MDM2 dual site inhibitors and their use for anti-tumor activity

Abstract

The application belongs to the technical field of biological medicine, and relates to an MDM2 double-site inhibitor and application of the inhibitor to anti-tumor activity, wherein the structure of the inhibitor is shown in general formula (1). The application is used in the preparation of drugs for preventing or treating breast cancer, liver cancer and osteosarcoma. The inhibitor has anti-proliferation activity on human breast cancer cells, human liver cancer cells and human osteosarcoma cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an MDM2 dual-site inhibitor and its anti-tumor activity. Background Technology

[0002] Reishi mushroom is the dried fruiting body of two fungi, *Ganoderma lucidum* Karst and *Ganoderma sinese*, belonging to the genus *Ganoderma* of the family Polyporaceae in the class Basidiomycetes. Modern pharmacological studies have shown that reishi mushroom possesses a wide range of physiological activities, such as antitumor, immunomodulatory, antiviral, and central nervous system protection. The chemical composition of reishi mushroom is complex, with over 400 compounds identified to date, mainly including triterpenes, polysaccharides, nucleosides, and sterols, of which over 300 are triterpenoids. The relative molecular mass of reishi triterpenoids is generally between 400 and 600, with complex structures, mostly highly oxidized lanosterane-type compounds. They possess activities such as antitumor, liver protection and detoxification, cholesterol reduction, and anti-HIV activity. Based on the different functional groups and side chain structures, they can be divided into several major categories, including ganoderic acids, alcohols, aldehydes, and lactones.

[0003] Currently, cancer remains a prevalent and common disease, posing a significant challenge to medicine. Chemotherapy is one of the primary methods of cancer treatment, but the low specificity of chemotherapy drugs against both normal and tumor cells, leading to toxic side effects and the development of drug resistance in tumor cells with long-term use, makes the search for novel anticancer drugs with high specificity and low toxicity a hot research direction for future cancer treatment.

[0004] The relationship between the p53-MDM2 signaling pathway and tumors has attracted attention in recent years. Excessive production of p53 protein in the human body can lead to impaired cell function or an increased tendency to form tumors. When p53 protein accumulates, the expression of MDM2 (Mouse Double Minute 2) protein in the downstream signaling pathway increases. The MDM2 structure includes a p53-binding domain, an acidic domain, a zinc finger domain, and a RING domain. Inhibition of the RING domain function leads to ubiquitin accumulation and subsequent ubiquitination and degradation of MDM2 itself. To maintain a balance and stability of p53 protein levels in cells, MDM2 can interact with the transcriptional activation domain of p53 to form a p53-MDM2 complex, thereby inhibiting p53 transcriptional activity. When cells are stimulated, MDM2 expression decreases while p53 expression increases. This increase in p53 induces MDM2 expression at the transcriptional level, forming a negative feedback regulatory loop.

[0005] Ganoderic acid A, a major component of Ganoderma lucidum triterpenes, is abundant and possesses antitumor activity, but it suffers from drawbacks such as low antitumor activity. Structural modification of natural compounds is an effective means of obtaining ideal active compounds and can expand the sources of drugs. Ganoderic acids possess various substituents, such as hydroxyl, carboxyl, and acetyl groups. These groups endow triterpenoids with diverse biological activities and also provide sites for structural modification, allowing for the development of more effective active compounds for anticancer drug development.

[0006] The existing technology, "Xu Yao, Xu Shulai, Liu Zhibin, et al. Research progress on extraction, purification and bioactivity of Ganoderma lucidum triterpenoids [J]. Food Industry Technology, 2022, 43(11): 458-464," discloses that triterpenoids extracted from Ganoderma lucidum fruiting bodies have cytotoxic, inhibitory, and apoptosis-inducing effects on various cancer cells, including human colon HT-29 cancer cells, breast cancer cells, lung cancer cells, and human nasopharyngeal carcinoma 5-8F cells. However, the extraction of triterpenoids from Ganoderma lucidum is costly, time-consuming, and labor-intensive.

[0007] This application synthesizes a novel MDM2 dual-site inhibitor, modifies the structure of ganoderic acid A, and applies it to the preparation of anticancer drugs. Summary of the Invention

[0008] To address the above problems, this invention provides an MDM2 dual-site inhibitor and its anti-tumor activity.

[0009] On the one hand, the present invention provides a dual-site inhibitor of MDM2.

[0010] The structure of the inhibitor is shown in the following general formula (1):

[0011]

[0012] In the formula, n is an integer from 1 to 10;

[0013] In the formula, R1 is one of the following substituents: hydrogen atom, halogen, nitro, methyl, and methoxy.

[0014] Preferably, n is an integer from 1 to 3.

[0015] Preferably, when R1 is a halogen, the halogen is F.

[0016] Preferably, the inhibitor is selected from B1-B14 and has the following structure:

[0017]

[0018]

[0019]

[0020]

[0021] On the other hand, the present invention provides the use of the above-mentioned inhibitor in the preparation of anti-tumor drugs.

[0022] Specifically, the drugs mentioned include, but are not limited to: anti-breast cancer drugs, anti-liver cancer drugs, or anti-human osteosarcoma drugs.

[0023] Preferably, the drug is an anti-liver cancer drug, and the drug includes B14, wherein the B14 inhibits the MDM2RING domain and / or the MDM2-p53 binding domain.

[0024] In another aspect, the present invention provides a drug comprising the above-mentioned inhibitors.

[0025] Specifically, the drug may be an anti-tumor drug.

