Application of pentacyclic triterpene compound

Abstract

The invention relates to the field of protein degradation, and relates to application of a pentacyclic triterpenoid compound. The invention discloses application of a pentacyclic triterpenoid compound as an E3 ligase ligand or combined oncogenic protein. The pentacyclic triterpenoid compound provided by the invention has an obvious effect of binding E3 ligase, and part of the compound has obvious binding specificity.

Description

Technical Field

[0001] The present invention relates to the field of protein degradation and relates to the application of a pentacyclic triterpene compound. Background Art

[0002] Cancer is the disease with the highest mortality rate worldwide, after cardiovascular disease, and the second leading cause of death worldwide. Besides early surgical resection and radiotherapy, chemotherapy remains the mainstay of cancer treatment. Chemotherapeutic drugs, targeted therapies, and their combination are currently the mainstays of treatment. With the advancement of translational medicine, targeted therapies have become a research hotspot in cancer treatment, with their reduced toxicity and side effects being one of their advantages in cancer treatment. Targeted therapies are mostly cell surface and intracellular kinase inhibitors, such as imatinib (EGFR inhibitor), vorinostat (HDAC inhibitor), bevacizumab (VEGF inhibitor), etoposide (topoisomerase II inhibitor), and gilteritinib (FLT3 inhibitor). These drugs exert their anti-tumor activity by targeting specific kinases, inhibiting tumor cell growth and inducing apoptosis. However, after a short period of efficacy, existing targeted drugs inevitably develop acquired resistance. Therefore, identifying new targets and therapeutic strategies to overcome resistance and reduce toxicity and side effects remains a key challenge in cancer treatment.

[0003] Target protein degradation has become an effective method for targeting undruggable pathogenic proteins. Using the heterobifunctional protein degradation technology of targeted chimeras (PROTACs), target protease inhibitors have been successfully converted into target protein degradation compounds, such as EZH2, MEK, HDAC6, and RTK protein degradation compounds, all of which can overcome acquired resistance to kinase inhibitors and improve tumor treatment efficacy. Currently, the ER PROTAC drug ARV-471 is being used clinically to treat patients with locally advanced or metastatic breast cancer who are estrogen receptor-positive (ER+). In addition to tumor treatment, PROTACs can also be applied in other fields. For example, the small molecule degrader DGY-08-097 for hepatitis C virus protease can overcome viral mutations that are resistant to traditional enzyme inhibitors. PROTACs are designed to synthesize E3 ligase ligand compounds, allowing them to bind to the corresponding E3 ligase, bringing them into proximity with the target protein, inducing ubiquitination and degradation of the protein by the endogenous 26S protease. Its degradation activity depends on the relevant E3 ligase. There are over 600 known E3 ligases in the human body, which can be divided into three categories: RING, HECT, and RBR. However, to date, less than 1% of these E3 ligases have small molecule ligands. To date, only four E3 ligases have been used in PROTAC development: VHL, CRBN, MDM2, and cIAP1. Most PROTACs use CRBN or VHL as their E3 ligase ligands. Expanding the repertoire of E3 ubiquitin ligases and ligands to target pathogenic proteins that these E3 ligases are unable to degrade is a major challenge facing PROTACs. Increasing evidence indicates that E3 ligase expression is cell-, tissue-, and species-specific. Discovering more specific E3 ligases and ligands for specific cells or tissues to develop more precisely targeted protein degraders is a major scientific challenge that PROTACs must address. Therefore, identifying specific E3 ligases and ligands for degrading diverse target proteins will greatly expand the feasibility, practicality, and selectivity of PROTAC technology and is crucial to its success. Summary of the Invention

[0004] The present invention aims to overcome the existing technical defects and provides an application of a pentacyclic triterpene compound.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The invention relates to an application of a pentacyclic triterpenoid compound, and an application of the pentacyclic triterpenoid compound as an E3 ligase ligand or a carcinogenic protein binding compound.

[0007] The E3 ligase is ITCH or SIAH2.

[0008] The oncogenic protein is NCOR1, HDAC3 or c-FLIP LFurthermore, pentacyclic triterpenoid compounds can be used to detect oncogenic proteins NCOR1, HDAC3 or c-FLIP L Application in.

[0009] The pentacyclic triterpene compound structural formula is

[0010]

[0011] The dotted line in the third ring of the pentacyclic triterpene compound may be present or absent depending on the choice of substituents.

[0012] The carbonyl pentacyclic triterpene compound is shown in Formula 1:

[0013]

[0014] In the formula, when X can be a carbonyl group, CH or CH2; Y can be CH or a carbonyl group, and the dotted line in the third ring can be present or absent depending on the selection of the substituent of X or Y.

[0015] The carbonyl pentacyclic triterpene compound is

[0016] 10a: methyl 2-cyano-3,11-dioxo-18β-oleanane-1,12-diene-30-carboxylate;

[0017] 10b: methyl 2-cyano-3-oxo-18β-oleanane-1,12-diene-30-carboxylate;

[0018] 10c: methyl 2-cyano-3,12-dioxo-18β-oleanane-1-ene-30-carboxylate;

[0019] 10d: 2-cyano-3,12-dioxo-18β-oleanane-1,9(11)-diene-30-carboxylic acid methyl ester or 10e: 2-cyano-3-oxo-18β-oleanane-1,9(11),12-triene-30-carboxylic acid methyl ester.

[0020]

[0021] The pentacyclic triterpenoid compound is prepared by dissolving the pentacyclic triterpenoid compound in dimethyl sulfoxide to prepare a 10-100 mM storage solution, which is stored at -20°C and diluted with anhydrous ethanol or aqueous solution when used.

[0022] The marker is a pentacyclic triterpenoid compound labeled with biotin, dissolved in dimethyl sulfoxide, prepared into a 30 mM storage solution, stored at -20°C, and diluted with anhydrous ethanol before use as a test compound.

[0023] The advantages of the present invention are:

[0024] The pentacyclic triterpene compound of the present invention has a significant binding effect on E3 ligase SIAH2 and ITCH, and its binding ability is better than that of the parent nucleus GA. Compound 10e has significant binding specificity for E3 ligase. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram showing the effect of using a pentacyclic triterpene compound combined with a cell lysis protein in an embodiment of the present invention.

[0026] Figure 2 This is a diagram showing the effect of using pentacyclic triterpenoid compounds combined with recombinant proteins in an embodiment of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and beneficial technical effect of the present invention clearer, the present invention is specifically described below by examples. It should be understood that the examples are only used to further illustrate the present invention, but the content of the present invention is not limited to the content involved in the examples and cannot be interpreted as limiting the scope of protection of the present invention. Those skilled in the art in this field can make some non-essential improvements and adjustments based on the content of the present invention mentioned above. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0028] Example 1: Preparation of 2-cyano-3,11-dioxo-18β-oleanane-1,12-diene-30-carboxylic acid methyl ester (10a)

[0029] Steps:

[0030] Step A: Preparation of methyl 3β-hydroxy-11-oxo-18β-oleanane-12-ene-30-carboxylate (5a)

[0031]

[0032] Dissolve 5.2 mmol of 3β-hydroxy-18β-oleanane-30-carboxylic acid (GA) in 200 ml of anhydrous methanol, add 15 mL of concentrated sulfuric acid dropwise, and reflux for 24 hours. Cool, freeze, filter, and wash with copious amounts of water until the filtrate is neutral. After drying, recrystallize from anhydrous methanol to obtain a white solid. Yield: 69%. mp 238-240°C. max / cm -1 (film KBr)3361,2946,2871,1724,1658,1620,1466,1387,1218,1157,1087,1043,994. 1H NMR(300MHz, CDCl3)5.67(1H,s,H-12),3.69(3H,s,-COOCH3),3.26~3.20(1H,m,H-3),2.34(1H,s,H-9),2.82~ 2.77(1H,m,H-18),1.37(3H,s),1.15(3H,s),1.14(3H,s),1.13(3H,s),1.01(3H,s),0.81(3H,s),0.80(3H,s). 13 C NMR(75MHz, CDCl3)200.3(C-11),176.9(C-30),169.2(C-13),128.5(C-12),78.7(C-3),61.8(C-9).MS(ESI)m / z:485.8[M+H] + ,507.6[M+Na] + .

