A c-3 substituted oleanolic acid benzamide derivative, its preparation method and application

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a C-3 substituted oleanolic acid benzamide derivative, a preparation method and application thereof. The C-3 substituted oleanolic acid benzamide derivative has a novel chemical structure, and has good inhibitory activity on influenza virus, especially on H1N1 influenza virus A in vitro and in vivo, which indicates that the compound can be prepared into an anti-influenza virus preparation, has a good application prospect in the prevention or / and treatment of influenza virus infection, and expands the application value of the C-3 substituted oleanolic acid benzamide derivative.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a C-3 substituted oleanolic acid benzylamide derivative, its preparation method, and its application. Background Technology

[0002] The constantly mutating influenza A virus poses a serious threat to human health worldwide. Each year, hundreds of thousands of people die globally from severe illness caused by influenza A, and it is also a leading cause of death among the elderly and those with weakened immune systems. Symptoms of influenza A include respiratory symptoms and fever; in severe cases, it can lead to pneumonia, and even respiratory failure, multiple organ failure, and death.

[0003] To date, antiviral drugs for influenza mainly fall into three categories: M2 proton channel inhibitors (amantadine and rimantadine), neuraminidase (NA) inhibitors (oseltamivir, peramivir, zanamivir, and lanamivir), and RNA-dependent RNA polymerase (RdRp) inhibitors. Influenza virus RdRp is a heterotrimeric complex composed of the polymerase acidic (PA), polymerase basic 1 (PB1), and polymerase basic 2 (PB2) subunits. Recently, two RdRp inhibitors have been marketed: the PA inhibitor baloxavir and the PB1 inhibitor favipira, demonstrating their promising potential as antiviral drugs for influenza. However, the available marketed antiviral drugs for influenza remain limited.

[0004] In order to cope with the continuous emergence of variant strains of influenza A virus, my country still needs to accelerate in-depth research on conserved potential drug targets in influenza A virus strains such as H1N1, and strengthen continuous scientific research on the development of broad-spectrum anti-influenza virus drugs, in order to obtain a variety of candidate drugs with novel mechanisms of action, so as to better protect people's health. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings and deficiencies of the limited availability of existing commercially available antiviral drugs for influenza, and to provide a C-3 substituted oleanolic acid benzylamide derivative.

[0006] The purpose of this invention is to provide a method for preparing the C-3 substituted oleanolic acid benzylamide derivative.

[0007] Another object of the present invention is to provide the application of the C-3 substituted oleanolic acid benzylamide derivative.

[0008] Another object of the present invention is to provide an antiviral drug for influenza.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention protects a C-3 substituted oleanolic acid benzylamide derivative, the structure of which is shown in Formula I below:

[0011]

[0012] In formula I, R is R 1 (m1) Substituted benzene ring, R 2 (m2) Substituted benzyl or R 3 (m3) Replace C 3～6 Cycloalkenyl, the R 1 Whether monosubstituted, polysubstituted, or unsubstituted, m1 represents R on the benzene ring. 1 The number of elements, m1 being any integer from 0 to 5; the R 1 Each is independently selected from hydrogen, hydroxyl, and C. 1～6 Alkoxy, halogen or C 1～6 alkyl;

[0013] The R 2 Whether monosubstituted, polysubstituted, or unsubstituted, m2 represents R on the benzene ring. 2 The number of elements, m2 is any integer from 0 to 5; the R 2 Each is independently selected from hydrogen, hydroxyl, and C. 1～6 Alkoxy, halogen or C 1～6 alkyl;

[0014] The R 3 Whether monosubstituted, polysubstituted, or unsubstituted, m3 represents the R on the cycloalkenyl group. 3 The number of elements, m3 is any integer from 0 to 5; the R 3 Each is independently selected from hydrogen, hydroxyl, and C. 1～6 Alkoxy, halogen or C 1～6 alkyl;

[0015] n is any integer from 0 to 6.

[0016] Specifically, the R 1 (m1) For substituents on the benzene ring, the hydrogen on the benzene ring is replaced by R 1 Monosubstituted, polysubstituted, or unsubstituted; m1 represents R on the benzene ring. 1The number of atoms, m1 is any integer from 0 to 5; when m1 = 0, it means that the H on the benzene ring is not substituted; when m1 = 1, it means that the H on the benzene ring is substituted by R. 1 Monosubstitution; when m1 = 2–5, it indicates that the H on the benzene ring is replaced by R. 1 Multiple substitutions, R at different substitution sites 1 Same or different.

[0017] Specifically, the R 2 (m2) The benzyl group is a substituent on the benzene ring, and the hydrogen on the benzene ring is replaced by R. 2 Monosubstituted, polysubstituted, or unsubstituted; m2 represents R on the benzene ring. 2 The number of atoms, m2 is any integer from 0 to 5; when m2 = 0, it means that the H on the benzene ring is not substituted; when m2 = 1, it means that the H on the benzene ring is substituted by R. 2 Monosubstitution; when m2 = 2–5, it indicates that the H on the benzene ring is replaced by R. 2 Multiple substitutions, R at different substitution sites 2 Same or different.

[0018] Specifically, the R 3 (m3) It is a substituent on the cycloalkenyl group, and the hydrogen on the cycloalkenyl group is replaced by R. 3 Monosubstituted, polysubstituted, or unsubstituted; m3 represents the R on the cycloalkenyl group. 3 The number of , m3 is any integer from 0 to 5; when m3 = 0, it means that the H on the cycloalkenyl group is not substituted; when m3 = 1, it means that the H on the cycloalkenyl group is substituted by R. 3 Monosubstituted; when m3 = 2–5, it indicates that the H on the cycloalkenyl group is replaced by R. 3 Multiple substitutions, R at different substitution sites 3 Same or different.

[0019] Preferably, R is R 1 (m1) Substituted benzene ring, R 2 (m2) Substituted benzyl or R 3 (m3) Replace C 5～6 Cycloalkenyl, the R 1 Each is independently selected from hydrogen, hydroxyl, and C. 1～6 alkoxy or halogen, said R 2 The R is selected from hydrogen or hydroxyl. 3 The numbers are selected from hydrogen or hydroxyl; m1, m2, and m3 are each independently selected from any natural number from 0 to 3; n is 0 or 1.

