An oleanolic acid derivative, its preparation method, and its application in antitumor activity.

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to an oleanolic acid derivative, its preparation method, and its application in antitumor activity. The oleanolic acid derivative is a compound shown in Formula 1, with substituents as described in the specification. The synthesized oleanolic acid derivative exhibits good antitumor activity when used alone or in combination with the first-line drug oxaliplatin. Specifically, compound A1-3, when used alone, reduced the survival rate of Hep 3B cells to 0.4%, while when used in combination with oxaliplatin, it achieved complete inhibition.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an oleanolic acid derivative, its preparation method, and its application in anti-tumor treatment. Background Technology

[0002] Liver cancer ranks fifth in global incidence, with 830,000 deaths from primary liver cancer in 2020. my country has become a high-risk region for hepatocellular carcinoma (CA CANCER J CLIN 2021, 71:209-249). Currently, chemotherapy regimens for HCC patients mainly include targeted therapy, immunotherapy, and combination therapy. Common treatment strategies for patients with liver function grades A and B include sorafenib, atezolizumab combined with bevacizumab, or systemic chemotherapy based on oxaliplatin (Chinese Society of Clinical Oncology (CSCO) Guidelines for the Diagnosis and Treatment of Primary Liver Cancer 2020). To date, oxaliplatin has been widely used in the treatment of various cancers (Drugs, 2000, 60:895-924), but its application in liver cancer has been less than satisfactory (Journal of Clinical Hepatobiliary Diseases, 2020, 36:194-197). Therefore, developing a drug that can synergistically treat liver cancer with oxaliplatin is urgently needed.

[0003] Oleanolic acid (OA) is a pentacyclic triterpenoid natural product widely found in food, medicinal herbs, and other plants. It possesses various pharmacological activities, including hepatoprotective, hypoglycemic, hypolipidemic, anti-ulcer, anti-HIV, and anti-tumor properties. The anticancer effects of oleanolic acid are observed throughout almost all stages of tumor development. In vitro experiments by Hsu et al. demonstrated that OA can inhibit tumor growth (Cancer Letters, 1997, 111:7-13); Ii J et al. found that OA inhibits tumor cell proliferation by blocking the cell cycle of tumor cells (World J Gastroenterol, 2002, 8:493-499). Studies have shown that caffeic acid, an analogue of cinnamic acid, can selectively inhibit the proliferation of liver cancer cells and reduce cell number by inducing apoptosis (Biochemical and biophysical research communications, 2018, 505:612-617).

[0004] Due to its low bioavailability, the biological activities of OA have not been fully and widely utilized. Therefore, the preparation of OA derivatives is an important way to address the low bioavailability and expand and improve the clinical efficacy of OA. Synthesized OA derivatives both domestically and internationally have shown good anticancer activity (Molecules, 2021, 26:4957; Cancer letters, 2014, 346:206-216; Molecules, 2021, 26:772). However, further research and development of more diverse OA derivatives are still needed. Summary of the Invention

[0005] Given the unsatisfactory clinical performance of existing drugs for treating liver cancer, the purpose of this invention is to provide an oleanolic acid derivative, its preparation method, and its application in anti-tumor treatment.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] An oleanolic acid derivative, wherein the oleanolic acid derivative is a compound shown in Formula 1.

[0008]

[0009] In the formula, R is an unsubstituted cinnamoyl or benzoyl group or a group substituted with at least one of the same or different substituents, wherein the substituent is -OMe or -OH.

[0010] Preferably, in the oleanolic acid derivative, R is a cinnamyl or benzoyl group substituted with at least one identical or different substituent, wherein the substituent is -OMe or -OH.

[0011] Further preferably, in the oleanolic acid derivative, R is a cinnamyl or benzoyl group substituted with 1-3 identical or different substituents, wherein the substituents are -OMe or -OH.

[0012] The chemical structural formula of the oleanolic acid derivative is shown below:

[0013]

[0014]

[0015] The preparation method of the above-mentioned oleanolic acid derivatives includes the following steps:

[0016] (1) TBDMSCl, triethylamine and DMAP were added sequentially to a dichloromethane solution of oleanolic acid (1), and the mixture was heated and stirred at 42°C for 4 h. The carboxyl-protected oleanolic acid (2) was then recrystallized from methanol.

[0017] (2) Add substituted cinnamic acid or substituted benzoic acid to dichloromethane, add DCC and DMAP at 0°C, and stir to react; then add carboxyl-protected oleanolic acid, stir to react at room temperature for 7 h to obtain the esterified product.

[0018] (3) The esterification product obtained in the previous step was reacted with tetrabutylammonium fluoride in tetrahydrofuran at room temperature for 3 hours, and then purified to obtain type A and type B oleanolic acid derivatives.

[0019]

[0020] The substituted cinnamic acid structure described in step (2) is synthesized via the following route: methoxy-substituted benzaldehyde (3) reacts with malonic acid in pyridine solvent and under piperidine catalysis to obtain methoxy-substituted cinnamic acid (4); intermediate 4 is demethylated by BBr3 at low temperature to obtain hydroxy-substituted phenylacrylic acid (5), and then the hydroxyl group in intermediate 5 is protected by TBDMSCl to obtain a hydroxy-protected cinnamic acid derivative (6).

[0021] The reaction formula is as follows:

[0022]

[0023] The substituted benzoic acid described in step (2) is synthesized via the following route: methoxy-substituted benzoic acid is demethylated by BBr3 under low temperature conditions to obtain hydroxy-substituted benzoic acid (8), and then the hydroxyl group in intermediate 8 is protected by TBDMSCl to obtain a hydroxy-protected benzoic acid derivative (9).

[0024] The reaction formula is as follows:

[0025]

[0026] The above-mentioned oleanolic acid derivatives and their application in combination with oxaliplatin to the survival rate of liver cancer cells.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] The derivatives of this invention use OA as the key core unit. A series of OA derivatives were synthesized by modifying the structure of natural products through ester bonds. The OA derivatives obtained in this invention, when used alone, showed significant inhibitory effects on the proliferation of hepatocellular carcinoma cells. Furthermore, some of the obtained OA derivatives exhibited synergistic effects in hepatocellular carcinoma proliferation inhibition activity tests when used in combination with the clinical drug oxaliplatin. This invention provides a feasible new approach for the development of subsequent hepatocellular carcinoma treatment drugs and offers a new direction for hepatocellular carcinoma treatment, demonstrating promising application prospects in the pharmaceutical field. Attached Figure Description

[0029] Figure 1 This is a bar chart showing the effect of oleanolic acid derivatives obtained in the embodiments of the present invention and their combination with oxaliplatin on the survival rate of Hep G2 cells;

[0030] Figure 2 The bar chart shows the effect of oleanolic acid derivatives obtained in the embodiments of the present invention and their combination with oxaliplatin on the survival rate of Hep G2 cells.

[0031] Figure 3 The bar chart shows the effect of oleanolic acid derivatives obtained in the embodiments of the present invention and their combination with oxaliplatin on the survival rate of Hep 3B cells.

[0032] Figure 4 The bar chart shows the effect of oleanolic acid derivatives obtained in the embodiments of the present invention and their combination with oxaliplatin on the survival rate of Hep 3B cells. Detailed Implementation

[0033] The compounds of the present invention and their preparation will be better understood in conjunction with the following examples, which are intended to illustrate rather than limit the scope of the invention.