[0033] Step B: Preparation of methyl 3,11-dioxo-18β-oleanane-12-ene-30-carboxylate (6a)

[0034]

[0035] Dissolve 21.2 mmol of methyl 3β-hydroxy-11-oxo-18β-oleanane-12-ene-30-carboxylate (5a) in 300 mL of acetone. Add an appropriate amount of Jones reagent dropwise under ice-cooling. Stir and react for 1 hour. Pour into water. The precipitated solid is filtered and dried to obtain compounds 2-6a to 2-6e. Yield: 79%. mp 245-246°C. max / cm -1 (film KBr)3428,2960,1725,1706,1655,1457,1387,1219,997. 1 H NMR(300MHz, CDCl3)5.71(1H,s,H-12),3.70(3H,s,-COOCH3),2.99~2.94(1H,m),2.65~2.61(1H,m),2.44 (1H,s,H-9),1.37(3H,s),1.27(3H,s),1.17(3H,s),1.15(3H,s),1.11(3H,s),1.07(3H,s),0.82(3H,s). 13 C NMR(75MHz, CDCl3)217.2(C-3),199.5(C-11),176.9(C-30),169.7(C-13),128.4(C-12),61.0(C-9).MS(ESI)m / z:483.7[M+H]+ ,505.6[M+Na] + .

[0036] Step C: Preparation of methyl 2-hydroxymethylene-3,11-dioxo-18β-oleanane-12-ene-30-carboxylate (7a)

[0037]

[0038] Add 10 mmol of 6a and 100 mL of ethyl formate to a 250 mL eggplant-shaped flask, stir to dissolve, then add 0.96 g (40 mmol) of NaH in batches over 30 min. React at room temperature for 1 h, slowly add anhydrous methanol, and add water after no bubbles are generated. Adjust the pH to 1-2 with concentrated hydrochloric acid, and extract with dichloromethane (30 mL x 3).

[0039] The organic layers were combined, washed with saturated brine and distilled water, and dried over anhydrous sodium sulfate overnight. A pale yellow oil was obtained. Separation by silica gel column chromatography with cyclohexane:acetone (v / v) = 20:1 afforded a white solid. Yield: 87%. mp 308-310°C. max / cm -1 (film KBr)3440,2949,2866,1733,1655,1461,1386,1326,1214,1157,1087,769. 1 H NMR(300MHz,d6-DMSO)14.69(1H,s,=CH-OH),8.64(1H,s,=CH-OH),5.73(1H,s,H-12),3.70(3H,s,-COOCH3),3.50~3.45(1 H,dd,H-1),2.44(1H,s,H-9),1.38(3H,s),1.21(3H,s),1.16(3H,s),1.16(3H,s),1.15(3H,s),1.06(3H,s),0.81(3H,s). 13 C NMR(75MHz,d6-DMSO)199.6(C-11),189.5(C-3),188.9(=CH-OH),176.9(C-3 0),169.9(C-13),128.6(C-12),105.8(C-2),59.6(C-9).MS(ESI)m / z:511.5

[0040] [M+H] + ,533.5[M+Na] + .

[0041] Step D: Preparation of 11-oxo-isoxazolo[2,3-d]-18β-oleanane-12-ene-30-carboxylic acid methyl ester (8a)

[0042]

[0043] To a 100 mL eggplant-shaped flask, 1.8 mmol of 7a, 0.36 g (5.1 mmol) of hydroxylamine hydrochloride, 0.04 g (0.49 mmol) of anhydrous sodium acetate, and 20 mL of glacial acetic acid were added. The mixture was refluxed for 1.5 h and poured into ice water. The precipitated solid was filtered, washed with water until neutral, dried by infrared spectroscopy, and separated by silica gel column chromatography using a developing solvent of petroleum ether:acetone (v / v) = 20:1 to obtain 8a as a white solid. Yield: 98%. mp: 228-230°C. max / cm -1 (film KBr)3433,2971,1727,1656,1621,1460,1387,1323,1272,1220,1160,1087,977,811. 1 H NMR(300MHz, CDCl3)8.03(1H,s,-CH=N-),5.77(1H,s,H-12),3.73(3H,s,-COOCH3),2.56(1H,s,H -9),1.41(3H,s),1.36(3H,s),1.27(3H,s),1.21(3H,s),1.18(3H,s),1.09(3H,s),0.86(3H,s). 13 C NMR (75MHz, CDCl3)199.4(C-11),176.9(C-30),172.2(C-3),169.9(C-13),150.4(-C=N-),128.5(C-12),109.0(C-2),60.1(C-9).HR-MS-FAB m / z[M+Na] + calcd for C 32 H 45 NNaO4:530.3241,found:530.3242.

[0044] Step E: Preparation of methyl 2-cyano-3,11-dioxo-18β-oleanane-12-ene-30-carboxylate (9a)

[0045]

[0046] 3.94 mmol of 8a was dissolved in a mixture of 60 mL of methanol and 125 mL of ether. 7.25 g (134 mmol) of sodium methoxide was added under ice-cooling. After stirring at room temperature for 45 min, the mixture was extracted with ether, washed with 5% hydrochloric acid, and the combined organic layers were dried over anhydrous sodium sulfate. Separation by silica gel column chromatography with cyclohexane:acetone (v / v) = 20:1 afforded a white solid. Yield: 95%. mp 258-260°C. max / cm -1 (film KBr)3399,2949,2205,1730,1661,1459,1388,1314,1218,1171,1086,979,879. 1 H NMR(300MHz,d6-DMSO)5.50(1H,s,H-12),3.64(3H,s,-COOCH3),2.99(1H,m,H-2),2.45(1H,s,H -9),1.35(3H,s),1.12(3H,s),1.11(3H,s),1.07(3H,s),1.05(3H,s),1.01(3H,s),0.77(3H,s). 13 C NMR(75MHz, CDCl3)207.4(C-3),199.0(C-11),176.9(C-30),170.9(C-13),12 8.4(C-12),118.8(-CN),79.6(C-2),61.3(C-9),59.7(C-5),52.1(-COOCH3), 51.8(C-18),48.4(C-14),44.0(C-4),41.2(C-20),40.3(C-8),37.7(C-19),3 7.6(C-10),35.8(C-22),31.8(C-17),31.6(C-7),31.1(C-21),28.6(C-28and C-29),28.3(C-1),27.9(C-23),26.5(C-15),26.4(C-16),23.3(C-27),19.9(C-24),18.6(C-25),18.2(C-26),15.4(C-6).HR-MS-FAB m / z[M+H] + calcd for C 32 H 46 NO4:508.3421,found:508.3423.

[0047] Step F: Preparation of methyl 2-cyano-3,11-dioxo-18β-oleanane-1,12-diene-30-carboxylate (10a)

[0048]

[0049] 3 mmol of compound 9a and DDQ (98%) (0.77 g, 3.32 mmol) were added to 80 mL of dry toluene and refluxed at 80°C for 30 min. Insoluble matter was removed by filtration, and the filtrate was evaporated to dryness to afford a yellow solid. Column chromatography using a cyclohexane:acetone (v / v) ratio of 15:1 afforded a white solid. Yield: 89%. mp 254-260°C. max / cm -1 (film KBr)3419,2943,2233,1720,1690,1649,1387,1281,1250,1219,1117,986,879. 1 H NMR(300MHz, CDCl3)8.41(1H,s,H-1),5.72(1H,s,H-12),3.71(3H,s,-COOCH3),2.61(1H,s,H-9),2.25(1H,dd, H-18),1.81(2H,m),1.51(3H,s),1.37(3H,s),1.24(3H,s),1.22(3H,s),1.20(3H,s),1.16(3H,s),0.74(3H,s). 13 C NMR(100MHz, CDCl3)198.1(C-3),197.6(C-11),176.8(C-30),172.6(C-13),171.8(C-1),127.8(C-12),114.9(- CN),113.3(C-2),54.4(C-9),51.9(C-5),51.8(-COOCH3),48.5(C-18),45.7(C-14),45.1(C-4),44.0(C-20),43. 6(C-8),41.1(C-19),39.7(C-10),37.7(C-22),31.8(C-17),31.6(C-7),31.1(C-21),28.6(C-28),28.3(C-29),2 7.6(C-23),26.5(C-15),26.3(C-16),23.4(C-27),21.5(C-24),19.6(C-25),19.0(C-26),18.1(C-6).HR-MS-FAB m / z[M+H] + calcd for C 32 H 44 NO4:506.3265,found:506.3265.

