A kind of preparation method of oleanolic acid glycoside

Abstract

The present invention belongs to the field of drug preparation, and in particular to a method for preparing oleanolic acid ester glycoside. The preparation method comprises first reacting oleanolic acid with a monosaccharide under the action of dicyclohexylcarbodiimide and 4-dimethylaminopyridine; then extracting the reactant to obtain a crude product; and recrystallizing the crude product. The yield and purity of the oleanolic acid ester glycoside prepared by the synthesis method of the present invention are both high, and the reaction steps are short, the cost is low, the post-processing is simple, and it is suitable for industrial production, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of drug preparation, and particularly relates to a method for preparing oleanolic acid ester glycoside. Background Art

[0002] Oleanolic acid is a pentacyclic triterpenoid compound isolated and extracted from the whole herb of Swertia vulgaris or the fruit of Ligustrum lucidum of the Gentianaceae family. It exists in a variety of plants as free bodies and glycosides. It has become a commonly used liver-protecting drug in clinical practice. It has a significant protective effect on acute and chronic liver damage in rats caused by carbon tetrachloride, can reduce elevated ALT and AST, alleviate inflammation, necrosis and interstitial inflammatory reactions, prevent fibrosis formation, promote the regeneration of liver cells, and accelerate the recovery of necrotic tissue.

[0003] However, oleanolic acid has poor water solubility, which affects its bioavailability. It is of great significance to develop and prepare derivatives with better water solubility and activity. Studies have shown that its chemical derivative oleanolic acid glycoside has obvious anti-inflammatory activity. Its structural formula is as follows:

[0004]

[0005] However, there are few reports on the synthesis of oleanolic acid glycosides.

[0006] Chinese invention patent application CN201310165693.5 discloses a preparation method, which adopts a biotransformation method: using oleanolic acid as a substrate, using a strain with a preservation number of NRRL1086, co-culturing the substrate and the strain in a culture medium for 4 to 7 days, terminating the reaction, extracting the fermentation broth with an organic solvent, concentrating the extract to obtain an extract, and separating the target compound by silica gel column chromatography.

[0007] The above method also requires bacterial culture, which is harsh and complicated. Therefore, it has always been a pursuit in the art to develop a method with mild reaction conditions and high product selectivity. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides a method for preparing oleanolic acid ester glycoside.

[0009] The technical solution adopted by the present invention is as follows:

[0010] A method for preparing oleanolic acid glycoside comprises the following steps:

[0011] (1) reacting oleanolic acid with monosaccharide in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine;

[0012] (2) extracting the reactant of step (1) to obtain a crude product;

[0013] (3) The crude product is recrystallized.

[0014] Preferably, in step (1), the monosaccharide is selected from one of glucose, mannose, arabinose, galactose, rhamnose and xylose, and the molar ratio of dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:1 to 6:1.

[0015] Preferably, in step (1), the molar ratio of 4-dimethylaminopyridine, oleanolic acid and monosaccharide is 1:1:1-1:4:4.8, and the molar ratio of dicyclohexylcarbodiimide and oleanolic acid is 1:1.2-1:0.5.

[0016] Preferably, the reaction in step (1) is a heating reaction, the reaction temperature is 35-40° C., and the reaction time is 3-6 h.

[0017] Preferably, the extraction in step (2) is performed by adding water to the reactants, separating the liquids, extracting the aqueous layer with dichloromethane, and combining the organic phases.

[0018] Preferably, the extraction is performed 1 to 3 times in step (2), and the volume ratio of the water layer to dichloromethane is 1:1 to 1:5.

[0019] Preferably, in step (2), the organic phase is filtered to remove the solvent to obtain a crude product.

[0020] Preferably, the solvent used for the recrystallization in step (3) is methanol, and the ratio of the crude product to methanol is 1:3 to 1:10.

[0021] Preferably, the recrystallization method in step (3) is stirred crystallization, and the recrystallization time is 4 to 96 hours.

[0022] Preferably, a solvent is added during the reaction in step (1), and the solvent is one or both of dichloromethane and acetone.