[0026] Preferably, the antitumor drugs include, but are not limited to: anti-breast cancer drugs, anti-liver cancer drugs, or anti-human osteosarcoma drugs.

[0027] More preferably, the drug may be an anti-breast cancer drug, which includes one or more of B1, B2, B5, and B11-B14.

[0028] More preferably, the drug may be an anti-liver cancer drug, and the drug includes one or more of B1-B10 and B12-B14.

[0029] More preferably, the drug may be an anti-human osteosarcoma drug, and the drug includes one or both of B7 or B9.

[0030] Specifically, the drug dosage form may be one or more of tablets, capsules, emulsions, suspensions, injections, or infusions.

[0031] Preferably, the drug dosage form may be a tablet or a capsule.

[0032] Specifically, the drug contains pharmaceutically acceptable excipients.

[0033] Preferably, the pharmaceutically acceptable excipients include one or more of fillers, binders, disintegrants, and wetting agents.

[0034] In another aspect, the present invention provides a method for preparing the above-mentioned inhibitor, the reaction route of which is as follows:

[0035]

[0036] Furthermore, the specific steps include:

[0037] (1) Synthesis of intermediate 1: Take R1-substituted indole compound, add anhydrous dimethylformamide (DMF) or tetrahydrofuran (THF) to dissolve it, add sodium hydride below 0°C, then add 3-bromopropyne, and after the reaction is complete, intermediate 1 is obtained.

[0038] (2) Synthesis of intermediate 2: Ganoderma lucidum acid A was weighed and added sequentially at room temperature with dichloromethane (DCM), 1-amino-11-azido-3,6,9-trioxaundecan or amino-diethylene glycol-azido, O-benzotriazole N,N,N',N' tetramethylurea tetrafluoroborate (TBTU), and N,N diisopropylethylamine (DIPEA). The reaction was carried out at room temperature to obtain reaction intermediate 2.

[0039] (3) Take intermediate 2 and add dichloromethane, intermediate 1 and copper 2-thiophenecarboxylate in sequence at room temperature to obtain the inhibitor.

[0040] For detailed preparation process, please refer to the description in paragraphs 0021-0027 of the specification with publication number CN112574273B.

[0041] The beneficial effects of this invention are:

[0042] This invention provides a dual-site inhibitor of MDM2 and its antitumor activity. The antitumor application can be in the preparation of drugs for the prevention or treatment of breast cancer, liver cancer, and / or osteosarcoma. Activity tests show that the inhibitor prepared by this invention has antiproliferative activity against human breast cancer cells, human liver cancer cells, and human osteosarcoma cells, and exerts a dual-site inhibitory effect on both the p53 and RING domains of MDM2, thereby exerting an antitumor effect. Attached Figure Description

[0043] Figure 1 The inhibitors B1-B14 exhibit antiproliferative activity against different tumor cell lines and normal cell lines.

[0044] Figure 2 This demonstrates the dual-site inhibitory activity of inhibitor B14 against MDM2. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0046] Ganoderma lucidum acid A is sourced from Chengdu Ruifensi Biotechnology Co., Ltd., batch number RFS-L06102009010.

[0047] Example 1: Synthesis of Inhibitor B1

[0048] (1) Synthesis of Intermediate 1: 305.2 mg (1.85 mmol) of 5-nitroindole was accurately weighed and added to a 50 mL round-bottom flask. 20 mL of dimethylformamide (DMF) was added sequentially at room temperature, followed by 88.8 mg (3.7 mmol) of sodium hydride below 0 °C. After reacting for 30 min, 3-bromopropyne was added. The reaction was detected by TLC using petroleum ether-ethyl acetate (4:1 v / v) as the developing solvent. After the reaction was complete, ethyl acetate was added for extraction. The mixture was shaken, allowed to stand, and separated. The ethyl acetate layer was evaporated to dryness. Column chromatography was performed with petroleum ether-ethyl acetate (50:1 v / v) solution. The target analyte was collected, and the solvent was evaporated to dryness, yielding 215.6 mg of yellow intermediate 1, with a yield of 51.9%.

[0049] (2) Synthesis of Intermediate 2: 100 mg (0.194 mmol) of ganoderic acid A was accurately weighed and added to a 50 mL round-bottom flask. At room temperature, 5 mL of dichloromethane, 77.2 μL (0.388 mmol) of 1-amino-11-azido-3,6,9-trioxaundecan, 74.8 mg (0.233 mmol) of TBTU, and 64.1 μL (0.388 mmol) of DIPEA were added sequentially. The mixture was stirred at room temperature for 30 min. The reaction was detected by TLC using a dichloromethane-methanol (10:1 v / v) solution as the developing solvent. After the reaction was complete, saturated NaCl solution was added, the mixture was shaken, allowed to stand, and separated. The lower layer was dried over Na2SO4 and filtered. Column chromatography was performed using a dichloroethane-methanol (20:1 v / v) solution. The target analyte was collected, and the solvent was evaporated to dryness, yielding 122 mg of a clear, oily intermediate 2, with a yield of 87.7%.

[0050] (3) Take 20 mg (0.0279 mmol) of intermediate 2 and add 2 mL of dichloromethane, 6.25 mg (0.0332 mmol) of intermediate 1 and 1.5 mg (0.0056 mmol) of copper 2-thiophenecarboxylate sequentially at room temperature. Stir at room temperature for 2.5 h. Use dichloroethane-methanol (8:1 v / v) as the developing solvent and detect the reaction by TLC. After the reaction is completed, perform column chromatography with dichloroethane-methanol (20:1 v / v) to collect the target analyte. Dry the solvent to obtain 24.2 mg of yellow solid inhibitor B1, with a yield of 95.2%.

