C-3-substituted pentacyclic triterpene benzamide derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a C-3-substituted pentacyclic triterpene benzamide derivative as well as a preparation method and application thereof. The invention discloses a C-3-substituted pentacyclic triterpenoid benzamide derivative with a novel structure, and the compounds show general strong inhibitory activity on novel coronaviruses and various mutant strains thereof, such as delta, Omext and the like. Specifically, the half inhibitory concentration (IC50) of all tested C-3 substituted pentacyclic triterpenoid benzylamide derivatives is lower than 10 mu M, which indicates that the C-3 substituted pentacyclic triterpenoid benzylamide derivatives have a remarkable inhibitory effect on novel coronavirus and mutant strains thereof. In view of the characteristic, the C-3 substituted pentacyclic triterpenoid benzamide derivative has the potential of being developed into a novel coronavirus resisting drug, and shows a good application prospect in the aspect of preventing and treating novel coronavirus infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to a C-3 substituted pentacyclic triterpene benzylamide derivative and a preparation method and application thereof. Background Art

[0003] So far, the medical community has not yet developed a specific drug that can completely cure COVID-19 infection. Currently, small molecule antiviral drugs such as Abidol, Molnupiravir and Remdesivir are widely used in clinical practice. These drugs can effectively inhibit the replication of the new coronavirus to a certain extent, thereby achieving treatment of the disease. Given that the new coronavirus has a high degree of variability and rapid mutation rate, and the existing anti-new coronavirus specific drugs are still limited, therefore, finding and developing compounds with broader-spectrum and more efficient anti-new coronavirus activity has become the current priority. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the high variability and rapid mutation rate of the new coronavirus, and the limited defects and shortcomings of existing anti-new coronavirus specific drugs, and to provide a C-3 substituted pentacyclic triterpene benzylamide derivative.

[0005] The object of the present invention is to provide a method for preparing the C-3 substituted pentacyclic triterpene benzylamide derivative.

[0006] Another object of the present invention is to provide the application of the C-3 substituted pentacyclic triterpene benzylamide derivative.

[0007] Another object of the present invention is to provide an anti-new coronavirus drug.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention protects a C-3 substituted pentacyclic triterpene benzylamide derivative, wherein the C-3 substituted pentacyclic triterpene benzylamide derivative has a structure shown in any one of formula I:

[0010]

[0011] Furthermore, the C-3 substituted pentacyclic triterpene benzylamide derivative also includes pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers or isotope-substituted compounds thereof.

[0012] The present invention also protects a method for preparing the C-3 substituted pentacyclic triterpene benzylamide derivative, comprising the following steps:

[0013] S1. Using oleanolic acid, ursolic acid or betulinic acid as raw materials, intermediate compound 1 is obtained through acetylation reaction, acyl chlorination reaction, condensation reaction and hydrolysis reaction in sequence:

[0014]

[0015] S2. Under ice bath conditions, the intermediate compound 1 obtained in step S1 and compound L-1 are mixed evenly, and a condensation reaction is carried out under an inert protection atmosphere and normal temperature conditions to obtain intermediate compound 2:

[0016]

[0017] S3. Under normal temperature conditions, the intermediate compound 2 obtained in step S2 is subjected to selective removal of the TBDMS (tert-butyldimethylsilyl, also abbreviated as TBS) protecting group to obtain intermediate compound 3:

[0018]

[0019] S4. Under ice bath conditions, the intermediate compound 3 obtained in step S3 and compound L-2 are mixed evenly, and a condensation reaction is carried out under an inert protection atmosphere and normal temperature conditions to obtain intermediate compound 4:

[0020]

[0021] S5. Under normal temperature conditions and an inert protection atmosphere, the intermediate compound 4 obtained in step S4 is subjected to selective removal of the Boc (tert-butoxycarbonyl) protecting group to obtain intermediate compound general formula 5:

[0022]

[0023] S6. Under ice bath conditions, the intermediate compound 5 obtained in step S5 and compound L-2 are mixed evenly, and a condensation reaction is carried out under an inert protection atmosphere and normal temperature conditions to obtain intermediate compound general formula 6:

[0024]

[0025] S7. The intermediate compound 6 is selectively deprotected from the TBDMS protecting group to obtain the C-3 substituted pentacyclic triterpenoid benzamide derivative as described in I:

[0026]

[0027] Furthermore, in step S1, the acetylation reagent used in the acetylation reaction includes one or more of acetic anhydride and acetyl chloride.

