) to obtain the light yellow solid product 32 (97 mg, 86%). 1 1H NMR (600 MHz, CDCl3) δ 12.29 (s, 1H), 8.77 (s, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.02 (s, 1H), 6.99–6.95 (m, 1H), 6.15 (s, 1H), 4.30–4.17 (m, 2H), 3.31 (d, J = 13.1 Hz, 1H), 3.22 (s, 1H), 3.15–3.08 (m, 2H), 2.30 (s, 3H), 1.44 (s, 3H), 1.31 (s, 3H), 1.27 (s, 3H), 1.19 (s, 3H), 1.04 (s, 3H), 1.04 (s, 3H), 0.92 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 207.59, 199.08, 175.50, 169.02, 167.94, 164.20, 142.50, 133.59, 131.18, 127.85, 125.17, 124.34, 123.67, 118.31, 50.09, 49.76, 48.99, 47.74, 46.06, 45.61, 42.45, 42.42, 36.25, 34.44, 33.43, 31.69, 30.69, 30.31, 29.86, 28.38, 27.40, 26.90, 24.70, 23.93, 22.25, 21.95, 21.04, 18.43。

[0183] Referring to the method of Example 4, compound 33 (light yellow solid, 13 mg, 62.5%) was prepared by the Curtius rearrangement reaction of compound 32 (20 mg, 0.032 mmol). 11H NMR (600 MHz, CDCl3) δ 7.96 (d, J = 8.3 Hz, 1H), 7.02 (s, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.32 (s, 1H), 5.94 (s, 1H), 4.33–4.17 (m, 2H), 3.55 (s, 1H), 3.31 (d, J = 13.3 Hz, 1H), 3.18–3.10 (m, 2H), 2.30 (s, 3H), 1.36 (s, 3H), 1.27 (s, 3H), 1.20 (s, 3H), 1.10 (s, 3H), 1.05 (s, 3H), 1.03 (s, 3H), 0.93 (s, 3H) ppm; 13 13C NMR (151 MHz, CDCl3) δ 200.22, 199.73, 175.63, 142.55, 135.82, 133.53, 131.19, 127.88, 125.14, 123.59, 123.27, 118.36, 50.08, 49.64, 49.06, 48.40, 45.75, 44.33, 42.47, 40.79, 36.29, 34.47, 33.49, 32.35, 30.72, 30.41, 29.89, 28.41, 28.11, 27.15, 24.63, 23.98, 22.16, 21.06, 18.58.

[0184] Example 27

[0185] (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-N-Cyclopropyl-4,4,6a,6b,11,11,14b-heptamethyl-8a-(5-methylindoline-1-carbonyl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropyrene-2-carboxamide Preparation

[0186]

[0187] Compound 32 (24 mg, 0.038 mmol), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate TBTU (18 mg, 0.056 mmol) and triethylamine (20 μL, 0.144 mmol) were dissolved in anhydrous tetrahydrofuran (1.0 mL). After stirring at room temperature for 15 minutes, cyclopropylamine (5.3 μL, 0.076 mmol) was added and the reaction was carried out at room temperature for 4 hours. After the reaction was detected to be complete by TLC, it was diluted with ethyl acetate, washed 3 times with water, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The crude product was separated by preparative thin-layer chromatography on a TLC plate with a thickness of 1 mm (n-hexane / ethyl acetate 3:1) to obtain compound 34 (white solid, 13.2 mg, 52%). 1 H NMR (600 MHz, CDCl3) δ 8.62 (s, 1H), 8.59 (s, 1H), 7.95 (dd, J = 8.4, 2.9 Hz, 1H), 7.02 (s, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.21 (s, 1H), 4.30–4.17 (m, 2H), 3.33 (d, J = 12.4 Hz, 1H), 3.21–3.16 (m, 1H), 3.14–3.08 (m, 2H), 2.86–2.83 (m, 1H), 2.30 (s, 3H), 1.35 (s, 3H), 1.29 (s, 3H), 1.25 (s, 3H), 1.15 (s, 3H), 1.05 (s, 3H), 1.04 (s, 3H), 0.93 (s, 3H), 0.80 (d, J = 6.1 Hz, 2H), 0.56–0.54 (m, 2H) ppm; 13 C NMR (151 MHz, CDCl3) δ 204.97, 199.28, 175.61, 164.58, 164.33, 133.55, 131.19, 127.87, 127.61, 125.16, 124.85, 118.35, 50.10, 49.86, 49.09, 47.69, 46.12, 45.98, 42.40, 41.91, 36.34, 34.50, 33.48, 31.64, 30.75, 30.41, 29.90, 28.44, 28.26, 27.60, 24.58, 23.99, 22.84, 22.30, 21.87, 21.05, 18.75, 6.57。