[0050] Example 2: Preparation of methyl 2-cyano-3-oxo-18β-oleanane-1,12-diene-30-carboxylate (10b)

[0051] Step A: Preparation of 3β-hydroxy-18β-oleanane-12-ene-30-carboxylic acid (1)

[0052]

[0053] Take 0.50g (1.8mmol) of HgCl2 and add 30mL of 7% hydrochloric acid. After dissolving, add 13.5g (0.206mol) of Zn powder. After stirring for 20 minutes, filter with suction and wash with a small amount of 1,4-dioxane. Pour the activated Zn powder into a solution of 5.04g (10.7mmol) of 18β-glycyrrhetinic acid in 100mL of 1,4-dioxane. Use an ice-water bath to maintain the temperature above 20°C. Add 1mL of concentrated hydrochloric acid dropwise. Pass HCl gas for 5 hours. TLC shows the reaction is complete. Let it stand and pour the supernatant into 200mL of distilled water. A large amount of solid precipitates. Filter with suction, wash the filter cake with water, and dry to obtain a white solid (4.65g, 95%). mp 314-317°C.ν max / cm -1 (film KBr)3437,2928,1705,1583,1467,1416,1384,1288,1028,995,919. 1 H NMR(300MHz,d6-DMSO)12.0(1H,s,-COOH),5.19(1H,t,H-12),4.33(1H,br s,-OH),3.03(1H,m,H-3),2.54~2.53(1H,t),1.14(3H,s),1.10(3H,s),0.94(3H,s),0.93(3H,s),0.91(3H,s),0.77(3H,s),0.71(3H,s). 13 C NMR(75MHz, CDCl3)178.1(C-30),144.5(C-13),122.2(C-12),76.9(C-3).MS(ESI)m / z:457.5[M+H] + .

[0054] Step B: Preparation of methyl 3β-hydroxy-18β-oleanane-12-ene-30-carboxylate (5b)

[0055]

[0056] The title compound was prepared according to the preparation method of Example 1, Step A, using 3β-hydroxy-18β-oleanane-12-ene-30-carboxylic acid (1) as the starting material to replace 3β-hydroxy-18β-oleanane-30-carboxylic acid (GA) in Example 1, Step A. Yield: 66%. mp 248-250°C. max / cm -1 (film KBr)3354,2944,1723,1465,1384,1218,1159,1088,1029,996. 1 H NMR(300MHz, CDCl3)5.27(1H,t,H-12),3.68(3H,s,-COOCH3),3.22(1H,m,H-3),1.14( 3H,s),1.13(3H,s),1.00(3H,s),0.96(3H,s),0.94(3H,s),0.79(3H,s),0.78(3H,s). 13 C NMR(75MHz, CDCl3)177.7(C-30),144.3(C-13),122.6(C-12),79.0(C-3).MS(ESI)m / z:471.3[M+H] + ,493.6[M+Na] + .

[0057] Step C: 3-Oxo-18β-oleanolic acid-12-ene-30-carboxylic acid methyl ester (6b)

[0058]

[0059] The title compound was prepared according to the preparation method of Example 1, Step B, using 3β-hydroxy-18β-oleanane-12-ene-30-carboxylic acid methyl ester (5b) in place of 3β-hydroxy-11-oxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (5a). Yield: 70%. mp 180-182°C. max / cm -1 (film KBr)3426,2948,1722,1705,1453,1431,1383,1214,1154,1088,1030,993. 1H NMR(300MHz, CDCl3)5.32~5.30(1H,t,H-12),3.69(3H,s,-COOCH3),2.57~2.51(1H,m),2.42~2.36( 1H,m),1.16(3H,s),1.13(3H,s),1.11(3H,s),1.08(3H,s),1.07(3H,s),1.00(3H,s),0.80(3H,s). 13 C NMR(75MHz, CDCl3)217.7(C-3),177.6(C-30),144.4(C-13),122.4(C-12).MS(ESI)m / z:469.1[M+H] + ,491.0[M+Na] + .

[0060] Step D: 2-Hydroxymethylene-3-oxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (7b)

[0061]

[0062] The title compound was prepared according to the preparation method of Example 1, Step C, using 3-oxo-18β-oleanolic acid-12-ene-30-carboxylic acid methyl ester (6b) in place of 3,11-dioxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (6a). Yield: 88%. mp 159-160°C. max / cm -1 (film KBr)3437,2964,2872,1731,1658,1461,1386,1217,1155,1055,986.1H NMR(300MHz, CDCl3)14.91(1H,s,=CH-OH),8.58(1H,s,=CH-OH),5.34~5.32(1H,t,H-12), 3.69(3H,s,-COOCH3),2.33~2.28(1H,d,H-1),1.20(3H,s),1.16(3H,s),1.14(3H,s),1.13 (3H,s),1.02(3H,s),0.94(3H,s),0.79(3H,s).13CNMR(75MHz,CDCl3)190.7(C-3),188.4( =CH-OH),177.7(C-30),144.3(C-13),122.4(C-12),105.8(C-2).MS(ESI)m / z:497.2[M+H] + .

[0063] Step E: Methyl isoxazolo[2,3-d]-18β-oleanane-12-ene-30-carboxylate (8b)

[0064]

[0065] The title compound was prepared according to the preparation method of Example 1, Step D, using 2-hydroxymethylene-3-oxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (7b) in place of 2-hydroxymethylene-3,11-dioxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (7a). Yield: 80%. mp 206-208°C. max / cm -1 (film KBr)3425,2948,1722,1641,1482,1463,1382,1316,1269,1221,1163,1087,864.1H NMR(300MHz,CDCl3)8.00(1H,s,

[0066] -CH=N-),5.35-5.33(1H,t,H-12),3.70(3H,s,-COOCH3),3.64(1H,m,H-1),1.32(3H ,s),1.23(3H,s),1.16(3H,s),1.13(3H,s),1.03(3H,s),0.92(3H,s),0.80(3H,s). 13 CNMR(75MHz, CDCl3)177.6(C-30),173.0(C-3),150.2(-C=N-),144.3(C-13),122.3(C-12),108.8(C-2).MS(ESI)m / z:494.0[M+H] + ,516.0[M+Na] + .

[0067] Step F: 2-cyano-3-oxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (9b)

[0068]

[0069] The title compound was prepared according to the preparation method of Example 1, Step E, using methyl isoxazolo[2,3-d]-18β-oleanane-12-ene-30-carboxylate (8b) as the starting material, replacing 11-oxo-isoxazolo[2,3-d]-18β-oleanane-12-ene-30-carboxylate (8a) in Example 1, Step E. Yield: 94%. mp 156-158°C. max / cm -1(film KBr)3432,2927,2859,2204,1719,1640,1456,1381,1222,1193,1167,1086. 1 H NMR(300MHz, CDCl3)5.30-5.28(1H,m,H-12),3.91(1H,m,H-2),3.69(3H,s,-COOCH3),2.37(1H, m),1.22(3H,s),1.18(3H,s),1.13(3H,s),1.12(3H,s),1.10(3H,s),1.02(3H,s),0.78(3H,s). 13 C NMR(100MHz, CDCl3)205.4(C-3),177.6(C-30),144.8(C-13),121.6(C-12),117.3(-CN),7 7.3(C-2),56.4(C-5),51.6(-COOCH3),48.5(C-18),46.9(C-4),44.3(C-20),42.8(C-19and C-14),39.6(C-9),38.3(C-8),37.8(C-10),36.6(C-22),32.1(C-7),31.9(C-17),31.3(C-21),28.6(C-28and C-29),28.2(C-1),27.9(C-23),26.8(C-16),25.9(C-15),25.1(C-27),2 3.3(C-11),21.4(C-24),19.1(C-6),16.5(C-25),15.1(C-26).HR-MS-FAB m / z[M+H] + calcdfor C 32 H 48 NO3:494.3629,found:494.3629.

[0070] Step G: 2-cyano-3-oxo-18β-oleanane-1,12-diene-30-carboxylic acid methyl ester (10b)

[0071]

[0072] The title compound was prepared according to the preparation method of Step F of Example 1, substituting 2-cyano-3-oxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (9b) for 2-cyano-3,11-dioxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (9a) in Step F of Example 1. Yield: 28%. mp 104-108°C. max / cm-1 (film KBr)3439,2929,2233,1729,1687,1455,1384,1244,1109. 1 H NMR(300MHz, CDCl3)7.76(1H,s,H-1),5.34(1H,t,H-12),3.70(3H,s,

[0073] -COOCH3),2.10(2H,m),1.25(3H,s),1.22(3H,s),1.16(3H,s),1.15(3H,s),1.14(3H,s),1.07(3H,s),0.79(3H,s). 13 C NMR(100MHz, CDCl3)198.1(C-3),177.5(C-30),170.0(C-1),145.2(C-13),121.1(C-12),115.0(-CN),113.8(C- 2),52.5(C-5),51.6(-COOCH3),48.3(C-18),44.9(C-4),44.2(C-20),42.6(C-19),42.0(C-14),41.1(C-9),40.8 (C-8),40.5(C-10),38.3(C-22),32.1(C-7),32.0(C-17),31.3(C-21),28.5(C-29),28.2(C-28),27.7(C-23),2 6.8(C-16),26.0(C-15),25.8(C-27),23.3(C-11),21.6(C-24),18.8(C-6),18.0(C-25),17.4(C-26).HR-MS-FAB m / z[M+Na] + calcd for C 32 H 45 NNaO3:514.3292,found:514.3291.