[0028] Furthermore, in step S1, the acyl chlorination reagent used in the acyl chlorination reaction includes one or more of oxalyl chloride and thionyl chloride.

[0029] Furthermore, in step S1, the condensation reaction is a condensation reaction of the product obtained by the chlorination reaction with benzylamine.

[0030] Furthermore, the condensation reaction also requires an organic base, and the organic base is preferably triethylamine.

[0031] Furthermore, in step S1, the hydrolysis reaction is carried out under alkaline conditions, and the alkaline conditions are provided by an alkaline reagent, and the alkaline reagent is preferably one or more of sodium hydroxide, potassium hydroxide, and lithium hydroxide.

[0032] Specifically, the reaction of step S1 is that the oleanolic acid, ursolic acid or betulinic acid is first subjected to an acetylation reaction under the action of an acetylation agent and a catalyst 4-dimethylaminopyridine to protect the C-3 hydroxyl group of pentacyclic triterpenes such as oleanolic acid, and then the C-17 carboxylic acid of the above pentacyclic triterpenes is activated into an acyl chloride using a reagent such as oxalyl chloride or thionyl chloride, followed by a condensation reaction with benzylamine, and finally the C-3 acetyl group is hydrolyzed and removed in a strong alkaline environment (preferably sodium hydroxide, potassium hydroxide or lithium hydroxide) to obtain an intermediate compound 1.

[0033] Furthermore, in step S1, the acetylation reaction time is 5 to 12 hours.

[0034] Furthermore, in step S1, the acyl chlorination reaction time is 10 to 24 hours.

[0035] Furthermore, in step S1, the condensation reaction time is 2 to 12 hours.

[0036] Furthermore, in step S1, the hydrolysis reaction time is 5 to 12 hours.

[0037] Furthermore, in step S2, the condensation reaction time is 12 to 24 hours.

[0038] Furthermore, in step S3, the reaction time for selectively removing the TBDMS protecting group is 6 to 12 hours.

[0039] Furthermore, in step S4, the condensation reaction time is 12 to 24 hours.

[0040] Furthermore, in step S5, the reaction time for selectively removing the Boc protecting group is 1 to 2 hours.

[0041] Furthermore, in step S6, the condensation reaction time is 12 to 24 hours.

[0042] Furthermore, in step S7, the time for selectively removing the TBDMS protecting group is 12 to 24 hours.

[0043] Furthermore, in step S1, the acetylation reaction further includes post-treatment, which includes reduced pressure concentration, washing, drying and filtering. Specifically, the product after the acetylation reaction is evaporated under reduced pressure to remove the solvent, the residue is dissolved in dichloromethane, and 1M hydrochloric acid, saturated NaHCO 3 The organic phase was washed with anhydrous Na 2 SO 4 The residue was dried, filtered and concentrated under reduced pressure to be used in the next reaction.

[0044] Furthermore, in step S1, the acyl chlorination reaction further includes post-treatment, and the post-treatment includes reduced pressure concentration, specifically, the product after the acyl chlorination reaction is concentrated under reduced pressure to obtain the product of the next reaction.

[0045] Furthermore, in step S1, the condensation reaction further includes post-treatment, and the post-treatment includes concentration under reduced pressure, specifically, concentrating the product after the condensation reaction under reduced pressure to remove the solvent.

[0046] Furthermore, in step S1, the hydrolysis reaction further includes post-treatment, which includes reduced pressure concentration, washing, drying and silica gel column chromatography, specifically, the product after the hydrolysis reaction is added with 1M hydrochloric acid solution to adjust the system pH to 7, and the reduced pressure concentration is used to remove most of the solvent, and the residue is dissolved in ethyl acetate, and then washed with water and saturated NaCl solution three times in sequence, and the organic phase is washed with anhydrous Na 2 SO 4 The mixture was dried, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 2 / 1 / 1) to obtain a white solid product, namely, intermediate compound 1.

[0047] Furthermore, in step S2, the condensation agent used in the condensation reaction is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or N,N-dicyclohexylcarbodiimide, and the catalyst is preferably 4-dimethylaminopyridine.

[0048] Preferably, the molar ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or N,N-dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:(0.05-0.15).

[0049] Furthermore, in step S2, the reaction solvent used in the condensation reaction is preferably dry dichloromethane, tetrahydrofuran or N,N-dimethylformamide.