[0023] Beneficial effects of the present invention:

[0024] (1) The method for synthesizing oleanolic acid glycoside of the present invention has short reaction steps, low cost, simple post-treatment, is suitable for industrial production, and has good application prospects.

[0025] (2) The yield and purity of oleanolic acid glycoside prepared by the method of the present invention are both high. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 For 28- O - β -Blank solution spectrum in the HPLC chromatogram of D-pyranose oleanolic acid ester;

[0027] Figure 2For 28- O - β HPLC chromatogram of 28-D-pyranose oleanolic acid glycoside O - β -D-glucopyranose oleanolic acid glycoside spectrum. DETAILED DESCRIPTION

[0028] Example 1

[0029] 28- O - β The preparation method of -D-pyranose oleanolic acid glycoside is as follows:

[0030] (1) Weigh oleanolic acid (10.23 mmol), dicyclohexylcarbodiimide (10.23 mmol) and 4-dimethylaminopyridine (3.41 mmol) and add them into a 100 mL three-necked flask in sequence. Add 30 mL of dichloromethane into the reaction flask, stir for 30 min, then add glucose (11.25 mmol) and react at 40 °C for 4 h.

[0031] (2) TLC tracking reaction. After the reaction is completed, 30 mL of water is added, the liquids are separated, and the aqueous layer is extracted twice with 30 mL of dichloromethane. The organic phases are combined, dried over anhydrous sodium sulfate, filtered, and the solvent is removed by vacuum rotary evaporation to obtain a crude product.

[0032] (3) The crude product was recrystallized by stirring with methanol (solid-liquid ratio of 5:1) for 12 h to obtain 2.436 g of white solid with a yield of 38.5% and a purity of 99.0%.

[0033] Example 2

[0034] 28- O - β The preparation method of -D-pyranose oleanolic acid glycoside is as follows:

[0035] (1) Weigh oleanolic acid (10.23 mmol), dicyclohexylcarbodiimide (10.23 mmol) and 4-dimethylaminopyridine (3.41 mmol) and add them to a 100 mL three-necked flask in sequence. Add 30 mL of dichloromethane to the reaction flask, stir for 30 min, then add glucose (11.25 mmol) and reflux at 40 °C for 5 h.

[0036] (2) TLC tracking reaction. After the reaction is completed, 30 mL of water is added, the liquid is separated, and the aqueous layer is extracted three times with 30 mL of dichloromethane. The extracts are combined to obtain the dichloromethane extract organic phase, which is dried over anhydrous sodium sulfate, filtered, and the solvent is removed by vacuum rotary evaporation to obtain a crude product.

[0037] (3) The crude product was recrystallized by stirring with methanol (solid-liquid ratio of 3:1) for 24 h to obtain 2.429 g of white solid with a yield of 38.4% and a purity of 98.8%.

[0038] Example 3

[0039] 28- O - β The preparation method of -D-pyranose oleanolic acid glycoside is as follows:

[0040] (1) Weigh oleanolic acid (12 mmol), dicyclohexylcarbodiimide (18 mmol) and 4-dimethylaminopyridine (6 mmol) and add them to a 100 mL three-necked flask in sequence. Add 50 mL of dichloromethane to the reaction flask, stir for 30 min, then add glucose (14 mmol) and react at 35°C for 4 h.

[0041] (2) TLC tracking reaction, after the reaction is completed, add 40 mL of water, separate the layers, extract the aqueous layer with 40 mL of dichloromethane, dry the organic phase of the extract with anhydrous sodium sulfate, filter, and remove the solvent by vacuum rotary evaporation to obtain a crude product;

[0042] (3) The crude product was recrystallized from methanol (solid-liquid ratio of 10:1) to obtain 2.709 g of white solid with a yield of 36.5% and a purity of 98.1%.

[0043] Structural confirmation

[0044] 1.1 Mass spectrometry and nuclear magnetic resonance confirmed that the white solid obtained in Examples 1 to 3 was 28- O - β -D-glucopyranose oleanolic acid glycoside.

[0045] HR-MS spectrum showed that the quasi-molecular ion m / z 617.4053 [MH] - (C 36 H 58 O 8 The calculated value is 617.4042).