[0188] Example 28

[0189] Preparation of (4aR,6aS,6bR,8aS,12aS,12bR,14bS)-N-morpholin-4-yl-4,4,6a,6b,11,11,14b-heptamethyl-8a-(5-methylindoline-1-carbonyl)-3,13-dioxo-3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-octadecahydropyrene-2-carboxamide

[0190]

[0191] Compound 35 (white solid, 28.3 mg, 85%) was prepared by reacting compound 32 (30 mg, 0.048 mmol) with morpholine (8.3 mg, 0.096 mmol) according to the method of Example 27. 1 H NMR (600 MHz, CDCl3) δ 7.95 (d, J = 8.3 Hz, 1H), 7.41 (s, 1H), 7.02 (s, 1H), 6.97 (dd, J = 8.0, 1.7 Hz, 1H), 5.96 (s, 1H), 4.27 (td, J = 9.4, 5.8 Hz, 1H), 4.19 (q, J = 9.0 Hz, 1H), 3.78–3.65 (m, 5H), 3.34–3.29 (m, 1H), 3.23 (t, J = 4.8 Hz, 3H), 3.16–3.07 (m, 2H), 2.30 (s, 3H), 1.42 (s, 3H), 1.30 (s, 3H), 1.20 (s, 3H), 1.15 (s, 3H), 1.05 (s, 6H), 0.93 (s, 3H) ppm; 13 C NMR (151 MHz, CDCl3) δ 200.34, 199.47, 175.54, 165.89, 153.33, 142.55, 134.03, 133.59, 131.18, 127.89, 125.17, 124.11, 118.36, 66.88, 66.85, 50.10, 49.74, 49.04, 48.22, 47.59, 45.98, 45.19, 42.50, 42.36, 41.64, 36.31, 34.49, 33.46, 31.84, 30.72, 30.37, 29.89, 29.84, 28.46, 27.42, 27.29, 24.54, 23.95, 22.28, 21.75, 21.05, 18.60.

[0192] Example 29

[0193] (4aS, 6aR, 6bS, 8aR, 12aS, 14aR, 14bS)-11-Cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-N-(2-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)-1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropicene-4a(2H)-carboxamide Preparation

[0194]

[0195] Referring to the method of Example 7, Compound 36 (white solid, 125 mg, 81%) was prepared by reacting Compound 1 (100 mg, 0.203 mmol) with biotinyl ethylenediamine hydrochloride (87.4 mg, 0.305 mmol), R f = 0.3 (DCM:MeOH = 20:1). 1 1H NMR (600 MHz, CDCl3) δ 8.11 (s, 1H), 7.51 (s, 1H), 7.28 (s, 1H), 6.44 (s, 1H), 6.01 (s, 1H), 5.73 (s, 1H), 4.52–4.50 (m, 1H), 4.32–4.30 (m, 1H), 3.43–3.35 (m, 3H), 3.30–3.27 (m, 1H), 3.08–3.04 (m, 2H), 2.97–2.93 (m, 1H), 2.88 (dd, J = 12.9, 4.9 Hz, 1H), 2.74 (d, J = 12.8 Hz, 1H), 2.20 (t, J = 7.6 Hz, 2H), 1.98–1.91 (m, 1H), 1.45 (s, 3H), 1.28 (s, 3H), 1.22 (s, 3H), 1.13 (s, 3H), 0.97 (s, 3H), 0.95 (s, 3H), 0.86 (s, 3H) ppm.

[0196] Example 30

[0197] (4aS, 6aR, 6bS, 8aR, 12aS, 14aR, 14bS)-11-Cyano-N-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-hexadecahydropicene-4a(2H)-carboxamide Preparation

[0198]

[0199] Referring to the method of Example 5, Compound 37 (yellow solid, 20 mg, 12%) was prepared by reacting Compound 1 (100 mg, 0.203 mmol) with 5 - aminofluorescein (211.7 mg, 0.407 mmol) under light protection, R f = 0.3 (DCM:MeOH = 10:1). 1 1H NMR (600 MHz, MeOD - d4): δ 8.41 (s, 1H), 7.24 (s, 1H), 7.14 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 2.2 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.89 (d, J = 8.6 Hz, 1H), 6.81 (dt, J = 8.8, 2.4 Hz, 1H), 6.72–6.65 (m, 2H), 6.58 (dd, J = 8.7, 2.4 Hz, 1H), 6.16 (s, 1H), 1.37 (s, 3H), 1.29 (s, 3H), 1.25 (s, 3H), 1.17 (s, 3H), 1.10 (s, 3H), 1.02 (s, 3H), 0.94 (s, 3H) ppm.