[0051] Inhibitor B1: Yellow solid, yield 95.2%. 1H-NMR(600MHz, CDCl3)δ:8.56(s,1H,Indole-H),8.08(m,1H Indole-H),7.55(s,1H,Triazole-H),7.46(d,1H,J=9.6Hz,Indole-H),7.37(d,J=2.9Hz,1H,Indole-H),6.70(d,J=2.9Hz,1H,Indole -H),6.39(m,1H,CONH),5.47(s,2H,CH2-N(Indole)),4.73-4.70(m,1H,H-7),4.60-4.57(m,1H,H-15),4.48-4.67(m,2H,CH2-N(Triazo le)),4.40-4.27(1H,m,OH-7),3.81-3.79(m,2H,OCH2CH2-N(Triazole)),3.76-3.43(overlap,11H,CH2(OCH2CH2O)2,OH-15),3.40-3. 31(2H,m,CONHCH2),1.25(s,3H,CH3),1.22(s,3H,CH3),1.10-1.07(overlap,9H,CH3×3),0.91(s,3H,CH3),0.81(d,3H,J=6.4Hz,CH3). 13 C-APT(150MHz,CDCl3)δ:216.4,208.6,198.6,174.7,158.5,142.1,140.7,139.0,13 7.6,130.1,127.0,122.3,117.3,116.4,108.6,103.6,71.2,69.4,69.3,69.2,69.0,6 8.7,68.2,67.8,52.9,50.7,49.4,48.9,47.6,47.1,46.0,45.7,45.6,41.3,38.1,36 .9,35.1,34.9,34.5,33.3,31.6,27.9,26.4,19.6,18.7,18.5,18.4,17.0,16.1.HRMS calculated for C 49 H 68 N6O 11 Na[M+Na] + m / z 936.4838, found 939.4836.

[0052] Example 2 Synthesis of Inhibitor B2

[0053] The only difference from Example 1 is that the starting material for the synthesis of intermediate 1 is 6-nitroindole, and the amount added is calculated according to the molar ratio; the yield of inhibitor B2 is 90.1%.

[0054] Inhibitor B2: Yellow solid, yield 90.1%. 1 H-NMR (600MHz, CDCl3) δ: 8.39 (m, 1H, Indole-H), 8.00 (dd, J=9.3, 2.1Hz, 1H, Indole-H), 7.66-7.65 (overlap, 2H, Triazole-H, Indole-H), 7.55 (d, 1H, J= 2.8Hz,Indole-H),6.62(dd,J=3.2,0.7Hz,1H,Indole-H),6.37(m,1H,CONH),5.51(s,2H,CH2-N(Indole)),4.75-4.72(m,1H,H-7),4.62-4.57(m,1H,H-15 ),4.51-4.49(m,2H,CH2-N(Triazole)),4.22-4.14(1H,m,OH-7),3.83(t,J=5.3Hz,2H,OCH2CH2-N(Triazole)),3.61-3.47(overlap,11H,CH2(OCH2CH2O) 2,OH-15),3.40-3.35(2H,m,CONHCH2),1.25(s,3H,CH3),1.23(s,3H,CH3),1.11-1.08(overlap,9H,CH3×3),0.93(s,3H,CH3),0.83(d,3H,J=6.3Hz,CH3). 13 C-APT(150MHz,CDCl3)δ:216.3,208.5,198.6,174.7,158.4,145.0,141.9,139.0,13 3.4,132.8,132.6,122.4,120.0,114.2,105.4,102.0,71.2,69.4,69.3,69.2,69.1,6 8.7,68.2,67.8,52.9,50.7,49.4,48.9,47.6,47.1,46.0,45.7,45.6,41.0,38.1,36 .9,35.2,34.9,34.5,33.3,31.6,27.9,26.3,19.6,18.7,18.5,18.4,17.0,16.2.HRMS calculated for C 49 H 68 N6O 11 Na[M+Na]+ m / z 936.4838, found 939.4836.

[0055] Example 3 Synthesis of Inhibitor B3

[0056] The only difference from Example 1 is that the starting material for intermediate 1 is 5-nitroindole, and the amount added is calculated according to the molar ratio; the starting material for intermediate 2 is ganoderic acid A and amino-diethylene glycol-azide; and the yield of inhibitor B3 is 94.2%.

[0057] Inhibitor B3: Yellow solid, yield 94.2%. 1 H-NMR (600MHz, CDCl3) δ: 8.53 (d, J=2.2Hz, 1H, Indole-H), 8.06 (dd, J=8.9, 1.9Hz, 1H, Indole-H), 7.60 (s, 1H, Triazole-H), 7.49 (d, J=9.1Hz, 1H, Indole -H),7.38(d,J=3.4Hz,1H,Indole-H),6.68(d,J=3.1Hz,1H,Indole-H),6.05(m,1H,CONH),5.48(s,2H,CH2-N(Indole)),4.75-4.49(m,1H,H-15),4.61-4. 57(m,1H,H-7),4.48(t,J=4.8Hz,2H,CH2-N(Triazole)),3.78(t,J=4.3Hz,2H,OCH2),3.43(t,J=5.3Hz,2H,CH2O),3.40-3.21(m,2H,CONHCH2),1.95-1.8 7(m,1H,H-17),1.45-1.40(m,1H,H-1),1.24(s,3H,CH3),1.22(s,3H,CH3),1 .10-1.05(overlap,9H,3×CH3),0.91(s,3H,CH3),0.80(d,J=6.4Hz,3H,CH3). 13C-APT(150MHz,CDCl3)δ:216.4,208.8,198.6,174.8,158.4,140.6,139.0 ,137.7,130.1,126.9,117.2,116.4,108.6,103.6,71.2,68.7,67.8,67.7 ,52.9,50.6,49.3,48.8,47.6,47.1,46.1,45.7,41.2,38.0,36.9,35.1,3 4.8,34.5,33.2,31.7,27.9,26.4,19.6,18.6,18.5,18.4,17.0,16.2.HRMS calculated for C 45 H 61 N6O9[M+H] + m / z 829.4495, found 829.4487.