[0046] ¹H NMR (600 MHz, DMSO-d 6 ) spectrum shows the characteristic signal of allylic hydrogen of C-12 of oleanolic acid d 5.08ppm (d, J = 4.9 Hz, 1H), C-3 hydroxylated methylene hydrogen signal d 3.05 ppm (dt, J = 10.6, 5.4Hz, 1H). The methyl hydrogen signals of C-23, C-24, C-25, and C-26 appear at d0.96~0.90 ppm, while the methyl hydrogen of C-29 and C-30 are d 0.74 ppm (d, J = 7.3 Hz, 6H). The C-1' hydrogen of the glucose moiety is d There is a double peak at 5.30 ppm (d, J =8.1 Hz, 1H), indicating that it is β Configuration. The hydrogen signals from C-2' to C-6' appear at d 3.68 ppm (ddd, J = 11.8, 5.4, 2.0 Hz, 1H) and d In the range of 3.28~3.12 ppm, C-5' hydrogen appears d 4.34 ppm (d, J = 5.1 Hz, 1H).

[0047] ¹³C NMR (150 MHz, DMSO-d 6 ) spectrum shows that in the parent skeleton of oleanolic acid, the carboxyl carbon of C-28 is shown in d 175.21 ppm, the olefinic carbons of C-12 and C-13 are located at d 121.66 ppm and d 143.47 ppm. The hydroxylated carbon signal of C-3 is located at d 76.84 ppm, the methyl carbon signal is distributed at d The saturated skeleton carbon signal is mainly located in the range of 15.18~28.23 ppm. d 25.55~47.14 ppm range. The glucose C-1' carbon signal appears in d 94.08 ppm, and the carbon signals from C-2' to C-6' are located at d 72.36 ppm, 76.66 ppm, 69.53 ppm, 77.73 ppm and 60.67 ppm.

[0048] The HMBC spectrum and HMQC spectrum further confirmed the connection mode between the sugar group and the parent skeleton. Combining the one-dimensional and two-dimensional NMR data, the structure of the compound was confirmed. O - β The NMR data of -D-pyranose oleanolic acid ester are shown in Table 1.

[0049] Table 1 28- O - β -NMR data results of D-pyranose oleanolic acid glycoside

[0050]

[0051] 1.2 High performance liquid chromatography confirmed that the white solid was 28- O - β -D-glucopyranose oleanolic acid glycoside.

[0052] Preparation of test solution: Take 28- O - β -D-pyranose oleanolic acid glycoside was dissolved in methanol to prepare a solution with a concentration of 10 mg / mL.

[0053] Chromatographic conditions: chromatographic column: Unitary-C18 (250 mm×4.6 mm, 5 μm); mobile phase: A is 10% acetonitrile in water; B is 80% acetonitrile in water; gradient elution: 0%→30% B (0-10 min), 30%→50% B (10-25 min), 50%→85% B (25-40 min), 85%→100% B (40-55 min), 100%→0% B (55-56 min), 0% B (56-65 min); flow rate: 1.5 mL·min -1 ; Detection wavelength: 203 nm; Column temperature: 25 °C; Injection volume: 10 μL.

[0054] The results of the test are as follows Figure 1-Figure 2 As shown. It can be seen that except for 28- O - β -D-pyranose oleanolic acid glycoside chromatographic peak, no other impurity peaks were found. The results showed that 28- O - β The purity of -D-pyranose oleanolic acid glycoside is 99.0%.

[0055] Example 4

[0056] 28- O - α The preparation method of -D-mannose oleanolic acid glycoside is as follows:

[0057] (1) Weigh oleanolic acid (10.23 mmol), dicyclohexylcarbodiimide (10.23 mmol) and 4-dimethylaminopyridine (3.41 mmol) and add them to a 100 mL three-necked flask in sequence. Add 30 mL of dichloromethane to the reaction flask, stir for 30 min, then add mannose (11.25 mmol) and reflux for 4 h.