[0020] More preferably, the R 1 Each is independently selected from hydrogen, hydroxyl, and C. 1～3 Alkoxy or halogen.

[0021] More preferably, the R 1 Each is independently selected from hydrogen, hydroxyl, and C. 1～3 Alkyl groups or fluorine.

[0022] More preferably, R is selected from phenolyl, hydroquinone, pyrogallol, C 1～3 Alkoxy-substituted phenyl, difluorophenyl, dihydroxy-substituted benzyl, or trihydroxy-substituted cyclohexenyl.

[0023] More preferably, R is selected from 4-hydroxy-substituted phenyl, 3,4-dihydroxy-substituted phenyl, 3,4,5-trihydroxy-substituted phenyl, 2,3-dihydroxy-substituted phenyl, 3,5-dihydroxy-substituted phenyl, 2,4-dihydroxy-substituted phenyl, 3,4-dimethoxy-substituted phenyl, 3,4-difluoro-substituted phenyl, 3,4-dihydroxy-substituted benzyl, or 3,4,5-trihydroxy-substituted cyclohexenyl.

[0024] Furthermore, the C-3 substituted oleanolic acid benzylamide derivative also includes its pharmaceutically acceptable salt, its crystal form, its solvate, its stereoisomer, or its isotopic substituted compound.

[0025] This invention protects a method for preparing the C-3 substituted oleanolic acid benzylamide derivative, comprising the following steps:

[0026] S1. Under normal temperature conditions, intermediate compound 1 was obtained by acetylation reaction using oleanolic acid as a raw material:

[0027]

[0028] S2. Under normal temperature conditions, the intermediate compound 1 obtained in step S1 is reacted with oxalyl chloride to generate an acyl chloride intermediate, then triethylamine is added as an acid-binding agent, and then condensed with benzylamine to generate intermediate compound 2:

[0029]

[0030] S3. At 45–50 °C, the intermediate compound 2 obtained in step S2 is hydrolyzed in the presence of an alkaline reagent to obtain intermediate compound 3:

[0031]

[0032] S4. Under ice bath conditions, intermediate compound 3 and compound P-1 obtained in step S3 are mixed evenly, and a condensation reaction is carried out under an inert protective atmosphere and at room temperature to obtain intermediate compound 4:

[0033]

[0034] S5. At room temperature, intermediate compound 4 obtained in step S4 is selectively deprotected by the TBDMS protecting group to obtain intermediate compound 5:

[0035]

[0036] S6. Under ice bath conditions, intermediate compound 5 and compound P-2 obtained in step S5 are mixed evenly, and a condensation reaction is carried out under an inert protective atmosphere and at room temperature to obtain intermediate compound 6-n:

[0037]

[0038] S7. Under normal temperature and an inert protective atmosphere, the intermediate compound 6-n obtained in step S6 is selectively deprotected by the Boc (tert-butyloxycarbonyl) protecting group to obtain the intermediate compound of general formula 7-n:

[0039]

[0040] S8. Under ice bath conditions, the intermediate compound obtained in step S7 is mixed thoroughly with compound P-3, and a condensation reaction is carried out under an inert protective atmosphere and at room temperature to obtain the intermediate compound of general formula 8-n:

[0041]

[0042] Wherein, R' is defined as: when R in equation (I) is R 1 (m1) Substituted benzene ring or R 2 (m2) Substitute benzyl, and R 1 R 2 If R is a hydroxyl group, then R' is the group formed by replacing the hydrogen of the hydroxyl group in R with Bn; when R is selected from R 3 (m3) Replace C 3～6 Cycloalkenyl, and R 3 If the hydroxyl group is hydroxyl, then R' is the group formed by replacing the hydrogen of the hydroxyl group in R with the group after TBDMS; otherwise, R' is defined the same as R.

[0043] S9. When R' does not contain TBDMS: Under normal temperature and hydrogen atmosphere, the intermediate compound 8-n obtained in step S8 is debenzyl protecting group to obtain the substituted oleanolic acid benzylamide derivative with C-3.

[0044] When R' contains TBDMS: Under normal temperature conditions, the intermediate compound 8-n obtained in step S8 is first selectively deprotected by the TBDMS protecting group, and then the benzyl protecting group is removed at -75 to -80°C in an inert protective atmosphere to obtain the oleanolic acid benzylamide derivative with C-3 substituted.

[0045]

[0046] The definitions of n and R are the same as those described above.

[0047] Furthermore, in step S1, the oleanolic acid undergoes an acetylation reaction under the action of an acetylation reagent and a catalyst.

[0048] Preferably, the acetylation agent is acetic anhydride or acetyl chloride, and the catalyst is preferably 4-dimethylaminopyridine.

[0049] Furthermore, the molar ratio of oleanolic acid, acetylation reagent, and catalyst is 1:(1.5-2):(0.1-0.2).

[0050] Furthermore, the reaction solvent used in the acetylation reaction is one or a combination of pyridine, dichloromethane, or the like.

[0051] Furthermore, in step S2, the reaction solvent used in the condensation reaction is preferably dichloromethane, tetrahydrofuran, or N,N-dimethylformamide.

[0052] Furthermore, the molar ratio of intermediate compound 1, oxaloyl chloride, triethylamine and benzylamine is 1:(3-5):(3-5):(1.5-2.5).

[0053] Further, in step S3, the alkaline reagent is selected from sodium hydroxide, potassium hydroxide, or lithium hydroxide, preferably sodium hydroxide.

[0054] Furthermore, in step S3, a catalyst is also required. The catalyst is a quaternary ammonium salt compound, preferably tetrabutylammonium iodide, benzyltriethylammonium chloride, tetrabutylammonium bromide or tetrabutylammonium chloride, and more preferably tetrabutylammonium iodide.

[0055] Furthermore, the molar ratio of the intermediate compound 2 to the catalyst is 1:(0.4 to 0.6).

[0056] Furthermore, in step S3, the reaction solvent used in the hydrolysis reaction is one or a combination of tetrahydrofuran and methanol.

[0057] Further, in step S4, the condensation reaction is carried out under the action of a condensing agent and a catalyst, wherein the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the acylation catalyst is preferably 4-dimethylaminopyridine.