[0058] Example 4 Synthesis of Inhibitor B4

[0059] The only difference from Example 1 is that the starting material for intermediate 1 is 6-nitroindole, and the amount added is calculated according to the molar ratio; the starting material for intermediate 2 is ganoderic acid A and amino-diethylene glycol-azide; and the yield of inhibitor B4 is 91.3%.

[0060] Inhibitor B4: Yellow solid, yield 91.3%. 1 H-NMR (600MHz, CDCl3) δ: 8.40 (m, 1H, Indole-H), 7.97 (dd, J = 8.4, 1.6Hz, 1H, Indole-H), 7.74 (s, 1H, Triazole-H), 7.64 (d, J=8.8Hz, 1H, Indole-H), 7.57 (d, J=3.2Hz, 1H, Indole-H), 6.61 (d, J=3. 2Hz,1H,Indole-H),6.13(m,1H,CONH),5.53(q,J=15.2Hz,2H,CH2-N(Indole)),4.74-4.70(m,1H, H-15),4.61-4.56(m,1H,H-7),4.50(t,J=4.8Hz,2H,CH2-N(Triazole)),3.79(t,J=4.4Hz,2H,OCH 2),3.50-3.43(m,2H,CH2O),3.43-3.20(m,2H,CONHCH2),1.95-1.87(m,1H,H-17),1.49-1.40(m,1H,H-1),1.24 (s,3H,CH3),1.22(s,3H,CH3),1.09-1.07(overlap,9H,3×CH3),0.91(s,3H,CH3),0.80(d,J=6.0Hz,3H,CH3). 13 C-APT(150MHz,CDCl3)δ:217.4,210.0,

[0061] 199.7,175.9,159.5,143.1,142.9,140.1,134.4,133.9,133.7,123. 4,121.1,115.2,106.5,103.1,72.3,69.8,68.9,58.8,53.9,51.7,50 .4,49.8,48.7,48.1,47.2,46.7,46.6,41.9,39.1,37.9,36.2,35.9,35.5,34.3,.2.8,28.9,27.4,20.7,19.7,19.5,19.4,18.0,17.2.HRMS calculated for C 45 H 60 N6O9Na[M+Na] + m / z 851.4314, found 851.4315.

[0062] Example 5 Synthesis of Inhibitor B5

[0063] The only difference from Example 1 is that the starting material for the synthesis of intermediate 1 is 7-nitroindole, and the amount added is calculated based on the molar ratio; the yield of inhibitor B5 is 89.1%.

[0064] Inhibitor B5: Yellow solid, yield 89.1%. 1H-NMR (600MHz, CDCl3) δ: 7.86 (dd, J=7.7, 1.0Hz, 1H, Indole-H), 7.78 (dd, J=7.7, 1.0Hz, 1H, Indole-H), 7.57 (s, 1H, Triazole-H), 7.46 (d, J=3.0Hz, 1H, I ndole-H),7.12(t,J=7.9Hz,1H,Indole-H),6.60(d,J=3.4Hz,1H,Indole-H),6.48(m,1H,CONH),5.56(s,2H,CH2-N(Indole)),4.72-4.59(m,1H,H-15),4. 60-4.57(m,1H,H-7),4.46-4.44(m,2H,CH2-N(Triazole)),3.80(t,J=4.9Hz,2H,OCH2),3.58-3.38(overlap,12H,NCH2CH2,OCH2CH2OCH2CH2O),1.98-1.9 2(m,1H,H-17),1.45-1.41(m,1H,H-1),1.25(s,3H,CH3),1.22(s,3H,CH3),1 .11-1.07(overlap,9H,3×CH3),0.90(s,3H,CH3),0.83(d,J=6.5Hz,3H,CH3). 13 C-APT(150MHz,CDCl3)δ:217.5,209.6,199.7,175.8,159.6,143.2,140.0,134.2 ,133.4,127.7,126.3,124.0,120.0,118.9,103.7,72.2,70.5,70.4,70.3,70.1,6 9.7,69.3,68.8,53.9,51.7,49.9,48.7,47.1,46.7,46.6,44.9,39.2,37.9,36.1, 35.9,34.3,32.6,28.9,27.4,20.7,19.8,19.5,19.4,18.0,17.2.HRMScalculated for C 49 H 68 N6O 11 Na[M+Na] + m / z 936.4838, found 939.4836.

[0065] Example 6 Synthesis of Inhibitor B6

[0066] The only difference from Example 1 is that the starting material for intermediate 1 is 7-nitroindole, and the amount added is calculated according to the molar ratio; the starting material for intermediate 2 is ganoderic acid A and amino-diethylene glycol-azide; and the yield of inhibitor B6 is 81.9%.