[0058] (2) TLC tracking reaction. After the reaction is completed, add 20 mL of water, separate the layers, extract the water layer with 20 mL of dichloromethane three times, combine the extracts to obtain the dichloromethane extract organic phase, dry it with anhydrous sodium sulfate, filter it, and remove the solvent by vacuum rotary evaporation to obtain a crude product.

[0059] (3) The crude product was recrystallized by stirring with methanol (solid-liquid ratio of 5:1) for 24 h to obtain 2.417 g of white solid with a yield of 38.2% and a purity of 98.7%.

[0060] 1 H NMR (400 MHz, pyridine-d5): d6.71 (d, J = 1.7Hz, 1H, 1′-H), 5.41(t, J = 3.4 Hz, 1H, 12-H), 4.88 (dt, 9.5 Hz, 5.0 Hz,1H, 4′-H), 4.69 (ddd, J =10.1 Hz, 9.5 Hz, 6.5 Hz, 1H, 3′-H), 4.67(ddd, J = 11.3 Hz, 6.0 Hz, 2.0 Hz,1H, 6′-H), 4.65 (ddd, J = 10.1 Hz,4.5 Hz, 1.6 Hz, 2′-H), 4.58 (ddd, J = 11.3Hz, 6.5 Hz, 5.0 Hz, 1H, 6′-H), 4.53 (ddd, J = 9.5 Hz, 5.0 Hz, 2.0 Hz, 1H, 5′-H), 3.46 (ddd, J = 10.9 Hz, 5.4 Hz, 5.4 Hz, 1H, 3-H), 3.19 (dd, J = 13.8 Hz,4.0 Hz, 1H,18-H), 2.06 (m, 1H,11-H), 2.03 (m, 2H, 2-H), 1.89 (m, 1H, 22-H),1.87 (m, 2H, 16-H), 1.85 (m, 1H, 15-H), 1.79 (m, 1H, 11-H), 1.77(m, 1H, 19-H), 1.66 (m, 1H, 9-H), 1.59 (m, 1H, 22-H), 1.55 (m, 1H,1-H), 1.55(m, 1H, 6-H), 1.49 (m, 1H, 7-H), 1.40 (m, 1H, 21-H), 1.36 (m, 1H, 7-H), 1.32 (m, 1H, 6-H), 1.26 (s, 3H, 23-H), 1.25 (m, 1H, 19-H), 1.23 (s, 3H, 27-H), 1.15 (m, 1H,21-H), 1.11 (m, 1H, 15-H), 1.06(s, 3H, 24-H), 1.02 (m, 1H, 1-H), 0.96 (s, 3H,30-H), 0.94 (s, 3H, 26-H), 0.91 (s, 3H, 29-H), 0.88 (s, 3H, 25-H), 0.84 (m,1H, 5-H);

[0061] 13C NMR (100 MHz, pyridine-d5): d 176.2 (C-28), 144.3 (C-13), 123.3(C-12), 95.6 (C-1′), 78.6 (C-5′), 78.3 (C-3), 73.4 (C-3′), 71.5 (C-2′), 68.6 (C-4′), 62.9 (C-6′), 55.9 (C-5), 48.3 (C-9), 47.6 (C-17), 46.4 (C-19), 42.4 (C-14), 42.2 (C-18), 39.9 (C-8), 39.6 (C-4), 39.2 (C-1),37.5 (C-10), 34.2 (C-21), 33.4 (C-7), 33.4 (C-29), 32.9 (C-22), 31.1(C-20), 28.9 (C-23), 28.3 (C-15), 28.3 (C-2), 26.3 (C-27), 24.2 (C-30), 23.9 (C-16), 23.7 (C-11), 18.9 (C-6), 17.7 (C-26), 16.8 (C-24), 15.8 (C-25).

[0062] Example 5

[0063] 28- O - β The preparation method of -D-arabinopyranose oleanolic acid glycoside is as follows:

[0064] (1) Weigh oleanolic acid (10.23 mmol), dicyclohexylcarbodiimide (10.23 mmol) and 4-dimethylaminopyridine (3.41 mmol) and add them to a 100 mL three-necked flask in sequence. Add 30 mL of dichloromethane to the reaction flask, stir for 30 min, then add arabinose (11.25 mmol) and reflux for 5 h.