[0034] This invention synthesizes a series of oleanolic acid derivatives and discovers their applications in tumor treatment, particularly liver cancer. The derivatives, used alone and in combination with oxaliplatin, demonstrate good survival-inhibiting activity against Hep G2 and Hep 3B cell lines. Further compounds A1-1, A1-3, and B1-1, when used in combination with oxaliplatin, significantly inhibit liver cancer cell activity. Moreover, A1-3, when used in combination with oxaliplatin, completely inhibits liver cancer cell survival. Due to its significant inhibitory effect, it has broad application prospects.

[0035] Example 1: 3β-O-(3,5-dihydroxycinnamoyl)oleanolic-12-en-28-acid (A1-1)

[0036]

[0037] Oleanolic acid (1) (5.00 g, 10.9 mmol) was weighed and added to a 250 mL round-bottom flask. 50 mL of dichloromethane was added and stirred to dissolve the precipitate. Then, tert-butyldimethylchlorosilane (2.00 g, 13.27 mmol) and triethylamine (2 mL, 14.39 mmol) were added, and the mixture was refluxed at 42 °C for 4 h. After the reaction was complete, the solvent was evaporated under reduced pressure until a large amount of solid precipitated. The solid was dissolved in hot methanol, recrystallized at low temperature, filtered, and the filter cake was repeatedly washed with methanol and dried to give 5.06 g of compound 2, with a yield of 80.95%.

[0038] The raw materials 3,5-dimethoxybenzaldehyde (11) (2.00 g, 12.04 mmol) and malonic acid (2.51 g, 24.08 mmol) were dissolved in 15 mL of pyridine, and then 3 mL of piperidine was added. The mixture was heated to 110 °C and refluxed for 3 h. After cooling to room temperature, 50 mL of water was added, and the pH was adjusted to 9-10 with saturated potassium carbonate solution. The mixture was repeatedly extracted with toluene (10 mL × 5) until the toluene layer was colorless. The pH of the aqueous layer was adjusted to 3-4 with dilute hydrochloric acid, and a large amount of solid precipitated out. The solid was filtered and dried to obtain 2.35 g of white solid compound (12), with a yield of 93.78%.

[0039] The white solid compound (12) (2.00 g, 9.61 mmol) obtained in the previous step was dissolved in 40 mL of dichloromethane. Under N2 atmosphere, a solution of 2 mol / L boron tribromide (3.70 mL, 38.42 mmol) in dichloromethane was added dropwise to a three-necked flask, and the reaction was carried out at room temperature for 24 h. The reaction solution was slowly poured into ice water to quench the reaction, extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by vacuum distillation to give 1.55 g of pale yellow solid compound (13), with a yield of 89.57%.

[0040] The pale yellow solid compound (13) (1.5 g, 8.33 mmol) obtained in the previous step was dissolved in 50 mL of dichloromethane. Then, tert-butyldimethylchlorosilane (5.02 g, 33.30 mmol), triethylamine (6.37 mL, 45.79 mmol), and DMAP (0.01 g, 0.1 mmol) were added sequentially, and the mixture was refluxed at 42 °C for 24 h. After cooling to room temperature, the mixture was concentrated under reduced pressure to obtain a large amount of solid. 50 mL of petroleum ether was added and stirred to dissolve the solid. The mixture was filtered, and the filter cake was repeatedly washed with petroleum ether. The filtrate was collected and concentrated under reduced pressure to obtain an oily primary product, which was dissolved in 20 mL of methanol. Weigh 1.5 g of potassium carbonate, dissolve it in an equal volume of water, mix and stir at room temperature for 1 h, quench with 3% w / w hydrochloric acid solution, extract with ethyl acetate (20 mL × 3), wash the organic phase with saturated brine, dry with anhydrous magnesium sulfate, filter, evaporate the solvent, and perform column chromatography (petroleum ether: ethyl acetate = 30:1, v / v) to give 2.43 g of white solid compound (14), yield 71.41%.

[0041] The white solid compound (14) obtained in the previous step (2.00 g, 4.89 mmol) was dissolved in 30 mL of dichloromethane. DCC (1.26 g, 6.12 mmol) and DMAP (0.45 g, 3.67 mmol) were weighed and dissolved in dichloromethane. The solutions were added dropwise at 0 °C, and the reaction was carried out at room temperature for 1 h. Compound 2 (0.70 g, 1.22 mmol) was then added, and the reaction was carried out at room temperature for 7 h. After the reaction was complete, the mixture was filtered, concentrated under reduced pressure, and the resulting pale yellow oily liquid was dissolved in hot n-hexane. The solution was cooled overnight at a low temperature, filtered, concentrated under reduced pressure, and subjected to column chromatography (petroleum ether: ethyl acetate = 50:1, v / v) to give 700 mg of white solid. The above-mentioned white solid was dissolved in 15 mL of tetrahydrofuran, and 0.1 mL of acetic acid was added and stirred until homogeneous. A tetrahydrofuran solution of TBAF (2 mL, 1 mol / L) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 3 h. The reaction was then stopped, and 20 mL of 5% dilute hydrochloric acid was slowly added. The mixture was extracted with ethyl acetate (20 mL × 3 times). The organic layers were combined and washed successively with 5% dilute hydrochloric acid, water, and saturated brine. The mixture was dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated to obtain 450 mg of a pale yellow oily crude product. Column chromatography (petroleum ether:ethyl acetate = 10:1, v / v) yielded 130 mg of a white solid, Al-1, with a yield of 17.23%.

[0042] ESI-MS m / z: 616.9 [MH] - .

[0043] 1H-NMR (600MHz, DMSO-d6) δ: 12.03 (s, 1H), 9.43 (s, 2H), 7.42 (d, J = 15.9Hz, 1H), 6.49 (d, J = 2.0Hz, 2H), 6.35 (d, J = 15.9Hz, 1H), 6.30 (s, 1H), 5.17 (d,J=3.7Hz,1H),4.52(dd,J=11.8,4.2Hz,1H),2.75(dd,J=13.9,4.5Hz,1H),1.91(dt,J=13.5,6.8Hz,1H),1.83(dd,J=9.0,3.6Hz,2H),1.69–1. 65(m,2H),1.63(d,J=13.9Hz,2H),1.61–1.55(m,4H),1.49(d,J=9.9Hz, 2H),1.44(dd,J=13.3,3.6Hz,2H),1.34(qd,J=13.8,7.3Hz,3H),1.24(d, J=7.7Hz,1H),1.12(s,3H),1.07(dd,J=13.3,3.7Hz,2H),1.01(d,J=13.3Hz,1H),0.92(s,3H),0.89(d,J=10.1Hz,9H),0.85(s,3H),0.74(s,3H).