[0200] Example 31

[0201] (4aS, 6aR, 6bS, 8aR, 12aS, 14aR, 14bS)-11 - Cyano - N-(2-(3-(3',6'-dihydroxy - 3 - oxo - 3H - spiro[isobenzofuran - 1,9'-xanthene]-5 - yl)thioureido)ethoxy)ethyl)-2,2,6a,6b,9,9,12a - heptamethyl - 10,14 - dioxo - 1,3,4,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b - hexadecahydropicene - 4a(2H)-carboxamide (Compound 39) Preparation

[0202]

[0203] Referring to the method of Example 5, Compound I-6 (white solid, 275 mg, 99.7%) was prepared by reacting Compound 1 (200 mg, 0.407 mmol) with tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (100 mg, 0.488 mmol). Then, Compound I-7 (275 mg, 0.407 mmol) was dissolved in dry dichloromethane (4 mL), and trifluoroacetic acid (623 μL, 8.14 mmol) was added under ice bath. The reaction mixture was transferred to room temperature and reacted overnight until TLC analysis indicated complete reaction. 10% sodium carbonate solution was slowly added under ice bath to neutralize, and the layers were separated. The aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried, concentrated, and the crude product was separated by Flash automatic column chromatography (dichloromethane / methanol 20:1) to obtain Compound 38 (white solid, 212 mg, 90%). 1 H NMR(600MHz,CDCl3)δ8.05(s,1H),6.70(s,1H),5.93(s,1H),3.44–3.39(m,4H),3.07–2.56(m,6H),1.41(s,3H),1.26(s,3H),1.16(s,3H),1.08(s,3H),0.93(s,3H),0.89(s,3H),0.80(s,3H)ppm; 13 C NMR(151MHz,CDCl3)δ199.28,196.66,177.26,168.78,166.05,162.52,123.88,114.43,72.33,69.51,51.00,49.43,47.58,46.24,46.21,45.84,44.94,42.56,42.05,41.55,39.19,36.45,36.06,34.56,33.87,33.26,31.62,31.38,30.58,28.63,27.72,26.87,26.50,26.37,26.32,24.66,23.06,22.79,21.65,21.50,18.18。

[0204] Compound 38 (100 mg, 0.173 mmol), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate TBTU (83.4 mg, 0.260 mmol) and N,N-diisopropylethylamine (60 μL, 0.346 mmol) were dissolved in N,N-dimethylformamide (3.0 mL). After stirring at room temperature for 15 minutes, fluorescein isothiocyanate (67.4 mg, 0.173 mmol) was added, and the reaction was carried out overnight in the dark at room temperature. After the reaction was detected to be complete by TLC, 20 mL of water was added for dilution, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The residue was separated by an automatic Flash column chromatography machine (dichloromethane / methanol 10:1) to obtain the crude product, and then separated by preparative thin-layer plate chromatography with a thickness of 1 mm (dichloromethane / methanol 10:1) to obtain Compound 39 (yellow solid, 50 mg, 30%). 1 H NMR (600 MHz, DMSO-d6) δ 10.21–10.05 (m, 2H), 8.64 (s, 1H), 8.32 (s, 1H), 8.09 (s, 1H), 7.78–7.66 (m, 2H), 7.19 (d, J = 8.2 Hz, 1H), 6.67 (d, J = 2.2 Hz, 2H), 6.62–6.52 (m, 4H), 6.18 (s, 1H), 5.76 (s, 1H), 3.74–3.55 (m, 4H), 3.49 (dd, J = 11.2, 5.2 Hz, 2H), 3.46–3.41 (m, 1H), 3.18–3.14 (m, 1H), 3.03 (d, J = 4.7 Hz, 1H), 2.86 (d, J = 13.6 Hz, 1H), 1.87 (t, J = 14.4 Hz, 1H), 1.82–1.76 (m, 2H), 1.65–1.54 (m, 6H), 1.39 (s, 3H), 1.24 (s, 3H), 1.23 (s, 3H), 1.13 (s, 3H), 1.01 (s, 3H), 0.90 (s, 3H), 0.87 (s, 3H) ppm; 1313C NMR (151 MHz, DMSO-d6) δ 199.23, 197.27, 180.46, 176.73, 168.95, 168.51, 168.29, 159.62, 151.93, 141.46, 129.06, 128.91, 123.31, 115.06, 112.77, 112.58, 109.76, 109.53, 102.25, 68.94, 68.02, 54.88, 48.64, 46.25, 45.42, 45.33, 44.33, 43.70, 42.52, 41.51, 35.32, 34.15, 33.22, 33.02, 31.15, 30.73, 30.19, 28.99, 28.39, 27.21, 26.08, 25.86, 24.26, 23.10, 22.07, 21.80, 21.27, 21.05, 17.54, 13.93。