[0050] Preferably, in step S2, the molar ratio of the intermediate compound 1, compound L-1 and the condensing agent is 1:(1-2):(2-3).

[0051] Further, in step S2, the condensation reaction further includes post-treatment, and the post-treatment includes dilution, washing, drying, filtering, concentrating under reduced pressure and silica gel column chromatography. Specifically, the mixture after the condensation reaction is diluted with dichloromethane, washed with 1M hydrochloric acid solution and saturated NaCl solution in sequence, the obtained organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is chromatographed on a silica gel column (V petroleum ether / V ethyl acetate / V dichloromethane = 8 / 1 / 1) to obtain a white solid product, i.e., intermediate compound 2.

[0052] Furthermore, in step S3, the reaction of selectively removing the TBDMS protecting group is carried out under the action of a fluorine-containing reagent catalyst.

[0053] Furthermore, the fluorine-containing reagent is tetrabutylammonium fluoride, pyridine hydrofluoride, cesium fluoride or a hydrate of the above fluorine-containing reagents.

[0054] Furthermore, in step S3, the reaction solvent used in the reaction of selectively removing the TBDMS protecting group is preferably tetrahydrofuran or dichloromethane.

[0055] Preferably, the molar ratio of the intermediate compound 2 to the fluorine-containing reagent is 1:(1.5-3), more preferably 1:(2-3).

[0056] Further, in step S3, the reaction of selectively removing the TBDMS protecting group also includes post-treatment, and the post-treatment includes vacuum concentration, washing, drying, filtering and silica gel column chromatography. Specifically, the mixture after the condensation reaction is vacuum concentrated, and then the residue is dissolved in ethyl acetate, and then washed with 1M hydrochloric acid solution and saturated NaCl solution in sequence, and the obtained organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is subjected to silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 2 / 1 / 1) to obtain a white solid product, i.e., intermediate compound 3.

[0057] Furthermore, in step S4, the condensation reaction requires the use of a condensation agent and a catalyst, the condensation agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or N,N-dicyclohexylcarbodiimide; the catalyst is 4-dimethylaminopyridine.

[0058] Preferably, the molar ratio of the condensing agent to the catalyst is 1:(0.05-0.10).

[0059] Furthermore, the molar ratio of the intermediate compound 3, the compound L-2 and the condensing agent is 1:(1.1-1.3):(1.3-1.7).

[0060] Furthermore, in step S4, the reaction solvent used in the condensation reaction is dry dichloromethane, tetrahydrofuran or N,N-dimethylformamide.

[0061] Further, in step S4, the condensation reaction further includes post-treatment, which includes dilution, washing, drying and silica gel column chromatography, specifically, diluting the mixture after the condensation reaction in dichloromethane, washing with 1M hydrochloric acid saturated NaCl solution three times in sequence, and anhydrous Na 2 SO 4 The reaction mixture was dried and purified by silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 6 / 1 / 1) to obtain a white solid product, namely intermediate compound 4.

[0062] Furthermore, in step S5, the reaction of selectively removing the Boc protecting group is carried out in an acidic solution, and the acidic solution is preferably a solution of trifluoroacetic acid or hydrogen chloride in ethyl acetate.

[0063] Furthermore, in step S5, the reaction solvent used in the reaction of selectively removing the Boc protecting group is preferably dichloromethane or tetrahydrofuran.

[0064] Furthermore, in step S5, the reaction of selectively removing the Boc protecting group also includes post-treatment, and the post-treatment includes terminating the reaction, diluting, washing, drying, filtering and concentrating under reduced pressure. Specifically, the product of the complete condensation reaction is added with a saturated sodium bicarbonate solution under an ice bath to terminate the reaction, and the mixture is diluted with dichloromethane, washed with water and NaCl solution three times in sequence, and the obtained organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid product, i.e., intermediate compound 5.

[0065] Further, in step S6, the condensation reaction is carried out under the action of a condensation agent, an anti-racemization additive and an organic base, the condensation agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or N,N-dicyclohexylcarbodiimide, the anti-racemization additive is preferably 1-hydroxy-7-azidobenzotriazole or 1-hydroxybenzotriazole, and the organic base is preferably N-methylmorpholine or N,N-diisopropylethylamine.

[0066] Preferably, the molar ratio of the condensing agent, the anti-racemization additive and the organic base is 1:(0.8-1.2):(2-3).