[0058] Preferably, the molar ratio of the condensing agent to the acylation catalyst is 1:(0.05 to 0.25).

[0059] Furthermore, in step S4, the reaction solvent used in the condensation reaction is preferably dichloromethane, tetrahydrofuran, or N,N-dimethylformamide.

[0060] Preferably, in step S4, the molar ratio of intermediate compound 3, compound P-1 and condensing agent is 1:(1.45~1.55):(2.25~2.75).

[0061] Furthermore, in step S5, the selective removal of the TBDMS (tert-butyldimethylsilyl ether) protecting group is carried out under the action of a catalyst, which is tetrabutylammonium fluoride or its hydrate or acetic acid.

[0062] Furthermore, in step S5, the reaction solvent used for the selective removal of the TBDMS protecting group is preferably tetrahydrofuran, dichloromethane, or N,N-dimethylformamide.

[0063] Preferably, the molar ratio of the intermediate compound 4 to the catalyst is 1:(4-6).

[0064] Further, in step S6, the condensation reaction is carried out under the action of a condensing agent and an acylation catalyst, wherein the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the acylation catalyst is preferably 4-dimethylaminopyridine.

[0065] Preferably, the molar ratio of the condensing agent to the acylation catalyst is 1:(0.05 to 0.25).

[0066] Furthermore, the molar ratio of intermediate compound 5, compound P-2 and condensing agent is 1:(1.8-2.2):(2.4-2.8).

[0067] Furthermore, in step S6, the reaction solvent used in the condensation reaction is dichloromethane, tetrahydrofuran, or N,N-dimethylformamide.

[0068] Furthermore, in step S7, the selective removal of the Boc protecting group is carried out under the action of a catalyst, namely trifluoroacetic acid.

[0069] Furthermore, in step S7, the reaction solvent used for the selective removal of the Boc protecting group is dichloromethane, tetrahydrofuran, or N,N-dimethylformamide.

[0070] Furthermore, the molar ratio of the intermediate compound 6-n to the catalyst is 1:(8-22).

[0071] Further, in step S8, the condensation reaction is carried out under the action of a condensing agent, an acylation catalyst, and an organic base. The condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), the acylation catalyst is preferably 1-hydroxy-7-azidobenzotriazole (HOAt) (mainly used to inhibit racemic reactions), and the organic base is preferably N-methylmorpholine (NMM) (to deprotonate carboxylic acids).

[0072] Preferably, the molar ratio of the condensing agent, the acylation catalyst, and the organic base is 1:1:(1.1 to 1.5).

[0073] Further, in step S8, the molar ratio of the intermediate compound 7-n, compound P-3, and condensing agent is 1:(1.8-2.2):(2.2-2.8).

[0074] Furthermore, in step S8, the reaction solvent used in the condensation reaction is dichloromethane, tetrahydrofuran, or N,N-dimethylformamide.

[0075] Furthermore, in step S9, when R' does not contain TBDMS, the reaction of removing the benzyl (Bn) protecting group from the hydroxyl group is carried out in the presence of the catalyst Pd / C (palladium on carbon).

[0076] Furthermore, when R' does not contain TBDMS, the mass ratio of the intermediate compound 8-n to Pd / C is 1:(0.1 to 0.2).

[0077] Furthermore, in step S9, the reaction solvent for removing the benzyl protecting group from the hydroxyl group is preferably one or a combination of tetrahydrofuran, methanol, or the like.

[0078] Furthermore, in step S9, when R' contains TBDMS, the selective removal of the TBDMS (tert-butyldimethylsilyl ether) protecting group is carried out under the action of a catalyst, which is tetrabutylammonium fluoride or its hydrate or acetic acid.

[0079] Preferably, when R' contains TBDMS, the molar ratio of the intermediate compound 8-n to the catalyst is 1:(4-6).

[0080] Furthermore, the reaction solvent used in the selective removal of the TBDMS protecting group is preferably tetrahydrofuran, dichloromethane, or N,N-dimethylformamide.

[0081] Furthermore, in step S9, when R' contains TBDMS, the selective removal of the Bn (benzyl) protecting group is carried out under the action of a catalyst, preferably boron trichloride.

[0082] Furthermore, in step S9, when R' contains TBDMS, the molar ratio of compound 8-n to catalyst in the selective removal of the Bn (benzyl) protecting group is 1:(2.2~2.8).

[0083] Furthermore, the gas in the inert protective atmosphere is selected from nitrogen, neon, argon, or helium.

[0084] Furthermore, the reaction in step S1 also includes post-treatment to obtain intermediate compound 1. The post-treatment includes vacuum evaporation, washing, drying, and silica gel column chromatography.

[0085] Specifically, the post-treatment involves removing the solvent by vacuum evaporation, dissolving the residue in DCM (dichloromethane), washing it several times (preferably three times) with 1M hydrochloric acid, saturated NaHCO3 solution, and saturated NaCl solution, drying it with anhydrous Na2SO4, and performing silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 6 / 1 / 1) to obtain intermediate compound 1.

[0086] Furthermore, the reaction in step S2 also includes post-processing to obtain intermediate compound 2. The post-processing includes vacuum evaporation, washing, drying, and silica gel column chromatography.

[0087] Specifically, the post-treatment involves removing the solvent by vacuum evaporation, dissolving the residue in DCM, washing it repeatedly (preferably three times) with water and saturated NaCl solution, drying it with anhydrous Na2SO4, and performing silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 4 / 1 / 1) to obtain intermediate compound 2.

[0088] Furthermore, the reaction in step S3 also includes post-processing to obtain intermediate compound 3. The post-processing includes vacuum evaporation, washing, drying, and silica gel column chromatography.

[0089] Specifically, the post-treatment involves reducing the pressure to remove most of the solvent, dissolving the residue in DCM, washing it repeatedly (preferably three times) with water and saturated NaCl solution, drying it with anhydrous Na2SO4, and performing silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 2 / 1 / 1) to obtain intermediate compound 3.

[0090] Furthermore, the reaction in step S4 also includes a post-treatment to obtain intermediate compound 4, the post-treatment including dilution, washing, drying and silica gel column chromatography.