[0065] (2) TLC tracking reaction. After the reaction is completed, 30 mL of water is added, the liquid is separated, and the aqueous layer is extracted three times with 30 mL of dichloromethane. The extracts are combined to obtain the dichloromethane extract organic phase, which is dried over anhydrous sodium sulfate, filtered, and the solvent is removed by vacuum rotary evaporation to obtain a crude product.

[0066] (3) The crude product was recrystallized by stirring with methanol (solid-liquid ratio of 5:1) for 24 h to obtain 2.311 g of white solid with a yield of 38.4% and a purity of 98.7%.

[0067] 1 H NMR (C5D5N-d5): d 6.23 (d, 1H, J=7.8 Hz, Aar-1-H), 5.27 (s, 1H), 4.65 (dd, 1H, J =7.6, 6.3 Hz), 4.35~4.28 (m, 2H), 4.24~3.82 (m, 2H), 3.16(dd, 1H, J= 10.4, 4.7 Hz), 2.83 (d, 1H, J=10.5 Hz);

[0068] 13 C NMR (100 MHz, pyridine-d5): d 177.2 (C-28), 144.1 (C-13), 122.9 (C-12), 99.3 (C-1′), 78.3 (C-3), 63.6 (C-5′), 70.1 (C-4′), 73.4 (C-3′), 73.4 (C-2′), 55.3 (C-5), 47.5 (C-9), 47.2 (C-17), 46.2 (C-19), 42.5 (C-14), 41.9 (C-18), 47.6 (C-8), 38.6 (C-4), 39.2 (C-1), 37.6 (C-10), 34.1 (C-21), 33.0 (C-7), 32.5 (C-22), 30.8 (C-20), 23.3 (C-23), 28.8(C-15), 27.3 (C-2), 26.0 (C-27), 23.9 (C-16), 23.7 (C-11), 18.5 (C-6), 17.1 (C-26), 23.3 (C-24), 15.9(C-25).

[0069] Example 6

[0070] 28- O - β The preparation method of -D-galactose oleanolic acid glycoside is as follows:

[0071] (1) Weigh oleanolic acid (10.23 mmol), dicyclohexylcarbodiimide (10.23 mmol) and 4-dimethylaminopyridine (3.41 mmol) and add them into a 100 mL three-necked flask in sequence. Add 30 mL of dichloromethane into the reaction flask, stir for 30 min, then add galactose (11.25 mmol) and reflux for 4 h.

[0072] (2) TLC tracking reaction. After the reaction is completed, add 20 mL of water, separate the layers, extract the water layer with 20 mL of dichloromethane three times, combine the extracts to obtain the dichloromethane extract organic phase, dry it with anhydrous sodium sulfate, filter it, and remove the solvent by vacuum rotary evaporation to obtain a crude product.

[0073] (3) The crude product was recrystallized by stirring with methanol (solid-liquid ratio of 5:1) for 24 h to obtain 2.423 g of white solid with a yield of 38.3% and a purity of 98.7%.

[0074] 1 H NMR (400 MHz, pyridine-d5): d6.30 (d, J = 8.1 Hz, 1H, 1′-H), 5.47(dd, J = 3.4 Hz, 3.4 Hz, 1H, 12-H), 4.70 (m, 1H, 3′-H), 4.67 (m, 1H, 2′-H),4.53 (ddd, J = 10.7 Hz, 6.5 Hz, 6.5 Hz, 1H, 6′-H), 4.43 (ddd, J = 10.7 Hz,5.4 Hz, 5.4 Hz, 1H, 6′-H), 4.27 (m, 1H, 4′-H), 4.23 (m, 1H, 5′-H), 3.47 (m,1H, 3-H), 3.24 (dd, J = 4.1 Hz, 4.1 Hz, 1H, 18-H), 2.37 (m, 1H, 2-H), 2.08(m, 2H, 11-H), 1.96 (m, 2H, 16- H), 1.85 (m, 2H, 2-H, 22-H), 1.79 (m, 1H, 22-H), 1.79 (m, 1H, 19- H), 1.69 (m, 1H, 9-H), 1.59 (m, 1H, 1-H), 1.56 (m, 1H,6-H), 1.49 (m, 1H, 7-H), 1.42 (m, 1H, 6-H), 1.39 (m, 1H, 7-H), 1.36 (m, 1H,21- H), 1.32 (m, 1H, 19-H), 1.26 (s, 6H, 23-H, 27-H), 1.17 (s, 3H, 26-H),1.14 (m, 2H, 15-H), 1.10 (m, 1H, 21-H), 1.05 (s, 3H, 24-H), 0.99 (m, 1H, 1-H), 0.95 (s, 3H, 25-H), 0.93 (s, 3H, 29-H), 0.89 (s, 3H, 30-H), 0.85 (m, 1H,5-H);