[0205] Example 32

[0206] In this example, the effect of the compound on the THP1-Blue TM ISG cell reporter gene was determined.

[0207] Experimental principle: Activation of innate immune signal receptors leads to the nuclear translocation of downstream IRF3, promoting the expression and secretion of embryonic alkaline phosphatase (SEAP). THP1-Blue TM ISG cells can be used to detect the intracellular interferon signaling by stably integrating the SEAP reporter gene induced by interferon regulatory factors. Quanti-Blue TM detection reagent can detect the activity of SEAP in the cell culture supernatant. Therefore, this reporter gene experiment can be used to characterize whether the compound affects the inflammation-related signaling pathway.

[0208] Experimental reagents and consumables: RPMI 1640 medium (Meilunbio), penicillin / streptomycin solution (Meilunbio), fetal bovine serum (Meilunbio), phorbol ester (MCE), cyclic dinucleotide (MCE), Quanti-Blue detection reagent (Invivogen), fully transparent 384-well plate (Nest Biotechnology).

[0209] Experimental method:

[0210] (1) Cell seeding and drug administration: The THP1-Blue TM ISG suspension cells in good growth condition were seeded at 1×10 6Inoculate at a density of / ml into a 384-well plate, 40 μL per well. After the cells adhere to the wall stimulated by PMA, perform drug administration treatment. Compounds are generally serially diluted 16-fold starting from 20 mM. After being diluted 20-fold with RPMI 1640 medium, add them to the wells to be administered at a volume of 5 μL per well, with DMSO as the control. After 1 h of compound treatment, stimulate with cyclic dinucleotide (5 μM, 5 μL per well), and use the DMSO well without cyclic dinucleotide as the control to reflect the stimulation level of the cells.

[0211] (2) Detection: After 24 h of cyclic dinucleotide treatment, add 16 μL of the detection reagent Quanti-Blue solution per well. Centrifuge the plate and incubate at 37 °C for 30 min. Finally, use a multi-functional microplate reader to detect the absorbance of the wells to be measured at 650 nm.

[0212] (3) Data processing: The formula for the relative inhibition rate is: RLU(%) = (reading 环二核苷酸+DMSO孔 - reading 环二核苷酸+化合物孔 ) / (reading 环二核苷酸+DMSO孔 - reading DMSO孔 ) * 100%. Import the data into GraphPad Prism 8 software for non-linear regression fitting and calculate the IC 50 value.

[0213] Experimental results: The effects of the compounds on the reporter gene activity of THP1-Blue TM ISG cells are shown in Table 1.

[0214] Table 1 Inflammatory inhibition activity IC TM of the compounds on THP1-Blue 50 ISG cells (nM)

[0215]

[0216] As can be seen from Table 1, the triterpenoid derivatives 1-29, 31, 32, 34, 38 in the present invention have good cell inflammatory inhibition effects. Among them, the IC 50 of Compound 19 reaches 25 nM, and it can be preferably used as a potential candidate drug for the treatment of autoinflammatory diseases.

[0217] The above is only a preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. Any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A pentacyclic triterpenoid derivative or its isomer, salt, ester or solvate, characterized in that, It has a general formula structure shown in Formula I: Wherein, R1 is one of the following groups: azido group, amino group, acyl chloride, -COOR3, -CONHR4, -NHCOR5, -CONR6R7; Among them, R2 is one of the following groups: hydrogen cyano group, methyl ester, ethyl ester, aldehyde group, carboxyl group, amino group, CONHR8, CONR9R 10 .