[0067] Furthermore, in step S6, the molar ratio of the intermediate compound 5, compound L-2 and the condensing agent is 1:(1.1-1.3):(1.2-1.5).

[0068] Furthermore, in step S6, the reaction solvent used in the condensation reaction is dry dichloromethane, tetrahydrofuran or N,N-dimethylformamide.

[0069] Furthermore, in step S6, the reaction of selectively removing the Boc protecting group also includes post-treatment, and the post-treatment includes dilution, washing, drying, filtering, concentrating under reduced pressure and silica gel column chromatography. Specifically, the product of the complete condensation reaction is diluted in dichloromethane, washed three times with 1M hydrochloric acid saturated NaCl solution in sequence, and the obtained organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is chromatographed on a silica gel column (V petroleum ether / V ethyl acetate / V dichloromethane = 3 / 1 / 1) to obtain a white solid product, i.e., intermediate compound 6.

[0070] Furthermore, in step S7, the reaction of selectively removing the TBDMS protecting group is carried out under the action of a fluorine-containing reagent, and the fluorine-containing reagent is tetrabutylammonium fluoride, pyridine hydrofluoride, cesium fluoride or a hydrate of the above fluorine-containing reagent.

[0071] Furthermore, in step S7, the reaction solvent used in the reaction of selectively removing the TBDMS protecting group is preferably tetrahydrofuran or dichloromethane.

[0072] Furthermore, the fluorine-containing reagent is tetrabutylammonium fluoride, pyridine hydrofluoride, cesium fluoride or a hydrate of the above fluorine-containing reagents.

[0073] Preferably, the molar ratio of the intermediate compound 6 to the fluorine-containing reagent is 1:(9-12), more preferably 1:(9.5-11).

[0074] Further, in step S7, the reaction of selectively removing the TBDMS protecting group also includes post-treatment, and the post-treatment includes reduced pressure concentration, dissolution, washing, drying, filtering, reduced pressure concentration and silica gel column chromatography. Specifically, the product of the complete condensation reaction is concentrated under reduced pressure and the residue is dissolved in ethyl acetate, and then washed with 1M hydrochloric acid solution and saturated NaCl solution in sequence. The obtained organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is chromatographed on a silica gel column (V dichloromethane / V methanol / V formic acid = 100 / 10 / 1) to obtain a white solid product, i.e., the target compound I.

[0075] Furthermore, the gas of the inert protective atmosphere is selected from one or more of nitrogen, argon, helium and neon.

[0076] The present invention also protects the use of the C-3 substituted pentacyclic triterpene benzylamide derivative in the preparation of anti-new coronavirus drugs.

[0077] Furthermore, the novel coronavirus is any one or more of the following coronaviruses or their variants: SARS-CoV-2 (novel coronavirus), Delta, and Omicron.

[0078] Among them, Delta and Omicron are two important variants of the new coronavirus.

[0079] The present invention also protects an anti-new coronavirus drug comprising one or more of the C-3 substituted pentacyclic triterpene benzylamide derivatives.

[0080] Compared with the prior art, the present invention has the following beneficial effects:

[0081] The present invention discloses a class of novel C-3 substituted pentacyclic triterpene benzylamide derivatives, which exhibit generally strong inhibitory activity against the novel coronavirus and its various mutants, such as Delta and Omicron. Specifically, the half inhibitory concentration (IC50) of all tested C-3 substituted pentacyclic triterpene benzylamide derivatives is 50 ) were all lower than 10μM, indicating that they have significant inhibitory effects on the new coronavirus and its mutants. In view of this characteristic, C-3 substituted pentacyclic triterpene benzylamide derivatives have the potential to be developed into anti-new coronavirus drugs. In the prevention and treatment of new coronavirus infection, this type of compound has shown good application prospects. DETAILED DESCRIPTION

[0082] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0083] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0084] Example 1

[0085] The preparation of compound Ⅰ-1, the synthetic route is shown below:

[0086]

[0087] The specific preparation method comprises the following steps:

[0088] The key reagents involved in the above synthesis steps a to g are: a. (1) Ac 2 O (acetic anhydride), DMAP (4-dimethylaminopyridine), Pyridine (pyridine);

[0089] (2)(COCl) 2 (oxalyl chloride, also known as oxalyl chloride), DCM (dichloromethane);

[0090] (3)TEA (triethylamine), BnNH 2 (benzylamine), DCM (dichloromethane);

[0091] (4) 4M NaOH, THF-MeOH (tetrahydrofuran-methanol solution);

[0092] b. EDC·HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), DMAP, DCM;

[0093] c.TBAF·3H 2 O (tetrabutylammonium fluoride trihydrate), THF (tetrahydrofuran);

[0094] d.EDC·HCl, DMAP, DCM;

[0095] e.CF 3 COOH, DCM;

[0096] f. EDC·HCl, HOAT (1-hydroxy-7-azobenzotriazole), NMM (N-methylmorpholine), DCM;

[0097] g.TBAF·3H2O,THF.