[0075] 13 C NMR (100 MHz, pyridine-d5): d176.7 (C-28), 144.5 (C-13), 123.2 (C-12), 96.5 (C-1′), 78.3 (C-3), 77.9 (C-5′), 75.9 (C-4′), 71.8 (C-3′), 70.4 (C-2′), 62.2 (C-6′), 56.2 (C-5), 48.4 (C-9), 47.3 (C-17), 46.5 (C-19), 42.5 (C-14), 41.9 (C-18), 40.33 (C-8), 39.6 (C-4), 39.2 (C-1), 37.6 (C-10), 34.4 (C-21), 33.5 (C-7), 33.5 (C-29), 32.9 (C-22), 31.0 (C-20), 28.9 (C-23), 28.6 (C-15), 28.5 (C-2), 26.3 (C-27), 24.1 (C-30), 23.9 (C-16), 23.7 (C-11), 19.2 (C-6), 17.9 (C-26), 16.9 (C-24), 15.9 (C-25).

[0076] Example 7

[0077] 28- O - α- L - The preparation method of rhamnosyl oleanolic acid glycoside is as follows:

[0078] (1) Weigh oleanolic acid (10.23 mmol), dicyclohexylcarbodiimide (10.23 mmol) and 4-dimethylaminopyridine (3.41 mmol) and add them into a 100 mL three-necked flask in sequence. Add 30 mL of dichloromethane into the reaction flask, stir for 30 min, then add rhamnose (11.25 mmol) and reflux for 4 h.

[0079] (2) TLC tracking reaction. After the reaction is completed, 20 mL of water is added, the liquid is separated, and the aqueous layer is extracted three times with 20 mL of dichloromethane. The extracts are combined to obtain the dichloromethane extract organic phase, which is dried over anhydrous sodium sulfate, filtered, and the solvent is removed by vacuum rotary evaporation to obtain a crude product.

[0080] (3) The crude product was recrystallized by stirring with methanol (solid-liquid ratio of 5:1) for 24 h to obtain 2.354 g of white solid with a yield of 38.2% and a purity of 98.7%.

[0081] 1H NMR (400 MHz, pyridine-d5): d 6.81 (d, J = 1.4 Hz, 1H, 1′-H), 5.44(t, J = 3.2 Hz, 1H, 12-H), 4.59 (dd, J = 3.2 Hz, 1.4 Hz, 1H, 2′-H), 4.52 (dd,J = 8.7 Hz, 3.1 Hz, 1H, 3′-H), 4.379 (t, J = 8.7 Hz, 1H, 4′-H), 4.37 (m, 1H,5′-H), 3.44 (dd, 10.7 Hz, 5.6 Hz,1H, 3-H), 3.15 (dd, J = 14.1 Hz, 3.6 Hz, 1H,18-H), 2.03 (m, 1H, 16- H), 2.00 (m, 1H, 2-H), 1.93 (m, 2H, 11-H), 1.85 (m,2H, 2-H, 22-H), 1.74 (m, 2H, 16-H, 19-H), 1.71 (d, J = 5.3 Hz, 3H, 6′-CH3),1.64 (m, 1H, 9-H), 1.59 (m, 1H, 22-H), 1.55 (m, 2H, 1-H, 6-H), 1.45 (m, 1H,7-H), 1.40 (m, 1H, 21-H), 1.35 (m, 1H, 6-H), 1.29 (m, 2H, 7-H, 19- H), 1.24(s, 3H, 23-H), 1.21 (s, 3H, 27-H), 1.16 (m, 1H, 21-H), 1.11 (m, 2H, 15-H),1.06 (s, 3H, 26-H), 1.00 (m, 1H, 1-H), 0.91 (s, 3H, 24-H), 0.91 (s, 3H, 25-H), 0.91 (s, 3H, 29-H), 0.87 (s, 3H, 30-H), 0.85 (m, 1H, 5-H);