[0044] Example 2: 3β-O-(2-hydroxycinnamoyl)oleanolic-12-en-28-acid (A1-2)

[0045] The difference from Example 1 is that the raw material 3,5-dimethoxybenzaldehyde in Example 1 was replaced with 2-methoxybenzaldehyde (2.00 g, 14.69 mmol) to prepare 2-methoxyphenylacrylic acid (2.30 g, 12.91 mmol) according to the above steps, with a yield of 87.87%; then, after demethylation with BBr3, 2-hydroxycinnamic acid (1.35 g, 8.22 mmol) was obtained with a yield of 73.27%; then, the hydroxyl group was protected with TBDMSCl to obtain 2-tert-butyldimethylcinnamic acid (1.72 g, 6.18 mmol) with a yield of 84.51%; 1.36 g of 2-tert-butyldimethylcinnamic acid was esterified with compound 2 (0.70 g, 1.22 mmol), and finally deprotected with TBAF to obtain 140 mg of white solid A1-2 with a yield of 19.05%.

[0046] ESI-MS m / z: 601.0 [MH] - .

[0047] 1H-NMR (600MHz, DMSO-d6)δ:12.01(s,1H),10.20(s,1H),7.86(d,J=16.1Hz,1H),7.62–7.58(m,1H),7.26–7.21(m,1H),6.90(d,J=8.2Hz,1H) ,6.82(t,J=7.5Hz,1H),6.57(d,J=16.1Hz,1H),5.17(d,J=3.9Hz,1H),4.53(dd,J=11.9,4.2Hz,1H),2.75(dd,J=14.0,4.7Hz,1H),1.97–1.8 9(m,1H),1.84(dd,J=9.1,3.5Hz,2H),1.70–1.65(m,2H),1.65–1.58(m,3H),1.57(s,2H),1.51(d,J=13.6Hz,2H),1.45(dd,J=13.3,10.2Hz, 2H),1.40–1.30(m,2H),1.30–1.21(m,2H),1.19–1.10(m,4H),1.10–0 .97(m,3H),0.93(s,3H),0.91–0.87(m,9H),0.85(s,3H),0.74(s,3H).

[0048] Example 3: 3β-O-(3-hydroxycinnamoyl)oleanolic-12-en-28-acid (A1-3)

[0049] The difference from Example 1 is that the raw material 3,5-dimethoxybenzaldehyde in Example 1 was replaced with 3-methoxybenzaldehyde (2.00 g, 14.69 mmol) to prepare 3-methoxyphenylacrylic acid (2.25 g, 12.63 mmol) according to the above steps, with a yield of 85.96%; then, after demethylation with BBr3, 3-hydroxycinnamic acid (1.4 g, 8.53 mmol) was obtained with a yield of 75.98%; then, the hydroxyl group was protected with TBDMSCl to obtain 3-tert-butyldimethylcinnamic acid (1.80 g, 6.46 mmol) with a yield of 88.44%; 1.36 g of 3-tert-butyldimethylcinnamic acid was esterified with compound 2 (0.70 g, 1.22 mmol), and finally deprotected with TBAF to obtain 120 mg of white solid A1-3 with a yield of 16.33%.

[0050] ESI-MS m / z: 601.1 [MH] - .

[0051] 1H NMR(600MHz,DMSO-d6)δ:12.03(s,1H),9.59(s,1H),7.53(d,J=15.9Hz,1H) ,7.21(t,J=7.8Hz,1H),7.13(d,J=7.7Hz,1H),7.03(t,J=2.0Hz,1H),6.83(d d,J=8.1,2.4Hz,1H),6.48(d,J=16.0Hz,1H),5.17(d,J=3.6Hz,1H),4.53(dd ,J=11.7,4.3Hz,1H),2.75(dd,J=14.0,4.6Hz,1H),1.92(td,J=13.5,3.9Hz, 1H),1.83(dd,J=9.1,3.6Hz,2H),1.70–1.65(m,2H),1.65–1.61(m,2H),1.61 –1.56(m,4H),1.53–1.47(m,2H),1.47–1.42(m,2H),1.35(dtd,J=34.9,13.6 ,12.9,3.5Hz,2H),1.28–1.23(m,1H),1.12(s,3H),1.10–1.03(m,2H),1.03– 0.99(m,1H),0.93(s,3H),0.89(d,J=14.6Hz,9H),0.85(s,3H),0.74(s,3H).

[0052] Example 4: 3β-O-(4-hydroxycinnamoyl)oleanolic-12-en-28-acid (A1-4)

[0053] The difference from Example 1 is that the raw material 3,5-dimethoxybenzaldehyde in Example 1 was replaced with 4-methoxybenzaldehyde (2.00 g, 14.69 mmol) to prepare 4-methoxyphenylacrylic acid (2.18 g, 12.23 mmol) according to the above steps, with a yield of 83.29%. After demethylation with BBr3, 4-hydroxycinnamic acid (1.45 g, 8.83 mmol) was obtained with a yield of 78.69%. Then, the hydroxyl group was protected with TBDMSCl to obtain 4-tert-butyldimethylcinnamic acid (1.70 g, 6.11 mmol) with a yield of 80.19%. 1.36 g of 4-tert-butyldimethylcinnamic acid was esterified with compound 2 (0.70 g, 1.22 mmol), and finally deprotected with TBAF to obtain 130 mg of white solid A1-4 with a yield of 17.69%.

[0054] ESI-MS m / z: 601.1 [MH] - .

[0055] 1H-NMR(600MHz,DMSO-d6)δ:12.03(s,1H),9.99(s,1H),7.55(d,1H),7.54(s,1H),7.52(s,1H),6.79(s,1H),6.78(s,1H),6.36(d,J=15.9Hz, 1H),5.17(d,J=3.9Hz,1H),4.52(dd,J=11.8,4.2Hz,1H),2.75(dd,J=14.1,4.6Hz,1H),1.92(d,J=4.0Hz,1H),1.83(dd,J=9.0,3.7Hz,2H),1 .67(d,J=13.7Hz,2H),1.63(d,J=13.0Hz,2H),1.58(t,J=10.6Hz,4H),1.49(d,J=9.1Hz,2H),1.44(d,J=13.0Hz,2H),1.41–1.29(m,3H),1.2 5(d,J=12.7Hz,1H),1.12(s,3H),1.08–1.04(m,1H),1.01(d,J=13.4Hz,1H),0.92(s,3H),0.89(d,J=10.7Hz,9H),0.84(s,3H),0.74(s,3H).

[0056] Example 5: 3β-O-(2,5-dihydroxycinnamoyl)oleanolic-12-en-28-acid (A1-5)

[0057] The difference from Example 1 is that the raw material 3,5-dimethoxybenzaldehyde in Example 1 was replaced with 2,5-dimethoxybenzaldehyde (2.00 g, 14.69 mmol) to prepare 2,5-dimethoxyphenylacrylic acid (2.08 g, 9.99 mmol) according to the above steps, with a yield of 83.00%. After demethylation with BBr3, 2,5-dihydroxycinnamic acid (1.48 g, 8.21 mmol) was obtained with a yield of 85.52%. The hydroxyl group was then protected with TBDMSCl to obtain 2,5-di-tert-butyldimethylcinnamic acid (2.34 g, 5.73 mmol) with a yield of 85.96%. 2.00 g of 2,5-di-tert-butyldimethylcinnamic acid was esterified with compound 2 (0.70 g, 1.22 mmol), and finally deprotected with TBAF to obtain 150 mg of white solid A1-5 with a yield of 19.88%.

[0058] ESI-MS m / z: 617.1 [MH] - .