[0074] Example 3: Preparation of methyl 2-cyano-3,12-dioxo-18β-oleanane-1-ene-30-carboxylate (10c)

[0075] Step A: Preparation of methyl 3β-acetoxy-12-oxo-18β-oleanane-30-carboxylate (2)

[0076]

[0077] The methyl ester of compound 1 (2.5 g, 5.5 mmol) was obtained by the methyl ester formation method of step B of Example 2. 20 mL / 100 mL acetic anhydride / pyridine was added and refluxed for 9 h to obtain 2.5 g (4.9 mmol) of a brown solid after protection of the 3-hydroxyl group. The solid was dissolved in 200 mL of glacial acetic acid and 20 mL of 30% H2O2 was added. The reaction was carried out at 100°C for 0.5 h. TLC showed that the reaction was complete. The solid was poured into ice water to precipitate a large amount of solid. The solid was filtered, washed with water until neutral, dried, and recrystallized from methanol to obtain white crystals (2.23 g, 77%), mp 298-300°C. max / cm -1 (film KBr)3437,2948,1729,1700,1464,1380,1324,1247,1223,1149,1083,1029,1000,610. 1 H NMR(300MHz, CDCl3)4.48(1H,dd,H-3),3.72(3H,s,-COOCH3),2.76(1H,d,H-13),2.57(1H,m,H-18),2.04(3 H,s,CH3COO-),1.14(3H,s),1.13(3H,s),0.93(3H,s),0.90(3H,s),0.87(3H,s),0.86(3H,s),0.85(3H,s). 13 C NMR(75MHz, CDCl3)212.2(C-12),177.6(C-30),170.4(CH3COO-),80.4(C-3).MS(ESI)m / z:529.5[M+H] + ,551.4[M+Na] + .

[0078] Step B: Preparation of methyl 3β-hydroxy-12-oxo-18β-oleanane-30-carboxylate (5c)

[0079]

[0080] Dissolve 5 mmol of methyl 3β-acetoxy-12-oxo-18β-oleanane-30-carboxylate in 100 mL of methanol, add 2.5 g of KOH, and reflux for 30 minutes. After completion, remove the solution and pour it into dilute hydrochloric acid, adjust the pH to neutral, filter, wash with water, and dry to obtain a pale yellow solid. Column chromatography using a cyclohexane:acetone (v / v) ratio of 30:1 as the eluent affords a white solid. Yield: 92%. mp: 242-244°C. max / cm -1(film KBr)3543,3441,2868,1710,1467,1388,1326,1230,1191,1155,1082,1045,993,922.1H NMR(300MHz,CDCl3)3.71(3H,s,

[0081] -COOCH3),3.19(1H,dd,H-3),2.76(1H,d,H-13),2.57(1H,m,H-18),1.13(3H,s) ,1.12(3H,s),0.99(3H,s),0.92(3H,s),0.87(3H,s),0.85(3H,s),0.78(3H,s). 13 C NMR(75MHz, CDCl3)212.3(C-12),177.6(C-30),78.6(C-3).MS(ESI)m / z:487.5[M+H] + .

[0082] Step C: Preparation of methyl 3,12-dioxo-18β-oleanane-30-carboxylate (6c)

[0083]

[0084] The title compound was prepared according to the preparation method of Example 1, Step B, using 3β-hydroxy-12-oxo-18β-oleanane-30-carboxylic acid methyl ester (5c) in place of 3β-hydroxy-11-oxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (5a). Yield: 85%. mp 94-96°C. max / cm -1 (film KBr)3430,2928,2853,1792,1704,1629,1460,1388,1221,1153,1112,1085,1001,769. 1 H NMR(300MHz, CDCl3)3.72(3H,s,-COOCH3),2.79(1H,d,H-13),2.59(1H,m,H-18),1.19 (3H,s),1.13(3H,s),1.09(3H,s),1.06(3H,s),1.01(3H,s),0.94(3H,s),0.87(3H,s). 13 C NMR(75MHz, CDCl3)216.7(C-3),211.5(C-12),177.5(C-30).MS(ESI)m / z:485.5[M+H]+.

[0085] Step D: Preparation of methyl 2-hydroxymethylene-3,12-dioxo-18β-oleanane-30-carboxylate (7c)

[0086]

[0087] The title compound was prepared according to the preparation method of Example 1, Step C, using 3,12-dioxo-18β-oleanane-30-carboxylic acid methyl ester (6c) in place of 3,11-dioxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (6a). Yield: 30%. mp 119-121°C. max / cm -1 (film KBr)3430,2927,2852,1729,1702,1638,1590,1458,1388,1322,1220,1165,1085,1055,1023,922,837. 1 H NMR(300MHz, CDCl3)14.90(1H,br,=CH-OH),8.61(1H,s,=CH-OH),3.73(3H,s,-COOCH3),2.82(1H,d,H-13) ,2.62(1H,m),1.42(3H,s),1.21(3H,s),1,19(3H,s),1.14(3H,s),1.13(3H,s),0.96(3H,s),0.88(3H,s). 13 C NMR (75MHz, CDCl3) δ: ppm 211.4 (C-12), 189.7 (C-3), 188.9 (=CH-OH), 177.5 (C-30), 105.1 (C-2). MS (ESI) m / z: 513.7 [M+H] + ,535.7[M+Na] + .

[0088] Step E: Preparation of 12-oxo-isoxazole[2,3-d]-18β-oleanane-30-carboxylic acid methyl ester (8c)

[0089]

[0090] The title compound was prepared according to the preparation method of Example 1, Step D, using 2-hydroxymethylene-3,12-dioxo-18β-oleanane-30-carboxylic acid methyl ester (7c) in place of 2-hydroxymethylene-3,11-dioxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (7a). Yield: 76%. mp: 120-122°C. max / cm -1(film KBr)3430,2927,2852,1729,1702,1641,1482,1462,1388,1323,1219,1165,1100,1085,1055,998,938,867,765,627. 1 H NMR(300MHz, CDCl3)8.00(1H,s,-CH=N-),3.73(3H,s,-COOCH3),2.83(1H,d,H-13),2.62(1H,m ),1.42(3H,s),1.23(3H,s),1.20(3H,s),1.14(3H,s),0.97(3H,s),0.88(3H,s),0.86(3H,s). 13 C NMR (75MHz,CDCl3)

[0091] 211.4(C-12),177.5(C-30),172.7(C-3),150.2(-C=N-),108.3(C-2).MS(ESI)m / z:509.7[M+H] + ,532.7[M+Na] + .

[0092] Step F: Preparation of methyl 2-cyano-3,12-dioxo-18β-oleanane-30-carboxylate (9c)

[0093]

[0094] The title compound was prepared according to the preparation method of Example 1, Step E, using 12-oxo-isoxazolo[2,3-d]-18β-oleanane-30-carboxylic acid methyl ester (8c) in place of 11-oxo-isoxazolo[2,3-d]-18β-oleanane-12-ene-30-carboxylic acid methyl ester (8a). Yield: 85%.mp

[0095] 265-267℃.ν max / cm -1 (film KBr)3425,2954,2205,1728,1632,1460,1381,1324,1221,1168,1085,1985,1002,925,770. 1 H NMR (300 MHz, CDCl3)

[0096] 3.72(3H,s,-COOCH3),2.79(1H,m,H-2),2.60(1H,m,H-13),2.25(2H,m,

[0097] H-11),1.25(3H,s),1.18(3H,s),1.16(3H,s),1.14(3H,s),1.10(3H,s),0.93(3H,s),0.86(3H,s). 13 C NMR(75MHz, CDCl3)210.3(C-3),204.7(C-12),177.5(C-30),116.9(-CN),79.0(C- 2),56.1(C-5),51.6(-COOCH3),50.2(C-13),48.5(C-4),46.8(C-20),44.1(C-8and C-9),42.2(C-14),41.3(C-10),38.5(C-18and C-22),37.0(C-11),35.8(C-19),34.2(C-17),32.1(C-21),31.3(C-7),30.6(C-29),28.8(C-1),27.8(C-23),27 .0(C-28),26.0(C-16),25.0(C-15),21.2(C-24),19.7(C-27),16.3(C-6),15.6(C-25),14.8(C-26).HR-MS-FAB m / z[M+Na] + calcd for C 32 H 47 NNaO4:532.3397,found:532.3397.