[0082] 13 C NMR (100 MHz, pyridine-d5): d176.0 (C-28), 143.6 (C-13), 123.5 (C-12), 95.5 (C-1′), 78.2 (C-3), 73.6 (C-4′), 73.0 (C-3′), 72.7 (C-5′), 71.6 (C-2′), 55.9 (C-5), 48.1 (C-9), 47.5 (C-17), 46.1 (C-19), 42.2 (C-14), 42.1 (C-18), 39.9 (C-8), 39.5 (C-1), 39.0 (C-4), 37.5 (C-10), 34.1 (C-21), 33.4 (C-7), 33.2 (C-22), 33.1 (C-29), 31.0 (C-20), 29.0 (C-23), 28.2 (C-15), 28.1 (C-2), 26.1 (C-27), 24.0 (C-11), 23.7 (C-30), 23.4 (C-16), 18.9 (6′-CH3), 18.9(C-6), 17.2(C-26), 16.7(C-24), 15.7(C-25).

[0083] Example 8

[0084] 28- O - β- D - The preparation method of xylopyranose oleanolic acid glycoside is as follows:

[0085] (1) Weigh oleanolic acid (10.23 mmol), dicyclohexylcarbodiimide (10.23 mmol) and 4-dimethylaminopyridine (3.41 mmol) and add them to a 100 mL three-necked flask in sequence. Add 30 mL of dichloromethane to the reaction flask, stir for 30 min, then add xylose (11.25 mmol) and reflux for 5 h.

[0086] (2) TLC tracking reaction. After the reaction is completed, add 20 mL of water, separate the layers, extract the water layer with 20 mL of dichloromethane three times, combine the extracts to obtain the dichloromethane extract organic phase, dry it with anhydrous sodium sulfate, filter it, and remove the solvent by vacuum rotary evaporation to obtain a crude product.

[0087] (3) The crude product was recrystallized by stirring with methanol (solid-liquid ratio of 5:1) for 24 h to obtain 2.323 g of white solid with a yield of 38.6% and a purity of 98.6%.

[0088] 1H NMR (400 MHz, pyridine-d5): d 6.22 (d, J = 6.8 Hz, 1H, 1′-H), 5.45(t, J = 3.1 Hz, 1H, 12-H), 4.37 (dd, J = 11.0 Hz, 4.0 Hz, 1H, 5′-H), 4.19−4.15 (m, 3H, 2′-H, 3′-H, 4′-H), 3.81 (dd, J = 11.0 Hz, 9.9 Hz, 1H, 5′-H),3.42 (dd, 10.6 Hz, 5.7 Hz, 1H, 3-H), 3.25 (dd, J = 14.0 Hz, 3.9 Hz, 1H, 18-H), 2.32 (m, 1H, 15-H), 2.09 (m, 2H, 16-H), 2.03 (m, 1H, 22-H), 1.96 (m, 2H,11-H), 1.84 (m, 2H, 15-H, 22-H), 1.80 (m, 1H, 19-H), 1.67 (m, 1H, 9-H), 1.53(m, 2H, 1-H, 6- H), 1.50 (m, 2H, 7-H), 1.35 (m, 2H, 6-H, 21-H), 1.29 (m, 1H,19-H), 1.24 (s, 3H, 27-H), 1.23 (s, 3H, 23-H), 1.17 (m, 1H, 2-H), 1.13 (m,1H, 21-H), 1.12 (s, 3H, 26-H), 1.05 (s, 3H, 24-H), 1.00 (m, 1H, 1-H), 0.95(s, 3H, 30-H), 0.93 (s, 6H, 25-H, 29-H), 0.84 (m, 1H, 5-H);