[0059] 1H-NMR (600MHz, DMSO-d6) δ: 12.03 (s, 1H), 9.50 (s, 1H), 8.87 (s, 1H), 7.79 (d, J = 16.1Hz, 1H), 6.91 (d, J = 2.7Hz, 1H), 6.73 (d, J = 8.7Hz, 1H), 6.69 (dd, J = 8.9,2.6Hz,1H),6.42(d,J=16.1Hz,1H),5.17(d,J=4.1Hz,1H),4.52(dd,J= 11.9,4.2Hz,1H),2.75(dd,J=14.0,4.5Hz,1H),1.92(td,J=13.5,3.9Hz,1H ),1.86–1.81(m,2H),1.68(d,J=12.9Hz,2H),1.63(d,J=13.9Hz,2H),1.58 (t,J=9.2Hz,4H),1.49(d,J=10.6Hz,2H),1.44(d,J=14.2Hz,2H),1.40(s,3 H),1.26(d,J=11.8Hz,1H),1.13(s,3H),1.07(d,J=12.7Hz,2H),1.01(d,J= 13.4Hz,1H),0.93(s,3H),0.89(d,J=8.1Hz,9H),0.85(s,3H),0.74(s,3H).

[0060] Example 6: 3β-O-(3,4-dihydroxycinnamoyl)oleanolic-12-en-28-acid (A1-6)

[0061] The difference from Example 1 is that the raw material 3,5-dimethoxybenzaldehyde in Example 1 was replaced with 3,4-dimethoxybenzaldehyde (2.00 g, 14.69 mmol) to prepare 3,4-dimethoxyphenylacrylic acid (2.00 g, 9.61 mmol) according to the above steps, with a yield of 79.81%. After demethylation with BBr3, 3,4-dihydroxycinnamic acid (1.43 g, 7.94 mmol) was obtained with a yield of 82.63%. The hydroxyl group was then protected with TBDMSCl to obtain 3,4-di-tert-butyldimethylcinnamic acid (2.33 g, 5.70 mmol) with a yield of 85.59%. 2.00 g of 3,4-di-tert-butyldimethylcinnamic acid was esterified with compound 2 (0.70 g, 1.22 mmol), and finally deprotected with TBAF to obtain 160 mg of white solid A1-6 with a yield of 21.21%.

[0062] ESI-MS m / z: 616.9 [MH] - .

[0063] 1H-NMR(600MHz,DMSO-d6)δ:12.02(s,1H),9.57(s,1H),9.10(s,1H),7.46(d d,J=20.8,15.8Hz,1H),7.04(d,J=2.1Hz,1H),6.99(dd,J=8.2,2.1Hz,1H),6 .75(d,J=8.1Hz,1H),6.24(d,J=15.9Hz,1H),5.20–5.15(m,1H),4.51(dd,J= 11.7,4.3Hz,1H),2.75(dd,J=13.8,4.6Hz,1H),1.92(td,J=13.4,3.9Hz,1H) ,1.83(dd,J=9.1,3.6Hz,2H),1.67(d,J=10.2Hz,2H),1.65–1.61(m,2H),1. 61–1.54(m,4H),1.49(d,J=11.8Hz,2H),1.47–1.42(m,2H),1.41–1.29(m,2H ),1.25(dd,J=14.1,5.1Hz,1H),1.12(s,3H),1.06(dd,J=13.2,4.1Hz,2H),1 .04–0.99(m,1H),0.92(s,3H),0.91–0.86(m,9H),0.84(s,3H),0.74(s,3H).

[0064] Example 7: 3β-O-(2-methoxycinnamoyl)oleanolic-12-en-28-acid (A2-1)

[0065]

[0066] The raw materials 2-methoxybenzaldehyde (15) (2.00 g, 14.7 mmol) and malonic acid (3.06 g, 29.38 mmol) were dissolved in 15 mL of pyridine, and 3 mL of piperidine was added dropwise. The mixture was heated to 110 °C and refluxed for 3 h. After cooling to room temperature, 50 mL of water was added, and the pH was adjusted to 9-10 with saturated potassium carbonate solution. The mixture was repeatedly extracted with toluene (10 mL × 5) until the toluene layer was colorless. The pH of the aqueous layer was adjusted to 3-4 with dilute hydrochloric acid, and a large amount of solid precipitated out. The solid was filtered and dried to obtain 2.35 g of white solid compound (16), with a yield of 89.78%.

[0067] The white solid compound (16) obtained in the previous step (1.80 g, 10.10 mmol) was dissolved in 30 mL of dichloromethane. DCC (2.61 g, 12.63 mmol) and DMAP (0.93 g, 7.58 mmol) were weighed and dissolved in dichloromethane. The dichloromethane solutions of DCC and DMAP were added dropwise at 0 °C. After the addition was complete, the reaction was allowed to proceed at room temperature for 1 h. Compound 2 (1.44 g, 2.53 mmol) was then added, and the reaction was allowed to proceed at room temperature for 7 h. After the reaction was complete, the mixture was filtered, concentrated under reduced pressure, and the resulting pale yellow oily liquid was dissolved in hot n-hexane. A white insoluble substance slowly precipitated out. The mixture was cooled overnight at a low temperature, filtered again, concentrated under reduced pressure, and subjected to column chromatography (petroleum ether: ethyl acetate = 50:1, v / v) to give 1.2 g of white solid. Dissolved in 15 mL tetrahydrofuran, 0.1 mL acetic acid was added and stirred until homogeneous. A tetrahydrofuran solution of TBAF (2 mL, 1 mol / L) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 3 h. The reaction was then stopped, and 20 mL of 5% dilute hydrochloric acid was slowly added. The mixture was extracted with ethyl acetate (20 mL × 3 times). The combined organic layers were washed successively with 5% dilute hydrochloric acid, water, and saturated brine. The mixture was dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated to obtain 450 mg of a pale yellow oily crude product. Column chromatography (petroleum ether:ethyl acetate = 10:1, v / v) yielded 300 mg of a white solid A2-1, with a yield of 19.22%.

[0068] ESI-MS m / z: 615.0 [MH] - .

[0069] 1H-NMR (600MHz, DMSO-d6) δ: 12.04 (s, 1H), 7.89 (d, J = 16.1 Hz, 1H), 7.69 (d, J = 7. 7Hz,1H),7.41(t,J=7.9Hz,1H),7.09(d,J=8.4Hz,1H),6.98(t,J=7.5Hz,1H),6. 57(d,J=16.1Hz,1H),5.17(d,J=3.6Hz,1H),4.54(dd,J=11.6,4.5Hz,1H),3.86 (s,3H),2.75(dd,J=13.7,4.6Hz,1H),1.91(dd,J=10.4,6.8Hz,1H),1.84–1.79( m,2H),1.67(d,J=12.4Hz,1H),1.65–1.61(m,2H),1.59(d,J=13.1Hz,2H),1.57 (d,J=12.6Hz,2H),1.49(d,J=12.5Hz,2H),1.46–1.41(m,2H),1.34(ddd,J=22.8 ,18.4,10.8Hz,2H),1.24(d,J=12.6Hz,2H),1.11(s,4H),1.09–1.04(m,2H),1. 02–0.97(m,1H),0.91(s,3H),0.89(d,J=11.2Hz,9H),0.85(s,3H),0.73(s,3H).