[0067] Inhibitor B6: Yellow solid, yield 81.9%. 1 H-NMR (600MHz, CDCl3) δ: 7.87 (d, J=7.6Hz, 1H, Indole-H), 7.78 (d, J=7.6Hz, 1H, Indole-H), 7.66 (s, 1H, Triazole-H), 7.48 (d, J=3.3Hz, 1H, Indole-H), 7.12(t,J=7.6Hz,1H,Indole-H),6.70(d,J=3.3Hz,1H,Indole-H),6.38(m,1H,CONH),5.57(q,J=15.9Hz,2H,CH2-N(Indole)),4.75-4.71(m,1H,H-15), 4.62-4.56(m,1H,H-7),4.51-4.52(m,2H,CH2-N(Triazole)),3.78-3.73(m ,2H,OCH2),3.50-3.46(m,2H,CH2O),3.46-3.26(m,2H,CONHCH2),1.98-1.90 (m,1H,H-17),1.49-1.41(m,1H,H-1),1.25(s,3H,CH3),1.22(s,3H,CH3),1. 12-1.05(overlap,9H,3×CH3),0.93(s,3H,CH3),0.83(d,J=6.5Hz,3H,CH3). 13 C-APT(150MHz,CDCl3)δ:216.4,208.5,198.6,175.1,158.4,142.2,139.0,13 5.5,133.2,132.5,126.8,128.2,123.1,119.2,117.9,102.7,71.2,68.8,67. 9,67.7,52.9,50.6,49.2,48.8,47.6,46.0,45.7,45.6,43.9,38.3,36.9,35. 1,34.9,34.5,33.3,31.6,27.8,26.3,19.6,18.7,18.5,18.4,17.0,16.2.HRMS calculated forC 45 H 60 N6O9Na[M+Na] +m / z 851.4314, found 851.4314.

[0068] Example 7 Synthesis of Inhibitor B7

[0069] The only difference from Example 1 is that the starting material for intermediate 1 is 4-methoxyindole, and the amount added is calculated according to the molar ratio; the starting material for intermediate 2 is ganoderic acid A and amino-diethylene glycol-azide; and the yield of inhibitor B7 is 91.9%.

[0070] Inhibitor B7: White solid, yield 91.9%. 1 H-NMR (600MHz, CDCl3) δ: 7.44 (s, 1H, Triazole-H), 7.13-7.11 (overlap, 2H, Indole-H), 7.00 (d, J = 8.2Hz, 1H, Indole-H), 6.62 (d, J = 3.1Hz, 1H, Ind ole-H),6.53(d,J=7.8Hz,1H,Indole-H),5.92(m,1H,CONH),5.43(s,2H,CH2-N(Indole)),4.75-4.69(m,1H,H-15),4.64-4.57(m,1H,H-7),4.48-4. 40(m,2H,CH2-N(Triazole)),3.94(s,3H,OCH3),3.76(t,J=5.1Hz,2H,OCH2),3.44-3.37(m,2H,CH2O),3.37-3.12(m,2H,CONHCH2),1.95-1.86(m,1 H,H-17),1.50-1.43(m,1H,H-1),1.26(s,3H,CH3),1.23(s,3H,CH3),1.11-1.06(overlap,9H,3×CH3),0.92(s,3H,CH3),0.83(d,J=6.3Hz,3H,CH3). 13C-APT(150MHz,CDCl3)δ:216.4,208.6,198.6,174.9,158.4,152.4,143..7,139.0,1 63.3,131.0,125.4,122.1,121.7,118.2,101.9,98.6,98.2,71.2,68.6,67.8,67.7,5 4.3,52.9,50.6,49.1,48.7,47.6,47.1,16.1,15.6,45.6,41.2,38.1,36.9,35.0,34 .8,34.5,33.3,31.6,27.8,26.3,19.6,18.7,18.5,18.4,16.9,16.2.HRMScalculated for C 46 H 63 N5O8Na[M+Na] + m / z 836.4569, found 836.4569.

[0071] Example 8 Synthesis of Inhibitor B8

[0072] The starting material for the synthesis of intermediate 1 was 7-methylindole, and the amount added was calculated based on the molar ratio; the starting material for the synthesis of intermediate 2 was ganoderic acid A and amino-diethylene glycol-azide; the yield of inhibitor B8 was 94.6%.

[0073] Inhibitor B8: White solid, yield 94.6%. 1H-NMR (600MHz, CDCl3) δ: 7.46 (d, J=7.2Hz, 1H, Indole-H), 7.16-10 (overlap, 2H, Triazole-H, Indole-H), 6.99 (t, J=7.3Hz, 1H, Indole-H), 7.89 (d, J =7.3Hz,1H,Indole-H),6.51(d,J=3.0Hz,1H,Indole-H),5.90-5.87(m,1H,CONH),5.67(s,2H,CH2-N(Indole)),4.74-4.71(m,1H,H-15),4.61-4.60( m,1H,H-7),4.44-4.37(m,2H,CH2-N(Triazole)),3.73(t,J=5.1Hz,2H,OCH2),3.37(t,J=5.5Hz,2H,CH2O),3.33-3.27(m,2H,CONHCH2),1.97-1.88(m ,1H,H-17),1.48-1.43(m,1H,H-1),1.25(s,3H,CH3),1.23(s,3H,CH3),1.10-1.07(overlap,9H,3×CH3),0.93(s,3H,CH3),0.83(d,J=6.4Hz,3H,CH3). 13 C-APT(150MHz,CDCl3)δ:217.4,209.6,199.7,175.9,159.4,146.8,140.1,134.6,13 2.1,129.9,129.7,124.8,122.7,121.0,120.1,119.3,102.8,82.3,69.7,68.9,68.8, 53.9,51.7,50.2,49.8,48.7,48.1,47.1,46.7,46.6,44.9,39.1,38.0,36.1,35.9,35 .5,34.3,32.7,28.9,27.4,20.7,19.8,19.7,19.5,19.4,18.1,17.3.HRMScalculated for C 46 H 63 N5O7Na[M+Na] + m / z 820.4619, found 820.4618.