[0091] Specifically, the post-treatment involves diluting the reacted mixture in dichloromethane, washing it several times (preferably three times) with 1M hydrochloric acid saturated NaCl solution, drying it with anhydrous Na2SO4, and performing silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 8 / 1 / 1) to obtain intermediate compound 4.

[0092] Furthermore, the reaction in step S5 also includes post-processing to obtain intermediate compound 5. The post-processing includes vacuum evaporation, washing, drying, and silica gel column chromatography.

[0093] Specifically, the post-treatment involves reducing the pressure to remove most of the solvent, dissolving the residue in ethyl acetate, washing it several times with 1M hydrochloric acid saturated NaCl solution, drying it with anhydrous Na2SO4, and performing silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 2 / 1 / 1) to obtain intermediate compound 5.

[0094] Furthermore, the reaction in step S6 also includes post-treatment to obtain intermediate compounds 6-n and 7-n. The post-treatment includes dilution, washing, drying, and silica gel column chromatography.

[0095] Specifically, the post-treatment involves diluting the reacted mixture in dichloromethane, washing it several times (preferably three times) with 1M hydrochloric acid saturated NaCl solution, drying it with anhydrous Na2SO4, and performing silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 6 / 1 / 1) to obtain intermediate compound 6.

[0096] Furthermore, the reaction in step S7 also includes post-treatment to obtain intermediate compound 7-n, the post-treatment including terminating the reaction, washing, and drying.

[0097] Specifically, the post-treatment involves terminating the reaction by adding a saturated sodium bicarbonate solution under an ice bath, diluting the resulting mixture in dichloromethane, washing it several times (preferably three times) with water and NaCl solution, and drying it with anhydrous Na2SO4 to obtain the intermediate compound 7-n.

[0098] Furthermore, the reaction in step S8 also includes post-treatment to obtain intermediate compound 8-n, the post-treatment including dilution, washing, drying and silica gel column chromatography.

[0099] Specifically, the post-treatment involves diluting the completely reacted mixture in dichloromethane, washing it three times with 1M hydrochloric acid saturated NaCl solution, drying it with anhydrous Na2SO4, and performing silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 3 / 1 / 1) to obtain the intermediate compound 8-n.

[0100] Furthermore, the reaction in step S9 also includes post-treatment to obtain the target compound C-3 as a substituted oleanolic acid benzylamide derivative. The post-treatment includes filtration, vacuum evaporation, and silica gel column chromatography.

[0101] Specifically, the post-processing involves filtration, concentration of the filtrate under reduced pressure, and silica gel column chromatography (V dichloromethane / V methanol / V formic acid = 100 / 10 / 1) to obtain the target compound.

[0102] This invention protects the use of the C-3 substituted oleanolic acid benzylamide derivative in the preparation of anti-influenza virus drugs.

[0103] Preferably, the influenza virus is an influenza A virus.

[0104] The present invention also protects an antiviral drug comprising one or more of the C-3 substituted oleanolic acid benzylamide derivatives.

[0105] Compared with the prior art, the present invention has the following beneficial effects: The C-3 substituted oleanolic acid benzylamide derivative of the present invention has a novel chemical structure and has good inhibitory activity against influenza viruses, especially influenza A virus H1N1, both in vivo and in vitro. This indicates that such compounds can be prepared into anti-influenza virus preparations and have good application prospects in the prevention and / or treatment of influenza virus infection, thus expanding the application value of C-3 substituted oleanolic acid amide derivatives. Attached Figure Description

[0106] Figure 1 A statistical graph showing the effect of intraperitoneal injection of different doses of compound I-1 on changes in mouse body weight after infection with influenza A virus.

[0107] Figure 2 A statistical chart showing the effect of intraperitoneal injection of different doses of compound I-1 on the survival rate of mice after infection with influenza A virus.

[0108] Figure 3 To illustrate the effect of intraperitoneal injection of different doses of compound I-1 on the changes in the lung index of mice after infection with influenza A virus, a statistical graph is presented. Compared with the data of the Model group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001, and ns indicates no significant difference.

[0109] Figure 4 A statistical graph showing the effect of intraperitoneal injection of different doses of compound I-1 on the viral protein content in mouse lung tissue after infection with influenza A virus.

[0110] Figure 5 Compound I-1 reacts with PA within a certain concentration range (239-716aa) A statistical chart of protein binding interactions.

[0111] Figure 6 Different concentrations of compound I-1 with PA C-PB1 N A statistical graph showing the relationship between the inhibition rates of protein binding. Detailed Implementation

[0112] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0113] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0114] Example 1: Preparation of Compound I-1

[0115] The synthetic route for the preparation of compound I-1 is shown in the following formula:

[0116]

[0117] a. Ac₂O (acetic anhydride), DMAP (4-dimethylaminopyridine), Pyridine; b. (1) (COCl)₂ (oxalyl chloride, also known as dichloromethane); (2) TEA (triethylamine), BnNH₂ (benzylamine), DCM; c. TBAI (tetrabutylammonium iodide), NaOH, THF-MeOH (tetrahydrofuran-methanol solution); d. EDC·HCl (1-ethyl-(3-dimethylamino) e. TBAF·3H2O (tetrabutylammonium fluoride trihydrate), THF; f. EDC·HCl, DMAP, DCM; g. CF3COOH, DCM; h. EDC·HCl, HOAT (1-hydroxy-7-azobenzotriazole), NMM (N-methylmorpholine), DCM; i. Pd / C (palladium on carbon), H2, MeOH-THF (methanol-tetrahydrofuran solution).

[0118] (1) Preparation of intermediate compound 1

[0119] Oleanolic acid (10 g, 21.90 mmol) was dissolved in 80 mL of pyridine. DMAP (0.27 g, 2.19 mmol) and acetic anhydride (3.14 mL, 32.87 mmol) were added at 0 °C. After stirring for 30 min, the mixture was transferred to room temperature and left overnight. The solvent was removed by vacuum distillation, and the residue was dissolved in DCM. The residue was washed three times successively with 1 M hydrochloric acid, saturated NaHCO3 solution, and saturated NaCl solution. After drying with anhydrous Na2SO4, the residue was subjected to silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 6 / 1 / 1) to obtain a white solid product, namely intermediate compound 1 (9.51 g, yield 87.06%).