[0098] Step G: Preparation of methyl 2-cyano-3,12-dioxo-18β-oleanane-1-ene-30-carboxylate (10c)

[0099]

[0100] The title compound was prepared according to the preparation method of Step F of Example 1, substituting 2-cyano-3,12-dioxo-18β-oleanane-30-carboxylic acid methyl ester (9c) for 2-cyano-3,11-dioxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (9a) in Step F of Example 1. Yield: 28%. mp 269-271°C. max / cm -1 (film KBr)3429,2935 2873,2232,1723,1684,1641,1611,1477,1452,1388,1305,1219,1193,1152,1116,1013,998,971,819. 1H NMR(400MHz, CDCl3)7.65(1H,s,H-1),3.73(3H,s,-COOCH3),2.82(1H,d,J=4.2H z,H-13),2.59(1H,m,H-19),2.47(1H,dd,J=4.8Hz,J=16.3Hz,H-11),2.37(1H,dd ,J=13.1Hz,J=16.3Hz,H-11'),2.01(1H,dd,J=4.8Hz,J=13.1Hz,H-9),1.25(3H,s ),1.22(3H,s),1.18(3H,s),1.16(3H,s),1.14(3H,s),0.96(3H,s),0.87(3H,s). 13 C NMR (100MHz, CDCl3)209.1(C-12),197.5(C-3),177.4(C-30),167.8(C-1),114.6(-CN and C-2),52.3(C-5),51.6(-COOCH3),50.3(C-13),44.9(C-4),44.1(C-20),43.0(C-9),42. 6(C-8),42.4(C-14),40.4(C-10),38.5(C-18),38.4(C-22),38.2(C-11),34.0(C-19),3 2.1(C-17),31.3(C-21),31.0(C-7),28.7(C-29),27.6(C-23),27.0(C-28),26.3(C-16) ,26.0(C-15),21.4(C-24),20.9(C-27),18.8(C-6),17.6(C-25),16.4(C-26).HR-MS-FAB m / z[M+Na] + calcdfor C 32 H 45 NNaO4:530.3241,found:530.3245.

[0101] Example 4: Preparation of 2-cyano-3,12-dioxo-18β-oleanane-1,9(11)-diene-30-carboxylic acid methyl ester (10d)

[0102] Step A: Preparation of methyl 3β-acetoxy-12-oxo-18β-oleanane-9(11)-ene-30-carboxylate (3)

[0103]

[0104] Dissolve 2.5 g (4.7 mmol) of 2 in 200 mL of glacial acetic acid, add a few drops of 40% HBr in acetic acid, and dissolve 0.3 mL of Br2 in 13 mL of glacial acetic acid. Add this solution dropwise to the solution at room temperature. Heat the mixture to 40°C for 15 minutes, then stir at room temperature for 24 hours until the reaction is complete. Pour the mixture into ice water to precipitate a white solid, filter it, wash it with water until neutral, and dry it to obtain 2.3 g of a white solid. Separate it by column chromatography using cyclohexane:acetone (v / v) = 50:1 as the eluent to obtain a white solid (1.73 g, 70%), mp 298-302°C. max / cm -1 (film KBr)3284,2964,1736,1656,1464,1388,1366,1209,1153,927,870. 1 H NMR(300MHz, CDCl3)5.80(1H,s,H-11),4.48(1H,dd,H-3),3.76(3H,s,-COOCH3),2.94(1H,d,H-13),2.22(1H,m,H-18 ),2.07(3H,s,CH3COO-),1.38(3H,s),1.22(3H,s),1.13(3H,s),0.97(3H,s),0.93(3H,s),0.92(3H,s),0.91(3H,s). 13 C NMR(75MHz, CDCl3)201.4(C-12),177.8(C-30),177.4(C-9),170.8(CH3COO-),123.0(C-11),79.6(C-3).MS(ESI)m / z:527.6[M+H] + ,549.6[M+Na] + .

[0105] Step B: Preparation of methyl 3β-hydroxy-12-oxo-18β-oleanane-9(11)-ene-30-carboxylate (5d)

[0106]

[0107] The title compound was prepared according to the preparation method of Example 3, Step B, using 3β-acetoxy-12-oxo-18β-oleanane-9(11)-ene-30-carboxylic acid methyl ester (3) as the starting material, replacing 3β-acetoxy-12-oxo-18β-oleanane-30-carboxylic acid methyl ester in Example 3, Step B. Yield: 85%. mp 221-223°C. max / cm -1(film KBr)3536,2949,2872,1722,1662,1598,1464,1382,1325,1226,1194,1162,1102,1084,1049,993,868. 1 H NMR(300MHz, CDCl3)5.79(1H,s,H-11),3.76(3H,s,-COOCH3),3.22(1H,dd,H-3),2.99(1H,d,H-13),2.22 (1H,m,H-18),1.39(3H,s),1.20(3H,s),1.12(3H,s),1,05(3H,s),0.98(3H,s),0.93(3H,s),0.84(3H,s). 13 C NMR(75MHz, CDCl3)201.5(C-12),178.4(C-30),177.4(C-9),122.9(C-11),77.9(C-3).MS(ESI)m / z:485.4[M+H] + .

[0108] Step C: Preparation of methyl 3,12-dioxo-18β-oleanane-9(11)-ene-30-carboxylate (6d)

[0109]

[0110] The title compound was prepared according to the preparation method of Example 1, Step B, using 3β-hydroxy-12-oxo-18β-oleanane-9(11)-ene-30-carboxylic acid methyl ester (5d) in place of 3β-hydroxy-11-oxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (5a). Yield: 86%. mp 221-223°C. max / cm -1 (film KBr)3435,2957,1732,1707,1659,1596,1456,1383,1324,1222,1194,1165,1151,1114,1084,993,867. 1 HNMR(300MHz,CDCl3)5.84(1H,s,H-11),3.76(3H,s,-COOCH3),3.03(1H,d,H-13),2.22(1H,m,H- 18),1.43(3H,s),1.33(3H,s),1.14(3H,s),1,13(3H,s),1.10(3H,s),0.99(3H,s),0.94(3H,s). 13C NMR(75MHz, CDCl3)215.9(C-3),201.1(C-12),177.4(C-30),176.6(C-9),124.2(C-11).MS(ESI)m / z:483.4[M+H] + ,505.4[M+Na] + .

[0111] Step D: Preparation of methyl 2-hydroxymethylene-3,12-dioxo-18β-oleanane-9(11)-ene-30-carboxylate (7d)

[0112]

[0113] The title compound was prepared according to the preparation method of Example 1, Step C, using 3,12-dioxo-18β-oleanane-9(11)-ene-30-carboxylic acid methyl ester (6d) in place of 3,11-dioxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (6a). Yield: 61%. mp 221-223°C. max / cm -1 (film KBr)3434,2951,2871,1729,1662,1596,1465,1377,1325,1222,1162,1081,754. 1 H NMR(300MHz, CDCl3)14.9(1H,br,=CH-OH),8.09(1H,s,=CH-OH),5.76(1H,s,H-11),3.78(3H,s,-COOCH3),3.08(1H,d,H-13),2.63(1H,d ,H-1),2.32(1H,d,H-1'),2.26(1H,m,H-18),1.44(3H,s),1.26(3H,s),1.21(3H,s),1,19(3H,s),1.15(3H,s),1.02(3H,s),0.96(3H,s). 13 C NMR (100MHz, CDCl3)201.1(C-3),190.0(C-12),188.1(=CH-OH),177.4(C-30),175.7(C-9),124.5(C-11),104.9(C-2).MS(ESI)m / z:511.5[M+H] + ,533.5[M+Na] + Step E: Preparation of 12-oxo-isoxazolo[2,3-d]-18β-oleanane-9(11)-ene-30-carboxylic acid methyl ester (8d)

[0114]

[0115] The title compound was prepared according to the preparation method of Step D of Example 1, except that 2-hydroxymethylene-3,12-dioxo-18β-oleanane-9(11)-ene-30-carboxylic acid methyl ester (7d) was substituted for 2-hydroxymethylene-3,11-dioxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (7a) in Step D of Example 1. Yield: 72%. mp 108-114°C. max / cm -1 (film KBr)3439,2928,2852,2234,1728,1690,1666,1466,1384,1325,1223,1166,1079,993,944,702.1H NMR(300MHz, CDCl3)8.09(1H,s,-CH=N-),5.94(1H,s,H-11),3.78(3H,s,-COOCH3),3.08(1H,d,H-13),2.80(1H,d,H-1),2.43 (1H,d,H-1'),2.26(1H,m,H-18),1.43(3H,s),1.37(3H,s),1.29(3H,s),1,19(3H,s),1.14(3H,s),1.03(3H,s),0.96(3H,s). 13 C NMR(75MHz, CDCl3)200.9(C-12),177.4(C-30),176.0(C-9),172.3(C-3), 150.2(-C=N-),124.7(C-11),108.5(C-2),51.6(C-13),49.6,48.0,45.8, 44.1,41.9,41.2,38.3,38.0,35.1,33.9,33.5,32.1,31.3,28.8,28.6,27 .1,26.9,26.4,26.2,24.6,23.4,22.0,21.4,18.3.HR-MS-FAB:m / z[M+Na] + calcd for C 32 H 45 NNaO4:530.3241,found:530.3242.