[0098] (1) Preparation of intermediate compound OA-1

[0099] Oleanolic acid (10 g, 21.90 mmol) was dissolved in 100 mL of dry pyridine, and acetic anhydride (3.14 mL, 32.87 mmol) and DMAP (0.27 g, 2.19 mmol) were added at 0°C, and the mixture was naturally returned to room temperature and stirred for 5 h. The solvent was evaporated under reduced pressure, and the residue was dissolved in DCM and treated with 1 M hydrochloric acid, saturated NaHCO 3 The organic phase was washed with anhydrous Na 2 SO 4 Dry, filter, and concentrate under reduced pressure. The residue is dissolved in 150 mL of dry dichloromethane, and oxalyl chloride (5.56 mL, 43.8 mmol) is added under ice bath. After stirring at room temperature for 10 h under nitrogen protection, the acid chloride intermediate is obtained. The prepared acid chloride intermediate is dissolved in 120 mL of dry DCM, and triethylamine (6.07 mL, 43.8 mmol) and benzylamine (3.49 mL, 31.94 mmol) are added under ice bath and stirred for 30 min. The mixture is transferred to room temperature and stirred for 2 h. The solvent is evaporated under reduced pressure, and the residue is dissolved in 150 mL of a mixed solution of 4M NaOH-tetrahydrofuran-methanol (V:V:V=1:1:1) under ice bath, and then stirred at room temperature for 6 h. 1M hydrochloric acid solution is added to adjust the system pH to 7, and most of the solvent is removed by concentration under reduced pressure. The residue is dissolved in ethyl acetate, and then washed with water and saturated NaCl solution 3 times in sequence. The organic phase is washed with anhydrous Na 2 SO 4The mixture was dried, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 2 / 1 / 1) to obtain a white solid product OA-1 (8.23 g, yield 68.9%).

[0100] (2) Preparation of intermediate compound OA-2

[0101] The intermediate OA-1 (3.0 g, 5.50 mmol) and (2S, 4R)-1-(tert-butoxycarbonyl)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-2-carboxylic acid, i.e. L-1 (2.85 g, 8.26 mmol) were dissolved in 50 mL of dry dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.64 g, 13.76 mmol) and 4-dimethylaminopyridine (134 mg, 1.10 mmol) were added successively at 0°C, stirred at 0°C for 30 min under nitrogen protection, then returned to room temperature and continued to stir overnight. The mixture was diluted with dichloromethane and washed with 1M hydrochloric acid solution and saturated NaCl solution successively. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (V petroleum ether / V ethyl acetate / V dichloromethane = 8 / 1 / 1) to obtain a white solid product OA-2 (4.13 g, yield 86%).

[0102] (3) Preparation of intermediate compound OA-3

[0103] Take the intermediate compound OA-2 (3.60g, 4.12mmol) and dissolve it in 50mL tetrahydrofuran, add tetrabutylammonium fluoride trihydrate (3.25g, 10.31mmol) at 0℃, transfer to room temperature and continue stirring and reacting for 6h. After concentration under reduced pressure, dissolve the residue in ethyl acetate, and then wash with 1M hydrochloric acid solution and saturated NaCl solution in sequence. The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is chromatographed on a silica gel column (V petroleum ether / V ethyl acetate / V dichloromethane = 2 / 1 / 1) to obtain a white solid product OA-3 (2.75g, yield 85%).

[0104] (4) Preparation of intermediate compound OA-4

[0105] The intermediate OA-3 (2.00 g, 2.62 mmol) and the fully TBS-protected Miscanthic acid L-2 (1.62 g, 3.14 mmol) were dissolved in 30 mL of dry dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.75 g, 3.93 mmol) and 4-dimethylaminopyridine (32 mg, 0.26 mmol) were added in sequence at 0°C. The mixture was naturally restored to room temperature, and N 2The mixture was diluted with dichloromethane and washed three times with 1M hydrochloric acid saturated NaCl solution, anhydrous Na 2 SO 4 After drying and chromatography on a silica gel column (V petroleum ether / V ethyl acetate / V dichloromethane = 6 / 1 / 1), a white solid product OA-4 (2.76 g, yield 83%) was obtained.