[0120] (2) Preparation of intermediate compound 2

[0121] Intermediate compound 1 (4 g, 8.02 mmol) was dissolved in 40 mL of dry dichloromethane. Oxaloyl chloride (2.71 mL, 32.08 mmol) was added, and the mixture was stirred at room temperature for 24 h. The solvent was removed under reduced pressure, and this process was repeated three times. The residue was dissolved in 30 mL of DCM, and the solvent was removed under reduced pressure. The acyl chloride intermediate was dissolved in 40 mL of DCM. Triethylamine (4.43 mL, 32.08 mmol) and benzylamine (1.75 mL, 16.04 mmol) were added under ice bath conditions, and the mixture was stirred for 30 min. The mixture was then transferred to room temperature and stirred overnight. The solvent was removed under reduced pressure, and the residue was dissolved in DCM. The mixture was washed three times with water and saturated NaCl solution, dried over anhydrous Na2SO4, and subjected to silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 4 / 1 / 1) to obtain a white solid product, namely intermediate compound 2 (4.37 g, yield 92.75%).

[0122] (3) Preparation of intermediate compound 3

[0123] Intermediate compound 2 (4.37 g, 7.44 mmol) was dissolved in 50 mL of a mixed solvent (V tetrahydrofuran / V methanol = 3 / 2), and TBAI (1.37 g, 3.72 mmol) was added. The system was adjusted to alkalinity by slowly adding 4N sodium hydroxide solution, and refluxed overnight at 50 °C. The pH of the system was adjusted to 7 by adding 1M hydrochloric acid, and most of the solvent was removed by concentration under reduced pressure. The residue was dissolved in DCM, washed three times successively with water and saturated NaCl solution, dried over anhydrous Na2SO4, and subjected to silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 2 / 1 / 1) to give a white solid product, intermediate compound 3 (3.65 g, yield 89.94%).

[0124] (4) Preparation of intermediate compound 4

[0125] (2R,4S)-1-(tert-butoxycarbonyl)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-2-carboxylic acid (1.42 g, 4.13 mmol) was dissolved in 30 mL of dry dichloromethane. 4-Dimethylaminopyridine (67 mg, 0.55 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.32 g, 6.88 mmol), and intermediate compound 3 (1.5 g, 2.75 mmol) were added sequentially at -20 °C (i.e., under ice bath conditions), and the mixture was stirred for 30 min. The reaction was carried out overnight at room temperature under N2 protection. The mixture was diluted in dichloromethane, washed three times sequentially with 1 M hydrochloric acid saturated NaCl solution, dried over anhydrous Na2SO4, and subjected to silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 8 / 1 / 1) to give a white solid product, intermediate compound 4 (2.11 g, yield 87.92%).

[0126] (5) Preparation of intermediate compound 5

[0127] Intermediate compound 4 (1.8 g, 2.06 mmol) was dissolved in 30 mL of tetrahydrofuran. Tetrabutylammonium fluoride trihydrate (3.31 g, 10.31 mmol) was added at 0 °C, and the mixture was stirred for 15 min. The mixture was then transferred to room temperature and reacted overnight. Most of the solvent was removed by concentration under reduced pressure. The residue was dissolved in ethyl acetate and washed three times with 1 M hydrochloric acid-saturated NaCl solution. The solution was dried over anhydrous Na₂SO₄ and subjected to silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 2 / 1 / 1) to obtain a white solid product, intermediate compound 5 (1.31 g, yield 84.47%).

[0128] (6) Preparation of intermediate compound 6

[0129] Dissolve 0.87 g (2.62 mmol) of 3,4-dibenzylbenzoic acid in 20 mL of dry dichloromethane. Add 31 mg (0.26 mmol) of 4-dimethylaminopyridine, 0.63 g (3.25 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1 g (1.31 mmol) of intermediate compound 5 sequentially at -20 °C (ice bath conditions). Stir for 30 min and react overnight at room temperature under N2 protection. Dilute the mixture in dichloromethane, wash three times with 1 M hydrochloric acid saturated NaCl solution, dry under anhydrous Na2SO4, and perform silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 6 / 1 / 1) to obtain a white solid product, intermediate compound 6 (734 mg, yield 53.18%).

[0130] (7) Preparation of intermediate compound 7

[0131] Intermediate compound 6 (400 mg, 0.38 mmol) was dissolved in 2.5 mL of dry dichloromethane, and trifluoroacetic acid (0.62 mL, 7.6 mmol) was added. The mixture was stirred at room temperature for 2 h under N2 protection. The reaction was terminated by adding saturated sodium bicarbonate solution in an ice bath. The mixture was diluted in dichloromethane and washed three times successively with water and NaCl solution. The product was dried over anhydrous Na2SO4 to obtain a white solid product, namely intermediate compound 7 (360 mg, yield 97.21%).

[0132] (8) Preparation of intermediate compounds 8

[0133] 3,4-Dibenzylbenzoic acid (127 mg, 0.76 mmol) was dissolved in 20 mL of dry dichloromethane. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (182 mg, 0.95 mmol), 1-hydroxy-7-azidobenzotriazole (129 mg, 0.95 mmol), intermediate compound 7 (370 mg, 0.38 mmol), and N-methylmorpholine (128 μL, 1.14 mmol) were added sequentially at -20 °C (ice bath conditions). The mixture was stirred for 10 min and reacted overnight at room temperature under N2 protection. The mixture was diluted in dichloromethane, washed three times sequentially with 1 M hydrochloric acid saturated NaCl solution, dried over anhydrous Na2SO4, and subjected to silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 3 / 1 / 1) to obtain a white solid product, intermediate compound 8 (200 mg, yield 40.77%).

[0134] (9) Preparation of compound I-1

[0135] Intermediate compound 8 (200 mg, 0.15 mmol) was dissolved in 6 mL of a mixed solvent (V tetrahydrofuran / V methanol = 1 / 1), and Pd / C (20 mg) was added. The mixture was reacted at room temperature in an H2 environment for 24 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure and subjected to silica gel column chromatography (V dichloromethane / V methanol / V formic acid = 100 / 10 / 1) to obtain a gray solid product, namely compound I-1 (80 mg, yield 57.31%).