[0089] 13 C NMR (100 MHz, pyridine-d5): d176.5 (C-28), 144.1 (C-13), 123.0 (C-12), 96.2 (C-1′), 78.3 (C-2′), 78.1 (C-3), 73.7 (C-3′), 71.0 (C-4′), 67.9 (C-5′), 56.0 (C-5), 48.2 (C-9), 47.3 (C-17), 46.3 (C-19), 42.1 (C-14), 41.8 (C-18), 40.0 (C-8), 39.3 (C-4), 39.1 (C-1), 37.4 (C-10), 34.1 (C-21), 33.4 (C-7), 33.2 (C-29), 32.7 (C-22), 31.0 (C-20), 28.8 (C-23), 28.4 (C-15), 28.2 (C-2), 26.1 (C-27), 24.0 (C-11), 23.5 (C-30), 23.6 (C-16), 19.0 (C-6), 17.8 (C-26), 16.6 (C-24), 15.7 (C-25).

[0090] Comparative Example 1

[0091] The difference between this comparative example and Example 1 is that the reaction time is 2 hours, and the rest is consistent with Example 1. The yield is 15.3% and the purity is 95.1%.

[0092] Comparative Example 2

[0093] The difference between this comparative example and Example 1 is that the molar ratio of 4-dimethylaminopyridine to oleanolic acid is 1:10, and the rest is consistent with Example 1. The yield is 18.3% and the purity is 95.5%.

[0094] Comparative Example 3

[0095] The difference between this comparative example and Example 1 is that the reaction is carried out at room temperature 25°C, and the rest is consistent with Example 1. The yield is 15.9% and the purity is 95.3%.

[0096] Comparative Example 4

[0097] The difference between this comparative example and Example 1 is that the reaction is carried out in an ice-water bath, and the rest is consistent with Example 1. The yield is 0.

[0098] Comparative Example 5

[0099] The difference between this comparative example and Example 1 is that dicyclohexylcarbodiimide is replaced by EDCI1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the rest is consistent with Example 1. The yield is 15.4% and the purity is 95.8%.

[0100] Comparative Example 6

[0101] The difference between this comparative example and Example 1 is that when 4-dimethylaminopyridine is replaced by an equal amount of potassium carbonate or triethylamine as a catalyst, the target product is not obtained.

[0102] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing oleanolic acid glycosides, characterized in that: The steps include: (1) reacting oleanolic acid with monosaccharide in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine; (2) extracting the reactant of step (1) to obtain a crude product; (3) Recrystallize the crude product; In step (1), the monosaccharide is selected from one of glucose, mannose, arabinose, galactose, rhamnose and xylose, and the molar ratio of dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:1 to 6:

1.

2. The preparation method according to claim 1, characterized in that: In step (1), the molar ratio of 4-dimethylaminopyridine, oleanolic acid and monosaccharide is 1:1:1-1:4:4.8, and the molar ratio of dicyclohexylcarbodiimide and oleanolic acid is 1:1.2-1:0.

5.

3. The preparation method according to claim 1, characterized in that: The reaction in step (1) is a heating reaction, the reaction temperature is 35-40°C, and the reaction time is 3-6h.

4. The preparation method according to claim 1, characterized in that: The extraction in step (2) is performed by adding water to the reactants, separating the layers, extracting the aqueous layer with dichloromethane, and combining the organic phases.

5. The preparation method according to claim 4, characterized in that: The number of extractions in step (2) is 1-3 times, and the volume ratio of the water layer to dichloromethane is 1:1-1:

5.

6. The preparation method according to claim 4, characterized in that: In step (2), the organic phase is filtered and the solvent is removed to obtain a crude product.

7. The preparation method according to claim 1, characterized in that: The recrystallization method in step (3) is stirred crystallization, and the recrystallization time is 4 to 96 hours.

8. The preparation method according to claim 1, characterized in that: During the reaction in step (1), a solvent is added, wherein the solvent is one or both of dichloromethane and acetone.

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