[0074] Example 9 Synthesis of Inhibitor B9

[0075] In the synthesis of intermediate 1, the starting material was indole, and the amount added was calculated based on the molar ratio; in the synthesis of intermediate 2, the starting materials were ganoderic acid A and amino-diethylene glycol-azide; the yield of inhibitor B9 was 89.5%.

[0076] Inhibitor B9: White solid, yield 89.5%. 1 H-NMR (600MHz, CDCl3) δ: 7.62 (d, J = 7.7Hz, 1H, Indole-H), 7.42 (s, 1H, Triazole-H), 7.38 (d, J = 7.7Hz, 1H, Indole-H), 7.21 (d, J = 3.2Hz, 1H, Indole-H), 7.2 2-7.17(m,1H,Indole-H),7.10(m,1H,Indole-H),6.52(d,J=3.2Hz,1H,Indole-H),5.86(m,1H,CONH),5.45(s,2H,CH2-N(Indole)),4.73-4.70(m,1H,H-15 ),4.60-4.57(m,1H,H-7),4.47-4.40(m,2H,CH2-N(Triazole)),3.75(t,J=5. 0Hz,2H,OCH2),3.41-3.36(m,2H,CH2O),3.35-3.11(m,2H,CONHCH2),1.96-1. 88(m,1H,H-17),1.48-1.43(m,1H,H-1),1.25(s,3H,CH3),1.22(s,3H,CH3),1 .10-1.06(overlap,9H,3×CH3),0.93(s,3H,CH3),0.82(d,J=6.3Hz,3H,CH3). 13 C-APT(150MHz,CDCl3)δ:216.3,108.6,198.6,174.8,158.3,143.6,139.0,13 4.8,127.7,126.9,122.0,120.8,120.1,118.7,108.5,100.9,71.2,68.6,67. 8,67.7,52.9,50.6,49.2,48.7,47.6,47.1,46.1,45.6,40.9,38.0,36.9,35. 1,34.8,34.5,33.2,31.6,27.9,26.3,19.6,18.7,18.5,18.4,16.9,16.2.HRMS calculated forC 45 H 61 N5O7Na[M+Na] +m / z 806.4463, found 806.4463.

[0077] Example 10 Synthesis of Inhibitor B10

[0078] The starting material for the synthesis of intermediate 1 was 6-fluoroindole, and the amount added was calculated based on the molar ratio; the starting material for the synthesis of intermediate 2 was ganoderic acid A and amino-diethylene glycol-azide; the yield of inhibitor B10 was 91.9%.

[0079] Inhibitor B10: White solid, yield 91.9%. 1 H-NMR (600MHz, CDCl3) δ: 7.54-7.50 (overlap, 2H, Indole-H, Triazole-H), 7.20 (d, J = 3.0Hz, 1H, Indole-H), 7.08 (dd, J = 9.7, 1.8Hz, 1H, Indole-H), 6.68 (td, J =9.1,1.8Hz,1H,Indole-H),6.49(d,J=3.0Hz,1H,Indole-H),5.96(m,1H,CONH) ,5.38(s,2H,CH2-N(Indole)),4.74-4.72(m,1H,H-15),4.61-4.59(m,1H,H-7), 4.46(t,J=4.3Hz,2H,CH2-N(Triazole)),3.77(t,J=4.4Hz,2H,OCH2),3.77(t,J=5.1Hz,2H,OCH2),3.53-3.36(overlap,12H,NCH2CH2,OCH2CH2OCH2CH2O),2.0 0-1.92(m,1H,H-17),1.48-1.43(m,1H,H-1),1.26(s,3H,CH3),1.23(s,3H,CH3) ,1.10-1.07(overlap,9H,3×CH3),0.92(s,3H,CH3),0.83(d,J=6.3Hz,3H,CH3). 13C-APT(150MHz, CDCl3)δ:217.4,209.8,199.6,175.9,160.5,159.4,158.9,144.1,140. 1,13509,13508,132.1,128.4,125.2,123.1,121.9,108.6,108.4,102.2,96.32,95.9,7 2.3,69.7,68.9,68.8,53.9,51.7,50.3,49.8,48.7,48.1,47.0,46.7,46.6,42.1,39.1, 38.0,36.2,35.9,35.5,34.3,32.7,28.9,27.4,20.7,19.7,19.5,19.4,18.0,17.2.HRMS calculated for C 45 H 60 FN5O7Na[M+Na] + m / z824.4369, found 824.4370.

[0080] Example 11 Synthesis of Inhibitor B11

[0081] The only difference from Example 1 is that the starting material for intermediate 1 is indole, and the amount added is calculated based on the molar ratio; the yield of inhibitor B11 is 84.3%.