[0136] Example 2: Preparation of compounds I-2 to I-9

[0137] Following the preparation process of compound I-1 obtained in Example 1 above, the R substituents and the number of carbon chain atoms n in the compound shown in Formula I were replaced as shown in Table 1 to obtain the corresponding product shown in Formula 1. It should be noted that when preparing compound I-2, the specific preparation example of step (9) is as follows:

[0138]

[0139] Intermediate compound 8-2 (500 mg, 0.33 mmol) was dissolved in 10 mL of tetrahydrofuran. Tetrabutylammonium fluoride trihydrate (535 mg, 1.69 mmol) was added at 0 °C, and the mixture was stirred for 15 min. The mixture was then transferred to room temperature and reacted overnight. Most of the solvent was removed by concentration under reduced pressure. The residue was dissolved in ethyl acetate and washed three times with 1 M hydrochloric acid-saturated NaCl solution. The mixture was dried over anhydrous Na₂SO₄ to obtain a mixture of yellow solids. The mixture was dissolved in 10 mL of dry dichloromethane, and approximately 0.85 mL of 1 M boron trichloride-dichloromethane solution was slowly added dropwise at -78 °C. The mixture was stirred at -78 °C for half an hour under N₂ protection. The reaction was quenched by adding saturated ammonium chloride solution, and the mixture was concentrated under reduced pressure. Silica gel column chromatography (Vdichloromethane / Vmethanol / Vformic acid = 100 / 10 / 1) yielded a purple solid I-2 (110 mg, yield 35.06%).

[0140] The other steps are basically the same as the preparation conditions of compound I-1 obtained in Example 1.

[0141] The results of the proton NMR spectra of the compounds obtained above are shown in Table 1 below.

[0142] Table 1. Appearance and 1H NMR data of the target compounds

[0143]

[0144]

[0145]

[0146]

[0147] Example 2 evaluates the cytotoxic effects of the compounds obtained in Example 1 and Example 2 on MDCK cells (CC). 50 ) and its activity against influenza A virus (A / WSN / 33 / H1N1) on cells (EC) 50 )

[0148] The C-3 substituted oleanolic acid benzylamide derivatives prepared in Examples 1 and 2 were used as test subjects to test their inhibitory activity against H1N1 influenza A virus strain (A / WSN / 33 / H1N1) on MDCK cells.

[0149] 1. Experimental Methods

[0150] MDCK cells were seeded in 96-well cell culture plates. After reaching a cell density of over 90%, the cells were washed twice with sterile PBS. The compounds were serially diluted 2-fold with FBS-free DMEM maintenance medium, resulting in nine different compound concentration groups: 0.39, 0.78, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 μM. A 0.1% DMSO solvent control group was also included. Each group had three replicate wells, with 200 μL per well. The cells were incubated at 37°C in a 5% CO2 incubator. After 48 hours, the culture was terminated, the supernatant was discarded, and CCK8 solution diluted with DMEM maintenance medium was added to each well. The cells were then incubated in the dark at 37°C in a 5% CO2 incubator. One hour later, the OD value was measured at 450 nm using a multi-functional microplate reader to calculate cell viability. The 50% cytotoxic concentration (CC50) of compound I-1 against MDCK cells was calculated using GraphPad Prism 9.0 software. 50 ).

[0151] The calculation formula is as follows:

[0152]

[0153] As above, after the MDCK cells have grown to a confluent monolayer, wash twice with PBS, and then dilute the A / WSN / 33 / H1N1 virus to 100 TCID using FBS-free DMEM basal maintenance medium. 50 Viral fluid was used, and a blank control group was set up. After culturing in a 37℃, 5% CO2 incubator for 1 hour, the supernatant was discarded. The compound was serially diluted twice with DMEM maintenance medium containing 0.5 μg / mL TPCK-trypsin, and seven concentration gradients of the compound drug were set up: solvent control group, virus control group, zanamivir positive control group, and 0.31-20 μM, 200 μL per well. The mixture was incubated in a 37℃, 5% CO2 incubator for 36-48 hours. The lesions in the virus control group were observed, and the maintenance supernatant was discarded. CCK8 solution diluted with DMEM maintenance medium was added to each well, and the mixture was incubated in a 37℃, 5% CO2 incubator in the dark. After 1 hour, the OD value was detected at 450 nm using a multi-mode microplate reader. The inhibition rate of the drug treatment group against the virus at each concentration was calculated to determine the half-maximal effective concentration (EC50). 50 The values ​​were calculated using a nonlinear regression function with GraphPad Prism 9.0 software.

[0154] The calculation formula is as follows:

[0155]

[0156] 2. Experimental Results

[0157] The measured results are shown in Table 2.

[0158] Table 2 shows the inhibitory activity of C-3 substituted oleanolic acid benzylamide derivatives against influenza A virus H1N1 (A / WSN / 33) in MDCK cells.

[0159] compound <![CDATA[EC 50 (μM)]]> <![CDATA[CC 50 (μM)]]> OA() 79.2±1.86 >100 I-1 0.76±0.12 111.01±12.11 I-2 10.53±1.26 78.31±9.21 I-3 1.36±0.27 93.95±7.41 I-4 2.13±0.58 90.77±7.11 I-5 4.57±0.39 103.63±10.39 I-6 2.63±0.81 99.31±8.44 I-7 18.21±0.65 76.59±6.97 I-8 25.82±2.03 123.63±10.21 I-9 35.60±1.91 109.21±9.67

[0160] As shown in Table 2, the compounds represented by Formula I all exhibited certain inhibitory activity against influenza A virus H1N1 in MDCK cells, and were all stronger than oleanolic acid (OA, EC). 50 =79 μM). Among them, compound I-1 inhibits influenza A virus H1N1 EC. 50 Below 1 μM, and with low toxicity to MDCK cells (CC). 50 The concentration (111 μM) is much lower than the concentration used, and therefore has broad application value in the preparation of anti-influenza A virus agents. Therefore, compound I-1 was used as a representative compound in subsequent experiments.

[0161] Example 4 evaluates the in vivo activity of compound I-1 against influenza A virus (A / WSN / 33 / H1N1).