[0106] (5) Preparation of intermediate compound OA-5

[0107] The intermediate compound OA-4 (1.36, 1.08 mmol) was dissolved in 8 mL of dry dichloromethane, and trifluoroacetic acid (1.86 mL, 1.28 mmol) was added. 2 Stir at room temperature for 2 h under protection. Add saturated sodium bicarbonate solution under ice bath to terminate the reaction, dilute the mixture with dichloromethane, wash with water and NaCl solution three times in sequence. Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a white solid product OA-5 (1.11 g, yield 89%).

[0108] (6) Preparation of intermediate compound OA-6

[0109] The intermediate compound OA-5 (740 mg, 0.76 mmol) and the fully TBS-protected Miscanthic acid L-2 (470 mg, 0.91 mmol) were dissolved in 10 mL of dry dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (182 mg, 0.95 mmol), 1-hydroxy-7-azidobenzotriazole (129 mg, 0.95 mmol) and N-methylmorpholine (256 μL, 2.28 mmol) were added in sequence at 0°C (i.e., under ice bath conditions). After stirring at 0°C for 30 min, N 2 The reaction was stirred at room temperature overnight under protection. The mixture was diluted with dichloromethane and washed three times with 1M hydrochloric acid saturated NaCl solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (V petroleum ether / V ethyl acetate / V dichloromethane = 3 / 1 / 1) to obtain a white solid product OA-6 (416 mg, yield 43%).

[0110] (7) Preparation of target compound I-1

[0111] The intermediate compound OA-6 (365 mg, 0.22 mmol) was dissolved in 6 mL of tetrahydrofuran, tetrabutylammonium fluoride trihydrate (693 mg, 2.2 mmol) was added at 0°C, and then 3 drops of acetic acid were added. The reaction system was returned to room temperature and continued to stir overnight. After concentration under reduced pressure, the residue was dissolved in ethyl acetate, and then washed with 1M hydrochloric acid solution and saturated NaCl solution in sequence. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was chromatographed on a silica gel column (V dichloromethane / V methanol / V formic acid = 100 / 10 / 1) to obtain a white solid product I-1 (165 mg, yield 77%).

[0112] According to the preparation process of the above compound I-1, the starting raw material oleanolic acid is replaced with ursolic acid or betulinic acid, etc., to obtain the corresponding products (I-2 and I-3) shown in formula I. The appearance and nuclear magnetic hydrogen spectrum test results of the above compounds are listed in Table 1. It can be seen from the above that the structures of the above three pentacyclic triterpene benzylamide derivatives are correct and are all compounds shown in formula I.

[0113] Table 1 Appearance and H NMR spectrum data of target compounds

[0114]

[0115]

[0116] Example 2 Activity Test

[0117] The C-3 substituted pentacyclic triterpene benzylamide derivatives prepared in Example 1 were used as test objects to test their inhibitory activity against the new coronavirus. The specific test method is as follows:

[0118] 1. In vitro virus inhibition experiment:

[0119] HEK293T cells in logarithmic growth phase were seeded into 96-well plates (4×10 5 / well) and cultured overnight. Take out the 96-well plate, replace the culture medium, add 0.5μg pcDNA3.1-SARS-CoV-2-Sipke plasmid and 1μg pNL4-3.Luc.RE-plasmid to each well, and co-transfect using PolyJet transfection reagent. After the above system was cultured at 37°C for 48h, the supernatant was collected and centrifuged at 3000r / min for 10min, then filtered with a 0.45μm sterile filter head, and the virus solution was stored at -80°C for later use. Gradient diluted C-3 substituted pentacyclic triterpene benzylamide derivatives and 1.3×10 4 TCID50 SARS-CoV-2 pseudovirus was placed in a 96-well plate and mixed at room temperature for 1 h. ACE2 / 293T cells (1×10 4 / well) and incubate for 48 hours. Measure the bioluminescence value to determine the infection of the virus. Calculate the inhibition rate based on the corresponding relationship between the bioluminescence value and the drug concentration, and draw a dose-response curve to determine the IC value of the saponin molecule against the virus. 50 value

[0120] The measured results are shown in Table 2.