[0116] Step F: Preparation of methyl 2-cyano-3,12-dioxo-18β-oleanane-9(11)-ene-30-carboxylate (9d)

[0117]

[0118] The title compound was prepared according to the preparation method of Step E of Example 1, substituting 12-oxo-isoxazolo[2,3-d]-18β-oleanane-9(11)-ene-30-carboxylic acid methyl ester (8d) for 11-oxo-isoxazolo[2,3-d]-18β-oleanane-12-ene-30-carboxylic acid methyl ester (8a) in Step E of Example 1. Yield: 82%. mp 172-174°C. max / cm -1 (film KBr)3384,2949,2209,1730,1662,1633,1597,1464,1377,1326,1302,1256,1221,1170,1081,989,865,707.1H NMR(400MHz, CDCl3)5.80(1H,s,H-11),3.75(3H,s,-COOCH3),3.03(1H,m,H-2),2.43(1H,d,J=15.2Hz,H-13),2 .21(2H,m,H-1),1.40(3H,s),1.23(3H,s),1.21(3H,s),1.13(3H,s),1.12(3H,s),0.97(3H,s),0.92(3H,s).13C NMR(100MHz, CDCl3)200.7(C-3),177.4(C-12),175.0(C-30),171.6(C-9),124.6(C -11),118.7(-CN),78.9(C-2),51.6(-COOCH3),48.4(C-13),48.0(C-5),45.6(C-8), 44.1(C-4),41.9(C-20),38.6(C-10),38.5(C-14),38.3(C-22),38.0(C-18),33.9( C-19),32.1(C-17),32.0(C-7),31.3(C-21),28.7(C-29),27.9(C-1),27.1(C-23and C-28),26.3(C-25),26.2(C-15),24.1(C-16),23.3(C-26),22.0(C-27),19.9(C-24),18.8(C-6).HR-MS-FAB m / z[M+H] + calcd for C 32 H 46 NO4:508.3421,found:508.3423.

[0119] Step G: Preparation of methyl 2-cyano-3,12-dioxo-18β-oleanane-1,9(11)-diene-30-carboxylate (10d)

[0120]

[0121] The title compound was prepared according to the preparation method of Step F of Example 1, substituting 2-cyano-3,12-dioxo-18β-oleanane-9(11)-ene-30-carboxylic acid methyl ester (9d) for 2-cyano-3,11-dioxo-18β-oleanane-12-ene-30-carboxylic acid methyl ester (9a) in Step F of Example 1. Yield: 45%. mp: 151-154°C. max / cm -1 (film KBr)3439,2928,2852,2234,1728,1690,1666,1466,1384,1325,1223,1166,1079,993,944,702. 1 H NMR(300MHz, CDCl3)8.04(1H,s,H-1),6.00(1H,s,H-11),3.76(3H,s,-COOCH3),3.06(1H,d,H-13),2.20( 1H,m,H-18),1.50(3H,s),1.47(3H,s),1.26(3H,s),1,17(3H,s),1.13(3H,s),1.00(3H,s),0.94(3H,s). 13 C NMR(75MHz, CDCl3)199.7(C-12),196.6(C-3),177.3(C-30),168.4(C-1),165.8(C- 9),124.3(C-11),114.6(-CN),114.4(C-2),51.7(-COOCH3),48.2(C-13),47.7(C-5) ,46.0(C-8),45.0(C-4),44.1(C-20),42.5(C-10),42.2(C-14),38.3(C-22),37.9(C -18),33.7(C-19),32.0(C-17),31.7(C-7),31.3(C-21),28.6(C-29),27.1(C-23and C-28),26.8(C-25),26.2(C-15),26.1(C-16),24.9(C-26),22.0(C-27),21.6(C-24),18.3(C-6).HR-MS-FAB m / z[M+Na] + calcd for C 32 H43 NNaO4:528.3084,found:528.3086.

[0122] Example 5: Preparation of 2-cyano-3-oxo-18β-oleanane-1,9(11),12-triene-30-carboxylic acid methyl ester (10e)

[0123] Step A: Preparation of methyl 3β-hydroxy-18β-oleanane-9(11),12-diene-30-carboxylate (5e)

[0124]

[0125] 2.50 g (5.02 mmol) of GA and 1.25 g of NaOH were dissolved in a mixture of 100 mL of THF and 100 mL of H₂O, and 12.5 g of NaBH₄ was slowly added. The reaction was refluxed at 64°C for 5.5 h. The reaction solution was poured into a 10% hydrochloric acid solution with stirring, resulting in the precipitation of a large amount of white solid. The pH was adjusted to 2-3, filtered, and dried. Column chromatography using petroleum ether:ethyl acetate (v / v) = 7:1 as the eluent afforded 4 as a white solid. The procedure of Example 3, Step A, was then followed to yield 5e (1.86 g, 75%), mp 160-162°C. max / cm -1 (film KBr)3373,2945,2869,1722,1636,1464,1377,1217,1159,1036,990. 1 HNMR(300MHz, CDCl3)5.61(1H,d,J=6.0Hz),5.59(1H,d,J=6.0Hz),3.70(3H,s,-COOC H3 ),3.24(1H,dd,H-3),1.19(3H,s),1.12(3H,s),1.12(3H,s),1.03(3H,s),0.99(3H,s),0.84(3H,s),0.81(3H,s). 13 C NMR(100MHz, CDCl3)177.6(C-30),154.5(C-9),146.0(C-13),121.4(C-12),115.7(C-11),78.6(C-3).MS(ESI)m / z:469.4[M+H] + .

[0126] Step B: Preparation of methyl 3-oxo-18β-oleanane-9(11),12-diene-30-carboxylate (6e)

[0127]

[0128] Yield: 80%.mp 205-208℃.ν max / cm -1 (film KBr)2987,2954,2868,1727,1709,1463,1382,1314,1217,1180,1155,1113,1079,989,832,821. 1 H NMR(300MHz, CDCl3)5.66(1H,d,J=6.0Hz),5.63(1H,d,J=6.0Hz),3.70(3H,s,-COOC H3 ),1.27(3H,s),1.16(3H,s),1.13(3H,s),1.12(3H,s),1.08(3H,s),1.01(3H,s),0.84(3H,s). 13 C NMR(100MHz, CDCl3)217.7(C-3),177.5(C-30),152.5(C-9),146.6(C-13),121.2(C-12),117.5(C-11).MS(ESI)m / z:467.3[M+H] + ,489.4[M+Na] + .

[0129] Step C: Preparation of 2-hydroxymethylene-3-oxo-18β-oleanane-9(11),12-diene-30-carboxylic acid methyl ester (7e)

[0130]

[0131] Yield: 35%.mp 91-93℃.ν max / cm -1 (film KBr)3439,2948,2867,2854,2362,2338,1730,1638,1589,1455,1379,1363,1314,1157,824. 1 H NMR (400MHz, CDCl3) 14.9 (1H, br, = CH-O H ),8.71(1H,s,=C H- OH),5.76(1H,d,J=6.0Hz),5.66(1H,d,J=6.0Hz),3.70(3H,s,-COOC H3),2.60(1H,d,H-1),2.30(1H,d,H-1'),2.10(1H,m,H-18),1.22(3H,s),1.16(3H,s),1.15(3H,s),1.13(3H,s),1.01(3H,s),0.85(3H,s). 13 C NMR(100MHz, CDCl3)189.9(C-3),189.1(= C H-OH),177.5(C-30),151.3(C-9),146.7(C-13),121.2(C-12),117.6(C-11),106.1(C-2).MS(ESI)m / z:495.3[M+H] + .