[0070] Example 8: 3β-O-(4-methoxycinnamoyl)oleanolic-12-en-28-acid (A2-2)

[0071] The difference from Example 7 is that 2-methoxybenzaldehyde in Example 7 was replaced with 4-methoxybenzaldehyde (2.00 g, 14.7 mmol), and 4-methoxyphenylacrylic acid (2.28 g, 12.80 mmol) was prepared according to the description in Example 7, with a yield of 87.11%. 1.8 g of 4-methoxyphenylacrylic acid was esterified with compound 2 (1.44 g, 2.53 mmol), and finally deprotected with TBAF to give 250 mg of white solid A2-2, with a yield of 16.02%.

[0072] ESI-MS m / z: 615.0 [MH] -

[0073] 1H-NMR (600MHz, DMSO-d6) δ: 12.00 (s, 1H), 7.57 (dd, J = 20.8, 16.0Hz, 1H), 6.94 (dd, J = 8.5, 5.5Hz, 2H), 6.44 (dd, J = 20.1, 16.0Hz, 1H), 5.14 (t ,J=3.7Hz,1H),4.50(dd,J=11.8,4.2Hz,1H),3.77(s,3H),2.72(dd,J=13.8,4.7Hz,1H),1.89(d,J=4.1Hz,1H),1.80(dd,J=9.1,3.6Hz,2H), 1.64(d,J=16.6Hz,2H),1.60(d,J=13.7Hz,2H),1.58–1.51(m,3H),1.46(q,J=7.6,6.3Hz,2H),1.41(dt,J=13.3,3.7Hz,2H),1.30(s,2H),1. 26–1.19(m,2H),1.09(s,3H),1.03(d,J=13.1Hz,2H),0.97(d,J=13.7Hz,2H),0.89(s,3H),0.86(d,J=14.6Hz,9H),0.82(s,3H),0.71(s,3H).

[0074] Example 9: 3β-O-(2,4-methoxycinnamoyl)oleanolic-12-en-28-acid (A2-3)

[0075] The difference from Example 7 is that 2-methoxybenzaldehyde in Example 7 was replaced with 2,4-dimethoxybenzaldehyde (2.00 g, 12.04 mmol), and 2,4-methoxyphenylacrylic acid (2.14 g, 10.28 mmol) was prepared according to the description in Example 7, with a yield of 85.40%. 2.1 g of 2,4-methoxyphenylacrylic acid was esterified with compound 2 (1.44 g, 2.53 mmol), and finally deprotected with TBAF to give 260 mg of white solid A2-3, with a yield of 15.89%.

[0076] ESI-MS m / z: 645.0 [MH] -

[0077] 1H-NMR (600MHz, DMSO-d6) δ: 12.03 (s, 1H), 7.80 (d, J=15.9Hz, 1H), 7.65 (d, J=8.7Hz, 1H), 6.62 (d, J=2.5Hz, 1H), 6.57 (dd, J=8.6, 2.5Hz, 1H), 6.44 (dd, J= 16.1,2.2Hz,1H),5.17(d,J=3.8Hz,1H),4.52(dd,J=11.8,4.2Hz,1H),3.86 (d,J=2.2Hz,3H),3.81(d,J=2.2Hz,3H),2.75(dd,J=14.3,4.5Hz,1H),1.92( td,J=13.5,4.0Hz,1H),1.86–1.80(m,2H),1.69–1.65(m,2H),1.63(d,J=13 .3Hz,2H),1.60–1.55(m,3H),1.52–1.47(m,2H),1.46–1.42(m,2H),1.37(d, J=27.4Hz,2H),1.32–1.23(m,2H),1.12(s,4H),1.09–1.03(m,2H),1.00(d,J =13.5Hz,1H),0.92(s,3H),0.88(d,J=6.3Hz,9H),0.84(s,3H),0.74(s,3H).

[0078] Example 10: 3β-O-(2,5-methoxycinnamoyl)oleanolic-12-en-28-acid (A2-4)

[0079] The difference from Example 7 is that 2-methoxybenzaldehyde in Example 7 was replaced with 2,5-dimethoxybenzaldehyde (2.00 g, 12.04 mmol), and 2,5-methoxyphenylacrylic acid (2.28 g, 10.95 mmol) was prepared according to the description in Example 7, with a yield of 90.98%. 2.1 g of 2,5-methoxyphenylacrylic acid was esterified with compound 2 (1.44 g, 2.53 mmol), and finally deprotected with TBAF to give 190 mg of white solid A2-4, with a yield of 11.61%.

[0080] ESI-MS m / z: 645.1 [MH] - .

[0081] 1H-NMR (600MHz, DMSO-d6) δ: 12.03 (s, 1H), 7.87 (d, J = 16.1Hz, 1H), 7.29 (d, J = 3.0Hz, 1H), 7.02 (d, J = 8.9Hz, 1H), 6.99 (dd, J = 9.1, 2.9Hz, 1H), 6.64 (d, J=16.1Hz,1H),5.17(d,J=3.4Hz,1H),4.54(dd,J=11.8,4.1Hz,1H),3.81( s,3H),3.75(s,3H),2.75(dd,J=13.9,4.5Hz,1H),1.92(td,J=13.6,3.8Hz, 1H),1.83(dd,J=8.8,3.6Hz,2H),1.70–1.65(m,2H),1.63(d,J=13.6Hz,2H ),1.59(t,J=8.6Hz,4H),1.50(s,2H),1.44(d,J=15.1Hz,2H),1.40–1.30(m ,3H),1.26(d,J=12.1Hz,1H),1.12(s,3H),1.09–1.04(m,2H),1.01(d,J=1 3.4Hz,1H),0.93(s,3H),0.89(d,J=13.2Hz,9H),0.85(s,3H),0.74(s,3H).

[0082] Example 11: 3β-O-(2,3-methoxycinnamoyl)oleanolic-12-en-28-acid (A2-5)

[0083] The difference from Example 7 is that 2-methoxybenzaldehyde in Example 7 was replaced with 2,3-dimethoxybenzaldehyde (2.00 g, 12.04 mmol), and 2,3-methoxyphenylacrylic acid (2.09 g, 10.04 mmol) was prepared according to the description in Example 7, with a yield of 83.40%. 2.1 g of 2,3-methoxyphenylacrylic acid was esterified with compound 2 (1.44 g, 2.53 mmol), and finally deprotected with TBAF to give 210 mg of white solid A2-5, with a yield of 12.83%.

[0084] ESI-MS m / z: 645.1 [MH] - .

[0085] 1H-NMR(600MHz,DMSO-d6)δ12.04(s,1H),7.86(d,J=16.5Hz,1H),7.37(s,1H),7.12(s,2H),6.5 8(d,J=16.2Hz,1H),5.17(s,1H),4.53(s,1H),3.82(s,3H),3.76(s,3H),2.75(s,1H),1.92(td, J=13.6,3.8Hz,1H),1.83(dd,J=8.8,3.6Hz,2H),1.60(m,7H),1.49(m,4H),1.34(m,2H),1.24(m ,2H),1.13(s,4H),1.07(m,3H),0.93(s,3H),0.89(d,J=13.2Hz,9H),0.86(s,3H),0.74(s,3H).