2. The pentacyclic triterpenoid derivative or its isomer, salt, ester or solvate according to claim 1, wherein R3 is one of the following groups: hydrogen methyl benzyl, trifluoroethyl benzotriazolyl; R4 is one of the following groups: substituted C1-C6 alkyl or unsubstituted C1-C6 alkyl, saturated alicyclic hydrocarbon group, substituted phenyl or unsubstituted phenyl, substituted benzyl or unsubstituted benzyl, substituted 5-6-membered heteroaryl or unsubstituted 5-6-membered heteroaryl; R5 is one of the following groups: substituted C1-C6 alkyl or unsubstituted C1-C6 alkyl, saturated alicyclic hydrocarbon group, substituted phenyl or unsubstituted phenyl, substituted benzyl or unsubstituted benzyl, substituted 5-6-membered heteroaryl or unsubstituted 5-6-membered heteroaryl; R6 and R7 are disubstituted groups on the N atom, or, a substituted C3-C8 ring or an unsubstituted C3-C8 ring formed by R6 and R7 and the heteroatom N: Among them, R is one of the following groups: halogen Trifluoromethyl Methyl Methoxy X is halogen, and it is any one of F, Cl, and Br; R8 is one of the following groups: substituted C1-C6 alkyl or unsubstituted C1-C6 alkyl, saturated alicyclic hydrocarbon group, substituted phenyl or unsubstituted phenyl, substituted benzyl or unsubstituted benzyl, substituted 5-6-membered heteroaryl or unsubstituted 5-6-membered heteroaryl; The R9 and R 10 are disubstituted groups on the N atom, or, R9 and R 10 form a substituted or unsubstituted C3-C8 ring together with the heteroatom N.

3. The pentacyclic triterpenoid derivative or its isomer, salt, ester or solvate according to claim 2, wherein The saturated alicyclic hydrocarbon group is any one of the groups in; and / or The unsubstituted C1-C6 alkyl group is any one of the groups in; and / or The substituted C1-C6 alkyl group is any one of the groups in 4. The pentacyclic triterpenoid derivative or its isomer, salt, ester or solvate according to any one of claims 1-3, characterized in that, The pentacyclic triterpenoid derivative is one of the following compounds:

5. A method for preparing the pentacyclic triterpenoid derivative according to any one of claims 1-4, characterized in that, It includes the following steps: The initial raw material oleanolic acid is converted into the general intermediate II through multiple steps of reaction, and then the general intermediate II is obtained through multiple steps of reaction to obtain the triterpenoid derivative shown in the general formula I.

6. The preparation method of the pentacyclic triterpenoid derivative according to claim 5, characterized in that, The preparation method includes the following synthetic route: and / or Wherein, II-1 is one of the intermediates shown in the general formula II; I-1, I-2, I-3 and I-4 are partial pentacyclic triterpenoid derivatives shown in the general formula I; Wherein, the Pd / C is wet palladium carbon with a mass fraction of 5%-10%; The solvents other than methanol, ethanol, ether and water are all ultra-dry solvents; The separation of all compounds is achieved by a Flash automatic column chromatography machine or preparative thin layer plate chromatography technology.

7. Use of the pentacyclic triterpenoid derivative or its isomer, salt, ester or solvate according to any one of claims 1-4 in the preparation of anti-inflammatory small molecule drugs. Use of a compound having an anti-inflammatory effect in a pentacyclic triterpenoid derivative or an isomer, salt, ester or solvate thereof according to any one of claims 1-4 in the preparation of an anti-inflammatory drug for treating autoimmune diseases, wherein the compound having an anti-inflammatory effect is preferably compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14, compound 15, compound 16, compound 17, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 24, compound 25, compound 26, compound 27, compound 28, compound 29, compound 31, compound 32, compound 34 and / or compound 38 in claim 4.

9. An anti-inflammatory small molecule pharmaceutical composition, characterized in that Comprising: The pentacyclic triterpenoid derivative or an isomer, salt, ester or solvate thereof according to any one of claims 1-4, and at least one pharmaceutically acceptable excipient.

10. An anti-inflammatory drug composition for treating autoimmune diseases, characterized in that, Comprising: A compound having an anti-inflammatory effect in the pentacyclic triterpenoid derivative or an isomer, salt, ester or solvate thereof according to any one of claims 1-4, and at least one pharmaceutically acceptable excipient; wherein the compound having an anti-inflammatory effect is preferably compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14, compound 15, compound 16, compound 17, compound 18, compound 19, compound 20, compound 21, compound 22, compound 23, compound 24, compound 25, compound 26, compound 27, compound 28, compound 29, compound 31, compound 32, compound 34 and / or compound 38 in claim 4.

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