[0162] 1. Experimental Methods

[0163] Forty-eight SPF-grade BALB / c mice (half male and half female) were randomly divided into six groups of eight each. These groups were: a solvent control group (Control), a virus group (Model), a positive control group treated with oseltamivir phosphate (OSP), and three treatment groups receiving compound I-1 at doses of 12.5, 25, and 50 mg / kg. After one week of acclimatization in the laboratory, the mice were anesthetized by inhalation of isoflurane. Except for the solvent control group, each mouse was intranasally inoculated with 50 μL of a 5×LD50 solution. 50 Influenza A virus (A / WSN / 33 / H1N1), 10 mg / kg oseltamivir phosphate solution or 12.5, 25, or 50 mg / kg compound I-1 were injected intraperitoneally 6 hours later. The solvent was 8% DMSO + 32% PEG300 + 5% Tween-80 + 55% physiological saline. After sterilization by filtration through a 0.22 μm filter membrane, the mice were continuously administered the drug for five days. The weight and mortality of the mice were recorded daily. When the weight of the mice decreased by 20% of their original weight, euthanasia was performed in accordance with animal ethics standards.

[0164] As above, 36 SPF-grade BALB / c mice (half male and half female) were randomly divided into 6 groups of 6 mice each. These groups were: solvent control group, virus group, oseltamivir positive control group, and compound I-1 treatment groups at three dose gradients of 12.5, 25, and 50 mg / kg. After one week of acclimatization in the laboratory, the mice were anesthetized by inhalation of isoflurane. Except for the solvent control group, each mouse was intranasally inoculated with 50 μL of 1000 TCID45 solution. 50 Influenza A virus (A / WSN / 33 / H1N1), mice were intraperitoneally injected with solvent, 10 mg / kg oseltamivir phosphate solution, or 12.5, 25, or 50 mg / kg compound I-1 6 hours later for three consecutive days. The weight of the mice was recorded, and lung tissue was collected. The weight of the intact lung tissue was recorded. The ratio of lung weight (mg) to body weight (g) was the lung index. Protein lysis buffer was added to the lung tissue, and after homogenization, proteins were extracted and Western blotting was performed. The expression levels of viral proteins in the lung tissue of each group were detected using viral protein antibodies PA, PB1, PB2, and NP.

[0165] 2. Experimental Results

[0166] Experimental results are as follows Figures 1-4 As shown, Figure 1 The study showed that intraperitoneal injection of different doses of compound I-1 had an effect on the weight changes of mice after infection with influenza A virus. The weight of mice in each dose group decreased more slowly than that in the virus group, and the weight of the treated groups began to recover on the eighth day after infection, indicating that the compound effectively protected the vital signs of infected mice. Figure 2 The study indicated the effect of intraperitoneal injection of different doses of compound I-1 on the survival rate of mice after infection with influenza A virus. All mice in the virus group died on the tenth day after infection, while the survival rate of mice in the high-dose I-1 group reached 87.5%. Compound I-1 protected mice from death caused by viral infection in a dose-dependent manner. Figure 3 The study indicated the effect of intraperitoneal injection of different doses of compound I-1 on the changes in the lung index of mice after infection with influenza A virus. The compound I-1 administration group showed a dose-dependent reduction in the increase in lung index caused by virus infection and alleviated the symptoms of pulmonary edema. Figure 4 This study investigated the effect of intraperitoneal injection of various doses of compound I-1 on viral protein levels in mouse lung tissue after infection with influenza A virus. The 12.5, 25, and 50 mg / kg doses all reduced viral protein expression and inhibited influenza A virus replication in the mouse lungs. Compound I-1 of this invention exhibits antiviral activity against A / WSN / 33 / H1N1 virus in mice. At doses of 12.5, 25, and 50 mg / kg, it alleviated weight loss in mice infected with influenza A virus to varying degrees, prevented some mice from dying from viral infection, and dose-dependently reduced the lung index, alleviated pulmonary edema, and decreased viral load in the lungs.

[0167] In summary, derivative I-1 has a good protective effect against mice infected with H1N1 influenza virus and can be prepared as an anti-influenza A virus drug for application, showing good application prospects in the prevention and / or treatment of influenza virus infection.

[0168] Example 5: Detection of the interaction between compound I-1 and the PA-C-terminal (239-716aa) protein-binding domain using surface plasmon resonance (SPR) technique.

[0169] 1. Experimental Methods

[0170] The purified PA-C-terminal (239-716aa) protein was diluted to 1 mg / mL with PBS and immobilized on the surface of a 3D photocrosslinked chip. After drying, crosslinking, activation, and nitrogen blowing, the chip was tightly sealed to the cap and stored at 4°C for later use. Compound I-1 was serially diluted to five concentration gradients from 0.65 to 10 μM as mobile phases and analyzed using a PlexArray HT I-10 instrument. PBS was used as the buffer, and 10 mM glycine solution at pH 2.0 was used as the regeneration solution. The data were fitted using BIAevolution software, and the dissociation constant K was calculated. D .

[0171] 2. Experimental Results

[0172] Experimental results are as follows Figure 5 As shown, the vertical axis represents the protein content bound to the compound, and the horizontal axis represents the content of the compound bound to PA. (239-716aa) Protein interaction time, compared with the PBS group, showed that compound I-1 was comparable to PA in the concentration range of 650-10000 nM. (239-716aa) The protein exhibits concentration-dependent binding, and the dissociation constant K was calculated. D =9.97×10 -9 M. Dissociation constant K D K is an equilibrium constant for a reaction, used to measure the tendency of a larger substance to reversibly separate (dissociate) into smaller components. It is a parameter describing the strength of intermolecular interactions. In molecular biology, K is... D Value less than 10 -8 M indicates strong intermolecular binding. The dissociation constant between compound I-1 of this invention and PA(239-716aa) protein is 9.97 × 10⁻⁶. -9 M indicates that the two have a high affinity and can effectively bind to the target site to inhibit the replication and transcription process of influenza virus, thereby playing an anti-influenza role.