[0086] Example 12: 3β-O-(3-hydroxybenzoyl)oleanolic-12-en-28-acid (B1-1)

[0087]

[0088] The starting material 3-methoxybenzoic acid (17) (2.00 g, 13.15 mmol) was dissolved in 40 mL of dichloromethane. Under N2 atmosphere, a 2 mol / L boron tribromide (2.53 mL, 26.29 mmol) solution in dichloromethane was added dropwise to a three-necked flask, and the reaction was carried out at room temperature for 24 h. The reaction solution was slowly quenched in ice water, extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by vacuum distillation to give 1.55 g of a pale yellow solid compound (18), with a yield of 85.37%.

[0089] The pale yellow solid compound (18) (2.00 g, 14.48 mmol) obtained in the previous step was dissolved in 50 mL of dichloromethane. Then, tert-butyldimethylchlorosilane (4.36 g, 28.96 mmol), triethylamine (5.03 mL, 36.20 mmol), and DMAP (0.01 g, 0.1 mmol) were added sequentially, and the mixture was refluxed at 42 °C for 24 h. After cooling to room temperature, the mixture was concentrated under reduced pressure to obtain a large amount of solid. Petroleum ether was added and stirred to dissolve the solid. The mixture was filtered, and the filter cake was repeatedly washed with petroleum ether. The filtrate was collected and concentrated under reduced pressure to obtain an oily primary product, which was dissolved in 20 mL of methanol. Weigh 2.0 g of potassium carbonate, dissolve it in an equal volume of water, mix and stir at room temperature for 1 h, quench with 3% w / w hydrochloric acid solution, extract with ethyl acetate (20 mL × 3), wash the organic phase with saturated brine, dry with anhydrous magnesium sulfate, filter, evaporate the solvent, and perform column chromatography (petroleum ether: ethyl acetate = 30:1, v / v) to give 2.52 g of white solid compound (19), yield 68.96%.

[0090] The white solid compound (19) obtained in the previous step (1.80 g, 7.14 mmol) was dissolved in 30 mL of dichloromethane. DCC (1.84 g, 8.92 mmol) and DMAP (0.65 g, 5.35 mmol) were weighed and dissolved in dichloromethane. The solutions were added dropwise at 0 °C, and the reaction was carried out at room temperature for 1 h. Compound 2 (1.02 g, 1.78 mmol) was added, and the reaction was carried out at room temperature for 7 h. After the reaction was complete, the mixture was filtered, concentrated under reduced pressure, and the resulting pale yellow oily liquid was dissolved in hot n-hexane. The solution was cooled overnight at a low temperature, filtered, concentrated under reduced pressure, and subjected to column chromatography (petroleum ether: ethyl acetate = 50:1, v / v) to give 900 mg of white solid. Dissolved in 15 mL tetrahydrofuran, 0.1 mL acetic acid was added and stirred until homogeneous. A tetrahydrofuran solution of 1.5 mL TBAF (1 mol / L) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 3 hours. The reaction was then stopped, and 20 mL of 5% dilute hydrochloric acid was slowly added. The mixture was extracted with ethyl acetate (20 mL × 3 times), and the organic layers were combined. The extract was washed successively with 5% dilute hydrochloric acid, water, and saturated brine. The mixture was dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated to obtain 450 mg of a pale yellow oily crude product. Column chromatography (petroleum ether:ethyl acetate = 6:1, v / v) yielded 200 mg of a white solid B1-1, with a yield of 19.48%.

[0091] ESI-MS m / z: 575.0 [MH] - .

[0092] 1H-NMR(600MHz,DMSO-d6)δ:12.02(s,1H),9.82(s,1H),7.38(d,J=7.8Hz,1H ),7.36(d,J=2.3Hz,1H),7.31(t,J=7.8Hz,1H),7.02(dd,J=8.0,2.5Hz,1H), 5.18(d,J=3.6Hz,1H),4.61(dd,J=11.5,4.8Hz,1H),2.76(dd,J=14.0,4.5Hz ,1H),1.92(dt,J=12.3,6.3Hz,1H),1.84(dd,J=9.1,3.6Hz,2H),1.73–1.68( m,1H),1.66(dd,J=9.4,5.7Hz,2H),1.62(d,J=4.4Hz,2H),1.59(d,J=9.4Hz ,2H),1.52(d,J=15.7Hz,2H),1.49–1.44(m,2H),1.44–1.37(m,2H),1.37–1. 29(m,1H),1.27(d,J=12.1Hz,1H),1.13(s,4H),1.10–1.04(m,2H),1.01(d,J =14.2Hz,1H),0.97(s,3H),0.95(s,3H),0.88(d,J=4.1Hz,9H),0.75(s,3H).

[0093] Example 13: 3β-O-(3,4-dihydroxybenzoyl)oleanolic-12-en-28-acid (B1-2)

[0094] The difference from Example 12 is that 3-methoxybenzoic acid in Example 12 was replaced with 3,4-dimethoxybenzoic acid (2.00 g, 10.98 mmol) to prepare 3,4-dihydroxybenzoic acid (1.49 g, 9.67 mmol) according to the method described in Example 7, with a yield of 88.06%; the hydroxyl group was protected by TBDMSCl to obtain 3,4-di-tert-butyldimethylbenzoic acid (1.89 g, 4.94 mmol), with a yield of 76.12%; 2.72 g of 3,4-di-tert-butyldimethylbenzoic acid was esterified with compound 2 (1.02 g, 1.78 mmol), and finally deprotected by TBAF to obtain 190 mg of white solid B1-2, with a yield of 18.01%.

[0095] ESI-MS m / z: 591.0 [MH] - .

[0096] 1H-NMR (600MHz, DMSO-d6) δ: 12.04 (s, 1H), 9.74 (s, 1H), 9.35 (s, 1H), 7.37 (s, 1H), 7.31 (d, J = 8.3Hz, 1H), 6.81 (d, J = 8.3Hz, 1H),5.19(d,J=3.9Hz,1H),4.55(dd,J=11.5,4.9Hz,1H),2.76(dd,J=13.9,4.8Hz,1H),1.98–1.90(m,1H),1.87–1.82(m,2 H),1.71–1.64(m,2H),1.64–1.57(m,5H),1.52(d,J=13.6Hz,2H),1.48(s,2H),1.41–1.31(m,2H),1.27(d,J=11.9Hz,2H), 1.13(s,4H),1.11–1.04(m,2H),1.02(d,J=13.4Hz,1H),0.97(s,3H),0.95(s,2H),0.89(s,7H),0.87(s,3H),0.75(s,3H).

[0097] Example 14: 3β-O-(3,5-dihydroxybenzoyl)oleanolic-12-en-28-acid (B1-3)

[0098] The difference from Example 12 is that 3-methoxybenzoic acid in Example 12 was replaced with 3,5-dimethoxybenzoic acid (2.00 g, 10.98 mmol) to prepare 3,5-dihydroxybenzoic acid (1.45 g, 9.41 mmol) according to the description in Example 7, with a yield of 85.70%; hydroxyl groups were protected with TBDMSCl to obtain 3,5-di-tert-butyldimethylbenzoic acid (1.84 g, 4.81 mmol), with a yield of 74.11%; 2.72 g of 3,4-di-tert-butyldimethylbenzoic acid was esterified with compound 2, and finally deprotected with TBAF to obtain 160 mg of white solid B1-3, with a yield of 15.16%.