[0132] Step D: Preparation of methyl isoxazole[2,3-d]-18β-oleanane-9(11),12-diene-30-carboxylate (8e)

[0133]

[0134] Yield: 31%.mp 147-149℃.ν max / cm -1 (film KBr)2969,2932,2867,1732,1482,1461,1384,1313,1266,1215,1151,1084. 1 H NMR(300MHz,CDCl3)8.04(1H,s,-C H =N-),5.92(1H,d,J=6.0Hz),5.67(1H,d,J=6.0Hz),3.70(3H,s,-COOC H3 ),2.79(1H,d,H-1),2.39(1H,d,H-1'),2.16-2.08(1H,m,H-18),1.33(3H,s), 1.25(3H,s),1.17(3H,s),1,15(3H,s),1.13(3H,s),1.02(3H,s),0.85(3H,s). 13 C NMR(100MHz, CDCl3)189.9(C-3),189.1(= C H-OH),177.6(C-30),172.8(C-3),151.9(C-9),150.3(- C=N-),146.7(C-13),121.2(C-12),117.8(C-11),109.5(C-2).MS(ESI)m / z:492.7[M+H] + .

[0135] Step E: Preparation of methyl 2-cyano-3-oxo-18β-oleanane-9(11),12-diene-30-carboxylate (9e)

[0136]

[0137] Yield: 28%.mp 119-121℃.ν max / cm -1 (film KBr)3419,2948,2868,2204,1729,1631,1455,1383,1180,1162,1108,1083,985. 1 H NMR (300MHz, CDCl3)5.68(1H,d,J=6.0Hz),5.66(1H,d,J=6.0Hz),3.72(3H,s,-COOC H3 ),2.82(1H,m,H-2),1.22(3H,s),1.19(3H,s),1.17(3H,s),1.15(3H,s),1.10(3H,s),1.00(3H,s),0.86(3H,s). 13 C NMR (100MHz, CDCl3)205.3(C-3),177.5(C-30),150.8(C-9),147.3(C-13),121.0,119.8(C-11,C-12),117.9(- C N),79.9(C-2).MS(ESI)m / z:492.5[M+H] + ,514.5[M+Na] + .

[0138] Step F: Preparation of 2-cyano-3-oxo-18β-oleanane-1,9(11),12-triene-30-carboxylic acid methyl ester (10e)

[0139]

[0140] Yield: 28%.mp 128-130℃.ν max / cm -1(film KBr)3418,2928,2854,2233,1728,1688,1538,1454,1379,1383,1242,1219,1108,993. 1 H NMR (400MHz, CDCl3)8.12(1H,s,H-1),5.89(1H,d,J=6.0Hz),5.73(1H,d,J=6.0Hz),3.71(3H,s,-COOC H3 ),2.15(1H,dd,H-18),1.45(3H,s),1.23(3H,s),1.19(3H,s),1.16(3H,s),1.13(3H,s),0.98(3H,s),0.84(3H,s). 13 C NMR (75MHz, CDCl3)197.7(C-3),177.4(C-30),169.8(C-1),148.9(C-9),144.6(C-13),120.5(C-12),118.8(C-11),115.0(- C N),113.6(C-2),51.7(-COO C H3),48.6(C-5),46.5(C-18),44.9(C-20),44.1(C-14),42.9(C-8),42.6(C-19),41.8 (C-4),40.5(C-10),38.1(C-22),31.5(C-17),31.1(C-7),30.9(C-21),28.4(C-28and C-29),28.3(C-23),27.6(C-16),27.0(C-15),25.5(C-27),21.4(C-24and C-25),19.6(C-26),18.4(C-6).HR-MS-FAB m / z[M+H] + calcd for C 31 H 44 NO3:490.3316,found:490.3313.

[0141] The biotin-labeled compounds 10e-B, 10d-B, 10a-B, and GA-B are pentacyclic triterpenoid compounds 10e, 10d, 10a, and GA, wherein a molecule of biotin is bound to the C30 position.

[0142]

[0143] Example 6: Preparation of a conjugate of 3β-hydroxy-18β-oleanane-30-acyl-ethylenediamine and biotin (GA-B)

[0144]

[0145] 250 mg (0.53 mmol) of 3β-hydroxy-18β-oleanane-30-carboxylic acid (GA) and 166 mg (0.58 mmol) of D-Biotin-ethylenediamine were weighed into a 25 mL eggplant-shaped flask and dissolved in 15 mL of dry N,N-dimethylformamide. 111 mg (0.58 mmol) of EDCI, 78 g (0.58 mmol) of HOBt, and 75 mg (0.58 mmol) of DIEA were then added sequentially. The mixture was stirred at 60°C for 6 h. After the reaction, the reaction mixture was poured into cold water and stirred for 30 min. The mixture was extracted with ethyl acetate, washed once with water, and once with saturated sodium chloride solution, and dried to obtain a white solid. The product was separated by silica gel column chromatography using dichloromethane:methanol (v / v) = 50:1 as the eluent to obtain a white solid.

[0146] Example 7: Preparation of a Conjugate of 2-Cyano-3,11-dioxo-18β-oleanane-1,12-diene-30-acyl-ethylenediamine and Biotin (10a-B) Step A: Preparation of 2-Cyano-3,11-dioxo-18β-oleanane-1,12-diene-30-carboxylic acid (10EA)

[0147]

[0148] 3 mmol of compound 10a and 4.5 g of anhydrous LiI (33 mmol) were added to 15 mL of dry DMF, refluxed at 154°C for 4 h, poured into ice-cold water, filtered, washed with water until neutral, and dried. After column chromatography with a dichloromethane:methanol (v / v) ratio of 50:1 as the eluent, a white solid was obtained.

[0149] Step B: Preparation of a conjugate of 2-cyano-3,11-dioxo-18β-oleanane-1,12-diene-30-acyl-ethylenediamine and biotin (10a-B)

[0150]

[0151] According to the preparation method of Example 6, the title compound was prepared by replacing 3β-hydroxy-18β-oleanane-30-carboxylic acid (GA) in Example 6 with 2-cyano-3,11-dioxo-18β-oleanane-1,12-diene-30-carboxylic acid (10EA).

[0152] Example 8: Preparation of a conjugate of 2-cyano-3,12-dioxo-18β-oleanane-1,9(11)-diene-30-acyl-ethylenediamine and biotin (10d-B)

[0153] Step A: Preparation of 2-cyano-3,12-dioxo-18β-oleanane-1,9(11)-diene-30-carboxylic acid (10ED)

[0154]

[0155] According to the preparation method of step A of example 7, the title compound was prepared by replacing 2-cyano-3,11-dioxo-18β-oleanane-1,9(11)-diene-30-carboxylic acid methyl ester (10a) in step A of example 7 with 2-cyano-3,11-dioxo-18β-oleanane-1,12-diene-30-carboxylic acid methyl ester (10d).

[0156] Step B: Preparation of a conjugate of 2-cyano-3,11-dioxo-18β-oleanane-1,12-diene-30-acyl-ethylenediamine and biotin (10d-B)

[0157]

[0158] According to the preparation method of Example 6, the title compound was prepared by replacing 3β-hydroxy-18β-oleanane-30-carboxylic acid (GA) in Example 6 with the raw material 2-cyano-3,12-dioxo-18β-oleanane-1,9(11)-diene-30-carboxylic acid (10ED).

[0159] Example 9: Preparation of a conjugate of 2-cyano-3-oxo-18β-oleanane-1,9(11),12-triene-30-acyl-ethylenediamine and biotin (10e-B) Step A: Preparation of 2-cyano-3-oxo-18β-oleanane-1,9(11),12-triene-30-carboxylic acid (10EE)

[0160]

[0161] According to the preparation method of Example 7 Step A, the title compound was prepared by replacing 2-cyano-3,11-dioxo-18β-oleanane-1,9(11),12-triene-30-carboxylic acid methyl ester (10e) with 2-cyano-3-oxo-18β-oleanane-1,9(11),12-triene-30-carboxylic acid methyl ester (10a) in Example 7 Step A.

[0162] Step B: Preparation of a conjugate of 2-cyano-3-oxo-18β-oleanane-1,9(11),12-triene-30-acyl-ethylenediamine and biotin (10e-B)

[0163]

[0164] According to the preparation method of Example 6, the raw material 2-cyano-3-oxo-18β-oleanane-1,9(11),12-triene-30-carboxylic acid (10EE) was used to replace 3β-hydroxy-18β-oleanane-30-carboxylic acid (GA) in Example 6 to prepare the title compound.

[0165] Example 10 Pentacyclic triterpenoid compounds bind to E3 ligases SIAH2 and ITCH in cells, and bind to oncogenic proteins NCOR1, HDAC3, and c-FLIP in cells L

[0166] Accurately weigh the biotin-labeled compounds 10e-B, 10d-B, 10a-B, and GA-B, respectively, and dissolve them in dimethyl sulfoxide to prepare 30 mM stock solutions. Store at -20°C and dilute with anhydrous ethanol to the appropriate concentration before use as test compounds.