[0173] Example 6: ELISA detection of compound I-1 inhibiting PA C -PB1 Ninteraction

[0174] 1. Experimental Methods

[0175] Add 400 ng of purified PA to each well of a 96-well plate. (239-716) After incubating the protein overnight at 4°C, it was blocked with 2% BSA for 1 hour, washed four times with PBS-T, and then serially diluted with compound I-1 solutions at concentration gradients of 0.16-10 μM using DMEM basal medium. This was then combined with 200 ng of purified GST-PB1. (1-25) Add protein to each well, and set the option to not add GST-PB1. (1-25) Protein solvent control group and GST-PB1 added (1-25) Protein and negative control group (without added compound). The 96-well plates were incubated at 37°C for 3 hours. After washing four times with PBS-T, monoclonal GST antibody and its corresponding secondary antibody were added sequentially. After the final wash, TMB reagent kit was used for color development, and the reaction was terminated with 1M sulfuric acid solution. The OD value was measured at 450 nm using a multi-functional microplate reader. The effect of each drug concentration on PA was calculated. C -PB1 N The inhibition rate of protein binding is used to determine the half-maximal effective concentration (IC50). 50 The value was calculated using a nonlinear regression function with GraphPadPrism 9.0 software.

[0176] The calculation formula is as follows:

[0177]

[0178] 2. Experimental Results

[0179] Experimental results are as follows Figure 6 As shown, different concentrations of compound I-1 and PA are illustrated. C -PB1 N The relationship between the inhibition rate of protein binding. Compound I-1 of the present invention inhibits PA... C -PB1 N Protein binding exhibits inhibitory activity, with an IC50 value of [missing information]. 50 The value was 0.96 μM, indicating that compound I-1 inhibited PA. C -PB1 N The binding of proteins can prevent the assembly of RNA polymerase, thereby inhibiting the viral replication process.

[0180] In summary, the C-3 substituted pentacyclic triterpenoid amide derivative provided by this invention has a significant inhibitory effect on influenza A virus H1N1 (A / WSN / 33) in vitro and in vivo, and can be prepared as an anti-influenza A virus drug for application, showing good application prospects in the prevention and treatment of influenza virus infection.

[0181] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A C-3 substituted oleanolic acid benzylamide derivative, characterized in that, The structure of the C-3 substituted oleanolic acid benzylamide derivative is shown in Formula I below: In Formula I, R is selected from phenolyl, hydroquinone, pyrogallol, C 1～3 Alkoxy-substituted phenyl, difluorophenyl, dihydroxy-substituted benzyl, or trihydroxy-substituted cyclohexenyl; The value of n is 0 or 1.

2. The C-3 substituted oleanolic acid benzylamide derivative according to claim 1, characterized in that, The R is selected from 4-hydroxy-substituted phenyl, 3,4-dihydroxy-substituted phenyl, 3,4,5-trihydroxy-substituted phenyl, 2,3-dihydroxy-substituted phenyl, 3,5-dihydroxy-substituted phenyl, 2,4-dihydroxy-substituted phenyl, 3,4-dimethoxy-substituted phenyl, 3,4-difluoro-substituted phenyl, 3,4-dihydroxy-substituted benzyl, or 3,4,5-trihydroxy-substituted cyclohexenyl.

3. The C-3 substituted oleanolic acid benzylamide derivative according to claim 1 or 2, characterized in that, The C-3 substituted oleanolic acid benzylamide derivatives also include their pharmaceutically acceptable salts.

4. The method for preparing the C-3 substituted oleanolic acid benzylamide derivative according to claim 1 or 2, characterized in that, Includes the following steps: S1. Using oleanolic acid as a raw material, intermediate compound 1 was obtained by acetylation at room temperature: ； S2. Under normal temperature conditions, the intermediate compound 1 obtained in step S1 is reacted with oxalyl chloride to generate an acyl chloride intermediate, then triethylamine is added as an acid-binding agent, and then condensed with benzylamine to generate intermediate compound 2: ； S3. At 45~50 °C, the intermediate compound 2 obtained in step S2 is hydrolyzed in the presence of an alkaline reagent to obtain intermediate compound 3: ； S4. Under ice bath conditions, intermediate compound 3 and compound P-1 obtained in step S3 are mixed evenly, and a condensation reaction is carried out under an inert protective atmosphere and at room temperature to obtain intermediate compound 4: ； S5. Under normal temperature conditions, intermediate compound 4 obtained in step S4 is selectively deprotected by the TBDMS protecting group to obtain intermediate compound 5: ； S6. Under ice bath conditions, intermediate compound 5 and compound P-2 obtained in step S5 are mixed evenly, and a condensation reaction is carried out under an inert protective atmosphere and at room temperature to obtain intermediate compound 6-n: ； S7. Under normal temperature and an inert protective atmosphere, the intermediate compound 6-n obtained in step S6 is selectively deprotected by the Boc protecting group to obtain the intermediate compound of general formula 7-n: ； S8. Under ice bath conditions, the intermediate compound obtained in step S7 is mixed thoroughly with compound P-3, and a condensation reaction is carried out under an inert protective atmosphere and at room temperature to obtain the intermediate compound of general formula 8-n: ； Wherein, R' is defined as follows: when R in Formula I is phenol, hydroquinone, pyrogallol, or dihydroxy-substituted benzyl, then R' is the group after the hydrogen of the hydroxyl group in R is replaced with Bn; when R is selected from trihydroxy-substituted cyclohexenyl, then R' is the group after the hydrogen of the hydroxyl group in R is replaced with TBDMS; in other cases, R' is defined in the same way as R. S9. When R' does not contain TBDMS: Under normal temperature and hydrogen atmosphere, the intermediate compound 8-n obtained in step S8 is debenzyl protecting group to obtain the substituted oleanolic acid benzylamide derivative with C-3. When R' contains TBDMS: Under normal temperature conditions, the intermediate compound 8-n obtained in step S8 is first selectively deprotected by the TBDMS protecting group, and then the benzyl protecting group is removed at -75~-80 °C in an inert protective atmosphere to obtain the oleanolic acid benzylamide derivative with C-3 substituted. ; Wherein, the definitions of n and R are the same as those in claim 1 or 2.

5. The use of the C-3 substituted oleanolic acid benzylamide derivative according to any one of claims 1 to 3 in the preparation of anti-influenza A virus drugs.

6. A drug for treating influenza A virus, characterized in that, It includes one or more of the C-3 substituted oleanolic acid benzylamide derivatives as described in any of claims 1 to 3.

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