[0121] Table 2C-3 Inhibitory activity of substituted pentacyclic triterpene benzylamide derivatives against novel coronavirus

[0122] Compound <![CDATA[pSARS-CoV-2(IC 50 ,μM)]]> <![CDATA[pDelta(IC 50 ,μM))]]> <![CDATA[pOmicron(IC 50 ,μM))]]> I-1 6.28 4.33 1.56 I-2 7.81 6.62 2.03 I-3 8.56 5.37 2.68

[0123] As can be seen from Table 2, the three C-3 substituted pentacyclic triterpene benzylamide derivatives shown in Formula I have strong inhibitory activity against the new coronavirus and its mutants Delta, Omicron, etc., and their inhibitory IC for three different pseudovirus strains is 50 All of them were lower than 10μM, especially the three target compounds mentioned above inhibited IC values ​​of Omicron, the main variant of the current novel coronavirus. 50 All were lower than 3 μM, indicating that the above compounds have broad-spectrum antiviral activity against the new coronavirus, can be prepared into anti-new coronavirus drugs for use, and have good application prospects in the prevention and treatment of new coronavirus infection.

[0124] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A C-3 substituted pentacyclic triterpene benzylamide derivative, characterized in that: The C-3 substituted pentacyclic triterpene benzylamide derivative has a structure shown in any one of formula I:

2. The C-3 substituted pentacyclic triterpene benzylamide derivative according to claim 1, characterized in that: The C-3 substituted pentacyclic triterpene benzylamide derivative also includes pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers or isotope-substituted compounds thereof.

3. The method for preparing the C-3 substituted pentacyclic triterpene benzylamide derivative according to claim 1, characterized in that: The steps include: S1. Using oleanolic acid, ursolic acid or betulinic acid as raw materials, acetylation reaction, chlorination reaction, condensation reaction and hydrolysis reaction are carried out in sequence to obtain intermediate compound 1: S2. Under ice bath conditions, the intermediate compound 1 obtained in step S1 and compound L-1 are mixed evenly, and a condensation reaction is carried out under an inert protective atmosphere at room temperature to obtain an intermediate compound 2: S3. Under normal temperature conditions, the intermediate compound 2 obtained in step S2 is selectively removed from the TBDMS protecting group to obtain the intermediate compound 3: S4. Under ice bath conditions, the intermediate compound 3 obtained in step S3 and compound L-2 are mixed evenly, and a condensation reaction is carried out under an inert protective atmosphere at room temperature to obtain an intermediate compound 4: S5. Under normal temperature and inert protective atmosphere, the intermediate compound 4 obtained in step S4 is selectively deprotected from the Boc protecting group to obtain an intermediate compound of formula 5: S6. Under ice bath conditions, the intermediate compound 5 obtained in step S5 is mixed evenly with compound L-2, and a condensation reaction is carried out under an inert protective atmosphere and room temperature conditions to obtain an intermediate compound of formula 6: S7. Selectively remove the TBDMS protecting group from the intermediate compound 6 to obtain the C-3 substituted pentacyclic triterpene benzylamide derivative as described in Ⅰ:

4. The preparation method according to claim 3, characterized in that: In step S1, the acetylation reagent used in the acetylation reaction includes one or more of acetic anhydride and acetyl chloride.

5. The preparation method according to claim 3, characterized in that: In step S2, the condensation agent used in the condensation reaction is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or a combination of N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine.

6. The preparation method according to claim 3, characterized in that: In step S3, the reaction of selectively removing the TBDMS protecting group is carried out under the action of a fluorine-containing reagent catalyst.

7. The preparation method according to claim 6, characterized in that: The fluorine-containing reagent is tetrabutylammonium fluoride, pyridine hydrofluoride, cesium fluoride or a hydrate of the above fluorine-containing reagents.

8. Use of the C-3 substituted pentacyclic triterpene benzylamide derivative according to claim 1 or 2 in the preparation of anti-new coronavirus drugs.

9. The use according to claim 8, characterized in that: The novel coronavirus is any one or more of the following coronaviruses or their variants: SARS-CoV-2, Delta, Omicron.

10. An anti-new coronavirus drug, characterized in that: Contains one or more of the C-3 substituted pentacyclic triterpene benzylamide derivatives described in claim 1 or 2.

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