[0082] Inhibitor B11: White solid, yield 84.3%. 1H-NMR (600MHz, CDCl3) δ: 7.38 (s, 1H, Triazole-H), 7.12-7.19 (overlap, 2H, Indole-H), 7.12 (t, J=7.9Hz, 1H, Indole-H), 6.99 (d, J=7.9Hz, 1H, Indole-H) ,6.52(d,J=7.9Hz,1H,Indole-H),6.60(d,J=3.1Hz,1H,Indole-H),6.41(m,1 H,CONH),5.40(s,2H,CH2-N(Indole)),4.75-4.70(m,1H,H-15),4.62-4.57(m ,1H,H-7),4.45-4.42(m,2H,CH2-N(Triazole)),3.90(s,3H,OCH3),3.80(t,J=5.2Hz,2H,OCH2),3.51-3.35(m,12H,NCH2CH2,OCH2CH2OCH2CH2O),1.98-1. 93(m,1H,H-17),1.48-1.42(m,1H,H-1),1.26(s,3H,CH3),1.23(s,3H,CH3),1 .11-1.08(overlap,9H,3×CH3),0.92(s,3H,CH3),0.83(d,J=6.4Hz,3H,CH3). 13 C-APT(150MHz, CDCl3)δ:217.4,109.6,199.7,175.8,159.6,153.4,144.6,140. 0,137.3,126.4,123.2,122.8,119.2,103.0,99.6,99.3,72.3,70.5,70.4,70.3 ,70.1,69.7,69.3,68.8,55.3,54.0,51.7,50.4,50.0,48.7,48.2,47.1,46.7,46.6,42.2,39.2,38.0,36.1,35.9,5.5,34.3,32.4,28.9,27.4,20.7,19.8,19.5,

[0083] 19.4,18.1,17.2.HRMS calculated for C 49 H 69 N5O9Na[M+Na] + m / z 894.4987, found894.4980.

[0084] Example 12 Synthesis of Inhibitor B12

[0085] The only difference from Example 1 is that the starting material for the synthesis of intermediate 1 is 7-methylindole, and the amount added is calculated based on the molar ratio; the yield of inhibitor B12 is 96.3%.

[0086] Inhibitor B12: White solid, yield 96.3%. 1 H-NMR (600MHz, CDCl3) δ: 7.45 (d, J=7.8Hz, 1H, Indole-H), 7.12 (s, 1H, Triazole-H), 7.10 (d, J=3.1Hz, 1H, Indole-H), 7.98 (t, J=7.2Hz, 1H, Indole-H) ,6.89(d,J=7.2Hz,1H,Indole-H),6.51(d,J=3.0Hz,1H,Indole-H),6.39(m,1H,CONH),5.66(s,2H,CH2-N(Indole)),4.74-4.75(m,1H,H-15),4.61-4. 58(m,1H,H-7),4.41-4.38(m,2H,CH2-N(Triazole)),3.77(t,J=5.1Hz,2H,OCH2),3.50-3.36(overlap,12H,NCH2CH2,OCH2CH2OCH2CH2O),1.99-1.93( m,1H,H-17),1.48-1.44(m,1H,H-1),1.27(s,3H,CH3),1.23(s,3H,CH3),1.12-1.08(overlap,9H,3×CH3),0.93(s,3H,CH3),0.84(d,J=6.5Hz,3H,CH3). 13 C-APT(150MHz,CDCl3)δ:217.4,209.6,199.7,175.8,159.5,146.6,14031,132.6,129.9 ,129.6,124.9,122.7,120.9,120.1,119.3,102.7,72.3,70.5,70.4,70.3,70.1,69.7,6 8.8,53.9,51.7,50.3,51.7,50.3,49.9,48.7,48.2,47.1,46.7,46.6,44.9,339.2,37.9 ,36.2,35.9,35.5,34.3,32.7,28.9,27.4,20.7,19.8,19.7,19.5,19.4,18.1,17.2.HRMS calculated for C 50 H 71 N5O9Na[M+Na]+ m / z 908.5144, found 908.5150.

[0087] Example 13 Synthesis of Inhibitor B13

[0088] The only difference from Example 1 is that the starting material for the synthesis of intermediate 1 is 4-methoxyindole, and the amount added is calculated according to the molar ratio; the yield of inhibitor B13 is 94.8%.

[0089] Inhibitor B13: white solid, yield 94.8%. 1 H-NMR (600MHz, CDCl3) δ: 7.61 (d, J=7.4Hz, 1H, Indole-H), 7.41 (s, 1H, Triazole-H), 7.39 (t, J=6.7Hz, 1H, Indole-H), 7.20-7.16 (overlap, 2H, Indol e-H),6.85(m,1H,Indole-H),6.50(d,J=3.0Hz,1H,Indole-H),6.41(m,1H,CONH),5.43(s,2H,CH2-N(Indole)),4.73-4.71(m,1H,H-15),4.60-4.57( m,1H,H-7),4.44-4.42(m,2H,CH2-N(Triazole)),3.79(t,J=5.2Hz,2H,OCH2),3.51-3.35(overlap,12H,NCH2CH2,OCH2CH2OCH2CH2O),1.98-1.90(m, 1H,H-17),1.48-1.44(m,1H,H-1),1.25(s,3H,CH3),1.23(s,3H,CH3),1.10-1.07(overlap,9H,3×CH3),0.92(s,3H,CH3),0.82(d,J=6.7Hz,3H,CH3). 13C-APT(150MHz,CDCl3)δ:217.5,209.6,199.7 175.8,159.6,144.5,140.0,135.8,128.8,127.9,123.2,119.7,109.5,1 02.0,72.2,70.5,70.4,70.3,70.1,69.7,69.3,68.8,53.9,51.7,50.4,4 HRMScalculated for C 50 H 71 N5O 10 Na[M+Na] + m / z 924.5093, found 924.5091.

[0090] Example 14 Synthesis of Inhibitor B14

[0091] The only difference from Example 1 is that the starting material for the synthesis of intermediate 1 is 6-fluoroindole, and the amount added is calculated based on the molar ratio; the yield of inhibitor B14 is 97.1%.