[0167] Cell lysis protein: Leukemia HL-60 cells were centrifuged at 1500r for 10 min, the supernatant was discarded, and the cells were resuspended in PBS and centrifuged at 3000r for 5 min. The supernatant was discarded and lysed in ice-cold 1% NP40 lysis buffer (P0013F, Beyotime) for 1 h. The cell fragments were centrifuged at 13000r for 15 min, and the protein supernatant was collected for protein quantification.

[0168] Interacting proteins of biotinylated compounds: Add 20 μM biotinylated compounds 10e-B, 10d-B, 10a-B, or GA-B (compounds diluted in absolute ethanol) or an equal volume of ethanol as a control to 400 μg of total protein. Preincubate at room temperature for 1 hour. Add 20 μL of anti-biotin magnetic beads and incubate overnight at 4°C to isolate the biotinylated proteins. Wash the beads four times with 1% NP40 buffer, add 20 μL of protein loading buffer, boil at 98°C for 5 minutes, and centrifuge at 13,000 rpm for 1 minute to collect the supernatant.

[0169] Western blot detection of interacting proteins: Prepare 8% or 12% separation gel, separate the above samples by SDS-PAGE electrophoresis, transfer to the membrane, block, wash with TBST, add 1:1000 dilution of primary antibody SIAH2 and other antibodies (RNF126, NEDD4 and NEDD8 were used as controls), shake overnight at 4°C, wash with TBST, add secondary antibody (1:5000 dilution) and incubate at room temperature for 1 hour, wash with TBST, add ECL luminescent solution and expose (see Figure 1 ).

[0170] Depend on Figure 1As shown, compounds 10e and 10d bind to the E3 ligases SIAH2 and ITCH in cell lysate proteins at 20 μM, while compounds 10a and GA do not bind at the same concentrations. These four compounds have no binding activity to other E3 ligases, RNF126, NEDD8, and NEDD8. This indicates that compounds 10e and 10d can specifically bind to the E3 ligases SIAH2 and ITCH and may serve as specific ligands for SIAH2 and ITCH.

[0171] HDAC3 and NCOR1 are specific substrate proteins of the E3 ligase SIAH2, c-FLIP L It is a substrate protein of the E3 ligase ITCH. Figure 1 It showed that 10e, 10d, and 10a could bind to NCOR1, HDAC3, and FLIP in cell lysate proteins. L Protein binding, GA does not bind to the above oncogenic proteins, which shows that compounds 10e, 10d, and 10a can bind to intracellular oncogenic proteins NCOR1, HDAC3, and c-FLIP L .

[0172] Example 10 Pentacyclic triterpenoid compounds bind to SIAH2 and ITCH recombinant proteins, and bind to recombinant oncogenic proteins NCOR1, HDAC3, and c-FLIP L

[0173] Purchase purified human SIAH2, ITCH, NCOR1, HDAC3, and c-FLIP expressed in E. coli L Proteins: SIAH2, c-FLIP L Recombinant proteins were purchased from Jiangsu Pubo Biotechnology Co., Ltd., ITCH recombinant protein was purchased from MedChemExpress, NCOR1 recombinant protein was purchased from Huamei Biotechnology, and HDAC3 recombinant protein was purchased from BPS Bioscience.

[0174] 2 μg of SIAH2, ITCH, NCOR1, HDAC3, c-FLIP LRecombinant proteins were added to recombinant protein buffer (20mM Tris-HCl, pH 7.0, 45mM NaCl, 5mM MgCl2, 0.14mM β-mercaptoethanol, 0.1mM DTT, 0.01% NP-40, 1% glycerol) with biotin-labeled compounds 10e-B, 10d-B, 10a-B, and GA-B (compounds diluted in anhydrous ethanol) or an equal volume of ethanol as a control and incubated at room temperature for 2 hours. The mixture was separated by SDS-PAGE electrophoresis, transferred to the membrane, blocked, washed with TBST, and incubated with anti-biotin antibody (Streptavidin, 1:2000 dilution) at room temperature for 1 hour. After TBST washing, the membrane was developed with ECL luminescence solution and exposed. Alternatively, primary antibodies against SIAH2, ITCH, NCOR1, HDAC3, and c-FLIP were added. L (1:1000 dilution) shake at 4 ° C overnight, wash the membrane with TBST, add secondary antibody (1:5000 dilution) and incubate at room temperature for 1 hour, wash the membrane with TBST, add ECL luminescent solution and develop and expose (see Figure 2 ).

[0175] like Figure 2 As shown, recombinant SIAH2 protein showed biotin binding activity with 20 μM biotin-labeled compounds 10e-B, 10d-B, and 10a-B, but GA-B showed no biotin-binding activity with SIAH2. Recombinant ITCH protein showed biotin-binding activity with 10e-B and 10d-B, while 10a-B and GA-B showed no biotin-binding activity with ITCH. E3 ligase recombinant binding assays showed that at a 20 μM concentration, 10d-B had the strongest binding to the recombinant proteins, while 10e-B had weaker binding than 10d-B and 10a-B had the weakest. GA-B had no binding activity with recombinant SIAH2 or ITCH.

[0176] like Figure 2 As shown, recombinant HDAC3 protein has biotin binding ability with 10e-B, 10d-B, and 10a-B, but no binding activity was detected with GA-B. L The protein has binding activity with 10e-B, 10d-B, 10a-B, and GA-B. In the binding experiment with recombinant HDAC3 and NCOR1, 10d-B has the strongest binding ability, followed by 10a-B, 10e-B has the weakest binding ability, and GA has the same binding ability with NCOR1 as 10e but does not bind to HDAC3. LIn the binding experiment, 10d-B had the strongest binding to GA-B, followed by 10e-B, and 10a-B was the weakest. The oncogenic protein recombinant binding experiment showed that 10d-B had the strongest binding; 10e-B and 10a-B had different binding abilities to different recombinant proteins, and both were weaker than 10d-B; GA-B had the strongest binding to c-FLIP. L The binding force is similar to that of 10d-B, and the binding force to other proteins is the weakest.

[0177] As shown in Examples 9 and 10, under the same concentration conditions, compound 10e can bind to SIAH2, ITCH, NCOR1, HDAC3, c-FLIP and other proteins in cell lysate. L The binding force was the strongest, and 10d was weaker; in the binding test with recombinant protein, 10d had the strongest binding activity, 10e and 10a were weaker, GA (except for c-FLIP L The above results show that 10e has specific binding to SIAH2 and ITCH in cells.

Claims

1. An application of a pentacyclic triterpene compound, characterized in that: Application of pentacyclic triterpenoids as E3 ligase ligands or binding oncogenic proteins.

2. The use of the pentacyclic triterpene compound according to claim 1, characterized in that: The E3 ligase is ITCH or SIAH2.

3. The use of the pentacyclic triterpene compound according to claim 1, characterized in that: The oncogenic protein is NCOR1, HDAC3 or c-FLIP L .

4. The use of the pentacyclic triterpene compound according to claim 1 or 2, characterized in that: The pentacyclic triterpene compound structural formula is The dotted line in the third ring of the pentacyclic triterpene compound may be present or absent depending on the choice of substituents.

5. The use of the pentacyclic triterpene compound according to claim 4, characterized in that: The carbonyl pentacyclic triterpene compound is shown in Formula 1: In the formula, X may be a carbonyl group, CH or CH2; Y may be CH or a carbonyl group. Depending on the choice of the substituent of X or Y, the dotted line in the third ring may be present or absent.

6. The use of the pentacyclic triterpene compound according to claim 5, characterized in that: The carbonyl pentacyclic triterpene compound is 10a: 2-cyano-3,11-dioxo-18β-oleanane-1,12-diene-30-carboxylic acid methyl ester; 10b: methyl 2-cyano-3-oxo-18β-oleanane-1,12-diene-30-carboxylate; 10c: methyl 2-cyano-3,12-dioxo-18β-oleanane-1-ene-30-carboxylate; 10d: 2-cyano-3,12-dioxo-18β-oleanane-1,9(11)-diene-30-carboxylic acid methyl ester or 10e: 2-cyano-3-oxo-18β-oleanane-1,9(11),12-triene-30-carboxylic acid methyl ester.

7. The use of the pentacyclic triterpene compound according to any one of claims 1 to 6, characterized in that: The pentacyclic triterpenoid compound is prepared by dissolving the pentacyclic triterpenoid compound in dimethyl sulfoxide to prepare a 10-100 mM storage solution, which is stored at -20°C and diluted with anhydrous ethanol or aqueous solution when used.

8. The use of the pentacyclic triterpene compound according to claim 7, characterized in that: The marker is a pentacyclic triterpenoid compound labeled with biotin, dissolved in dimethyl sulfoxide, prepared into a 30 mM storage solution, stored at -20°C, and diluted with anhydrous ethanol before use as a test compound.

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