[0099] ESI-MS m / z: 591.0 [MH] - .

[0100] 1H-NMR (600MHz, DMSO-d6) δ: 12.03 (s, 1H), 9.59 (s, 2H), 6.83 (d, J = 2.3Hz, 2H), 6.43 (t, J = 2.3Hz, 1H), 5.19 (d, J = 3.6Hz, 1H) ,4.57(dd,J=11.2,5.0Hz,1H),2.76(dd,J=13.9,4.6Hz,1H),1.93(d,J=4.0Hz,1H),1.84(dd,J=9.1,3.6Hz,2H),1.73–1.66 (m,2H),1.66–1.62(m,3H),1.61–1.56(m,2H),1.50(d,J=12.4Hz,2H),1.48–1.43(m,2H),1.33(s,2H),1.27(d,J=12.6Hz, 2H),1.13(s,4H),1.10–1.04(m,2H),1.01(d,J=13.6Hz,1H),0.97(s,3H),0.94(s,3H),0.88(d,J=8.8Hz,9H),0.75(s,3H).

[0101] Example 15: 3β-O-(3,4-dimethoxybenzoyl)oleanolic-12-en-28-acid (B2-1)

[0102]

[0103] 3,4-Dimethoxybenzoic acid (1.00 g, 5.49 mmol) was dissolved in 30 mL of dichloromethane. DCC (1.42 g, 6.86 mmol) and DMAP (0.5 g, 4.12 mmol) were weighed and dissolved in dichloromethane. These solutions were added dropwise to the reaction mixture at 0 °C and reacted at room temperature for 1 h. Compound 2 (0.78 g, 1.37 mmol) was then added, and the reaction was continued at room temperature for 7 h. After the reaction was complete, the mixture was filtered, concentrated under reduced pressure, and the resulting pale yellow oily liquid was dissolved in hot n-hexane. The solution was cooled overnight at a low temperature, filtered again, concentrated under reduced pressure, and subjected to column chromatography (petroleum ether:ethyl acetate = 50:1, v / v) to give 500 mg of a white solid. Dissolved in 15 mL tetrahydrofuran, 0.1 mL acetic acid was added and stirred until homogeneous. A tetrahydrofuran solution of 1 mL TBAF (1 mol / L) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 3 hours. The reaction was then stopped, and 20 mL of 5% dilute hydrochloric acid was slowly added. The mixture was extracted with ethyl acetate (20 mL × 3 times), and the organic layers were combined. The extract was washed successively with 5% dilute hydrochloric acid, water, and saturated brine. The mixture was dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated to obtain 300 mg of a pale yellow oily crude product. Column chromatography (petroleum ether:ethyl acetate = 10:1, v / v) yielded 120 mg of a white solid B2-1, with a yield of 14.12%.

[0104] ESI-MS m / z: 619.0 [MH] - .

[0105] 1 H-NMR(600MHz,DMSO-d6)δ:12.03(s,1H),7.59(d,J=8.5Hz,1H),7.44(s,1H),7.09(d,J=8.3Hz,1H),5.18(s, 1H),4.61(d,J=11.3Hz,1H),3.84(s,3H),3.81(s,3H),2.80–2.73(m,1H),2.00(s,1H),1.93(d,J=12.1Hz,2H) ,1.85(s,2H),1.66(s,2H),1.64–1.60(m,4H),1.51(s,2H),1.46(s,2H),1.24(s,3H),1.19(d,J=7.2Hz,1H),1 .14(s,4H),1.09(d,J=9.7Hz,2H),1.03(s,1H),0.98(s,3H),0.96(s,3H),0.89(d,J=6.9Hz,9H),0.76(s,3H).

[0106] Example 16: 3β-O-(4-methoxybenzoyl)oleanolic-12-en-28-acid (B2-2)

[0107] The difference from Example 15 is that 3,4-dimethoxybenzoic acid in Example 15 was replaced with 4-methoxybenzoic acid (0.83 g, 5.48 mmol). The ester condensation of 4-methoxybenzoic acid with compound 2 (0.78 g, 1.37 mmol) was carried out as described in Example 15, and then deprotected by TBAF to give 120 mg of white solid B2-2, with a yield of 14.84%.

[0108] ESI-MS m / z: 589.0 [MH] - .

[0109] 1H-NMR(600MHz,DMSO-d6)δ:12.03(s,1H),7.90(d,J=8.3Hz,2H),7.05(d,J=8.3Hz,2H),5.18(s,1H),4.65–4.56(m,1 H),3.83(s,3H),2.75(d,J=13.7Hz,1H),1.91(d,J=13.8Hz,1H),1.84(d,J=8.7Hz,2H),1.70(d,J=14.9Hz,1H),1.65( d,J=7.8Hz,2H),1.61(d,J=17.7Hz,4H),1.50(s,2H),1.46(d,J=13.2Hz,2H),1.42–1.26(m,3H),1.24(d,J=11.0Hz, 1H),1.13(s,4H),1.07(d,J=12.9Hz,2H),1.02(s,1H),0.97(s,3H),0.94(s,3H),0.87(d,J=8.9Hz,9H),0.74(s,3H).

[0110] Example 17: 3β-O-(2,3-dimethoxybenzoyl)oleanolic-12-en-28-acid (B2-3)

[0111] The difference from Example 15 is that 3,4-dimethoxybenzoic acid in Example 15 was replaced with 2,3-dimethoxybenzoic acid (1.00 g, 5.48 mmol). The ester condensation of 2,3-dimethoxybenzoic acid with compound 2 (0.78 g, 1.37 mmol) was carried out as described in Example 15, and then deprotected by TBAF to obtain 150 mg of white solid B2-3, with a yield of 17.66%.

[0112] ESI-MS m / z: 619.0 [MH] - .

[0113] 1H-NMR (600MHz, DMSO-d6)δ:12.04(s,1H),7.28–7.22(m,1H),7.15(d,J=5.1Hz,2H),5.19(s,1H),4.67(dd,J=11.4,4.9H z,1H),3.84(s,3H),3.76(s,3H),2.76(d,J=13.6Hz,1H),1.92(d,J=13.7Hz,1H),1.85(d,J=8.6Hz,2H),1.72(d,J=13.8H z,1H),1.70–1.64(m,3H),1.64–1.58(m,3H),1.51(s,2H),1.47(d,J=13.7Hz,2H),1.42–1.30(m,2H),1.30–1.23(m,2H) ,1.14(s,4H),1.08(d,J=13.0Hz,2H),1.02(d,J=13.0Hz,1H),0.94(s,3H),0.92(s,3H),0.91–0.86(m,9H),0.75(s,3H).

[0114] Example 18: 3β-O-(3,5-dimethoxybenzoyl)oleanolic-12-en-28-acid (B2-4)

[0115] The difference from Example 15 is that 3,4-dimethoxybenzoic acid in Example 15 was replaced with 3,5-dimethoxybenzoic acid (1.00 g, 5.48 mmol). It was esterified with compound 2 (0.78 g, 1.37 mmol) as described in Example 15, and finally deprotected by TBAF to obtain 160 mg of white solid B2-4, with a yield of 18.84%.

[0116] ESI-MS m / z: 619.0 [MH] - .

[0117] 1H-NMR(600MHz,DMSO-d6)δ:12.04(s,1H),7.05(s,2H),6.79(s,1H),5.18(s,1H),4.63(d,J=11.3Hz,1H),3.84– 3.75(m,6H),2.76(d,J=13.6Hz,1H),2.02–1.97(m,1H),1.84(d,J=8.4Hz,2H),1.73(d,J=11.8Hz,1H),1.64(s, 2H),1.63–1.57(m,4H),1.50(s,2H),1.46(s,2H),1.36(d,J=41.6Hz,2H),1.26(d,J=12.9Hz,2H),1.18(d,J=3. 9Hz,1H),1.15–1.10(m,4H),1.08(s,2H),1.00–0.96(m,3H),0.95–0.93(m,3H),0.91–0.86(m,9H),0.75(s,3H).

[0118] Example 19: 3β-O-(3-methoxybenzoyl)oleanolic-12-en-28-acid (B2-5)

[0119] The difference from Example 15 is that 3,4-dimethoxybenzoic acid in Example 15 was replaced with 3-methoxybenzoic acid (0.83 g, 5.48 mmol). It was esterified with compound 2 (0.78 g, 1.37 mmol) as described in Example 15, and then deprotected by TBAF to give 160 mg of white solid B2-5, with a yield of 19.78%.

[0120] ESI-MS m / z: 589.1 [MH] - .

[0121] 1H-NMR(600MHz,DMSO-d6)δ:12.03(s,1H),7.54(d,J=7.6Hz,1H),7.44(d,J=7.1Hz,2H),7.23(d,J=8.2Hz,1H),5.18(s,1H),4 .64(dd,J=11.6,4.8Hz,1H),3.81(s,3H),2.79–2.70(m,1H),1.92(t,J=13.6Hz,1H),1.84(d,J=8.8Hz,2H),1.73(d,J=12.7H z,1H),1.65(d,J=14.2Hz,2H),1.60(t,J=9.7Hz,4H),1.51(d,J=13.6Hz,2H),1.44(d,J=12.2Hz,2H),1.41–1.34(m,2H),1.2 9(dd,J=31.6,12.4Hz,2H),1.13(s,4H),1.10–1.05(m,2H),1.02(s,1H),0.97(s,3H),0.94(s,3H),0.88(s,9H),0.75(s,3H).

[0122] Example 20: 3β-O-(2,4-dimethoxybenzoyl)oleanolic-12-en-28-acid (B2-6)

[0123] The difference from Example 15 is that 3,4-dimethoxybenzoic acid in Example 15 was replaced with 2,4-dimethoxybenzoic acid (1.00 g, 5.48 mmol). The ester condensation of 2,4-dimethoxybenzoic acid with compound 2 (0.78 g, 1.37 mmol) was carried out as described in Example 15, and then deprotected by TBAF to obtain 180 mg of white solid B2-6, with a yield of 21.19%.

[0124] ESI-MS m / z: 619.0 [MH] - .

[0125] 1H-NMR(600MHz,DMSO-d6)δ:12.03(s,1H),7.68(d,J=8.6Hz,1H),6.62(d,J=2.2Hz,1H),6.58(dd,J=8.8,2.3Hz,1H),5.18(d,J=3.6Hz,1H) ,4.54(dd,J=10.0,6.2Hz,1H),3.82(s,4H),3.80(s,3H),2.75(dd,J=14.0,4.6Hz,1H),1.92(d,J=4.1Hz,1H),1.83(dd,J=9.0,3.6Hz,2H) ,1.70–1.65(m,1H),1.63(d,J=12.4Hz,3H),1.58(dd,J=15.5,5.8Hz,3H),1.51(d,J=13.9Hz,2H),1.48–1.43(m,2H),1.42–1.32(m,2H),1 .31–1.24(m,2H),1.13(s,4H),1.07(dt,J=12.6,5.3Hz,2H),1.03–0.98(m,1H),0.92(d,J=3.6Hz,6H),0.89(d,J=8.9Hz,9H),0.74(s,3H).

[0126] The experimental method for testing the drug activity of the compounds in this invention is as follows.

[0127] Preheat 2 mL of 0.25% trypsin solution in a 37°C water bath. Select Hep G2 and Hep 3B cells cultured to the logarithmic growth phase using standard methods, remove their culture medium, add digestion solution to cover the cells, let stand, and observe under a microscope until the intercellular spaces are clearly visible. Then add the previously removed culture medium to stop digestion. Gently pipette the cells until they detach, forming a cell suspension. Transfer the suspension to a centrifuge tube, centrifuge, remove the supernatant, resuspend the cells in a medium containing approximately 10 mL of serum (high glucose DMEM + 10% fetal bovine serum + a mixture of 1% penicillin and streptomycin), and seed them into cell culture dishes. Incubate at 37°C in a 5% CO2 incubator, changing the culture medium regularly.

[0128] Hep G2 and Hep 3B cells in the logarithmic growth phase were digested and diluted to a concentration of 0.5 × 10⁻⁶. 4Cell suspension at concentrations of cells / mL was seeded into 96-well plates at 100 μL per well and incubated at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was aspirated, and 100 μL of serum-free blank medium was added as a control group. The experimental groups were treated with either a 10 μM solution of the test compound diluted with culture medium, a 10 μM positive control OA or oxaliplatin solution, or a mixture of both (the mixture consisted of the test compound solution and oxaliplatin solution at a 1:1 volume ratio). Each group was divided into three replicates, and incubation continued for 24 h. Then, under dark conditions, 15 μL of MTT at a final concentration of 0.5 mg / mL was added to each well, and incubation continued for 4 h. After incubation, the supernatant was aspirated, and 150 μL of DMSO was added to each well. The plates were shaken for 5 min until the blue-purple crystals were completely dissolved. The OD value was measured at 490 nm using a microplate reader, and the average value was recorded. Cell viability was then calculated using formula (1).

[0129] Calculate cell viability using the following formula:

[0130]

[0131] The results are shown in Table 1-2.

[0132] Table 1. Inhibitory activity of OA derivatives on the proliferation of Hep G2 cell line

[0133]

[0134] Table 2. Inhibitory activity of OA derivatives on the proliferation of Hep 3B cells.

[0135]

[0136]

[0137] As shown in Tables 1 and 2 above, some of the synthesized compounds exhibit significant inhibitory activity against Hep G2 and Hep 3B cells. Furthermore, the inhibitory activity was significantly enhanced when used in combination with oxaliplatin compared to oxaliplatin alone.

[0138] When R is 3-hydroxycinnamoyl, compound A1-3 exhibits good inhibitory activity against Hep 3B cells, with a cell survival rate of 0.3942%. When combined with oxaliplatin, it achieves complete inhibition of the Hep 3B cell line.

Claims

1. An oleanolic acid derivative, characterized in that, Oleanolic acid derivatives are 。 2. An application of the oleanolic acid derivative as described in claim 1, characterized in that: The application of the oleanolic acid derivatives as described in claim 1 in the preparation of antitumor drugs.

3. The application of the oleanolic acid derivatives according to claim 2, characterized in that, The application of the oleanolic acid derivatives of claim 1 in combination with antitumor drugs in the preparation of antitumor drugs.

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