[0092] Inhibitor B14: white solid, yield 97.1%. 1H-NMR (600MHz, CDCl3) δ: 7.52-7.50 (m, 1H, Indole-H), 7.47 (s, 1H, Triazole-H), 7.18 (d, J=3.1Hz, 1H, Indole-H), 7.06 (dd, J=9.9, 2.2Hz, 1H, Indol e-H),6.85(m,1H,Indole-H),6.49(d,J=3.0Hz,1H,Indole-H),6.42(m,1H,CONH),5.36(s,2H,CH2-N(Indole)),4.74-4.71(m,1H,H-15),4.61-4.58( m,1H,H-7),4.47-4.45(m,2H,CH2-N(Triazole)),3.80(t,J=4.9Hz,2H,OCH2),3.55-3.33(overlap,12H,NCH2CH2,OCH2CH2OCH2CH2O),1.96-1.89(m, 1H,H-17),1.49-1.42(m,1H,H-1),1.25(s,3H,CH3),1.23(s,3H,CH3),1.10-1.07(overlap,9H,3×CH3),0.92(s,3H,CH3),0.83(d,J=6.0Hz,3H,CH3). 13 C-APT(150MHz,CDCl3)δ:217.4,209.6,199.7,175.8,160.5,159.6,158.9,143.9,140.1,135 .9,135.8,131.8,128.4,125.2,123.3,121.9,121.8,108.5,108.4,102.2,96.1,95.9,72.3,7 0.5,70.3,70.1,69.7,69.3,68.8,53.9,51.7,50.4,49.9,48.7,48.2,47.1,46.7,46.6,42.1, 39.2,37.9,36.1,35.9,35.5,34.3,32.7,28.9,27.4,20.7,19.8,19.5,19.4,18.0,17.2.HRMS calculated for C 49 H 68 FN5O9Na[M+Na] + m / z912.4893, found 912.4890.

[0093] Example 15 Antiproliferative activity of inhibitor B1-B14 against different tumor cell lines and normal cell lines

[0094] Cell viability assay procedures: The cell lines used in this study were purchased from Wuhan Pronosei Life Sciences Co., Ltd. HepG2 cells were cultured in DMEM medium (Sole Pro) containing 10% fetal bovine serum (PAN) and 1% penicillin-streptomycin (Hyclone) under a humid environment of 37℃ and 5% CO2. SJSA-1 and MCF-7 cell lines were cultured in PRMI 1640 medium (Gibco) containing 10% fetal bovine serum (PAN) and 1% penicillin-streptomycin (Hyclone). HK-2 cells were cultured in DMEM / F12 (Gibco) (1:1) medium. Cell viability was determined using the MTT assay. The MTT reagent kit was purchased from Beyotime. MCF-7, HepG2, SJSA-1, and HK-2 cells (7×10⁶ cells / year) were cultured in a humid environment of 5% CO2. 3 Cells were seeded per well in 96-well plates and cultured in serum-free medium for 24 hours. Then, they were treated with 0.1% DMSO, 25, 50, or 100 μM ganoderic acid A derivatives (B1-B14), or ganoderic acid A (GAA) for 48 hours (MCF-7, HepG2, HK2) or 72 hours (SJSA-1). After 48 or 72 hours, 10 μL of MTT (5 mg / mL) was added, and the plates were incubated at 37°C for 4 hours. Then, 100 μL of dissolving buffer was added. After complete dissolution of the crystals, absorbance was measured at 540 nm under a microscope. Cell viability was calculated as: [OD value of drug-treated wells - OD value of blank wells] / [OD value of control wells - OD value of blank wells] × 100%.

[0095] The results are as follows Figure 1 As shown.

[0096] Figure 1 Figure A shows the antiproliferative activity of B1-B14 against the MCF-7 cell line (human breast cancer cells);

[0097] Figure 1 Figure B in the middle shows the antiproliferative activity of B1-B14 against the HepG2 cell line (human liver cancer cells);

[0098] Figure 1 Figure C shows the antiproliferative activity of B1-B14 against the SJSA-1 cell line (human osteosarcoma cells);

[0099] Figure 1 Figure D shows the antiproliferative activity of B1-B14 against the HK-2 cell line (human renal cortical proximal tubular epithelial cells).

[0100] The results showed that inhibitors B1, B2, B5, and B11-B14 all had high anti-proliferative activity against human breast cancer cells; inhibitors B1-B10 and B12-B14 all had high anti-proliferative activity against human liver cancer cells; and inhibitors B7 and B9 had high anti-proliferative activity against human osteosarcoma cells.

[0101] Example 16: Inhibitory activity test of compound B14 against the two-site domain of MDM2

[0102] (1) Inhibitory activity test of B14 against the MDM2RING domain

[0103] The effect of different concentrations of compound B14 on MDM2 protein in the HepG2 cell line was determined using Western blotting.

[0104] Western Blot Procedure: HepG2 cells were treated with 12.5, 25, and 50 μM compound B14 for 24 hours. The culture medium was discarded, and the cells were washed three times with pre-chilled PBS. Then, RIPA lysis buffer (Beyotime) containing PMSF and a phosphatase inhibitor (Beyotime) was added, and the cells were incubated on ice for 30 minutes to lyse. Cells were collected using a cell scraper and transferred to pre-chilled EP tubes, sonicated on ice for 10 minutes, and then centrifuged at 12000g for 30 minutes at 4°C to extract total protein. The concentration of each sample was calculated using the BCA protein quantification method (BCA kit purchased from Beyotime), and samples of different concentrations were diluted to the same factor using RIPA lysis buffer. Loading buffer was added, and the cells were heated at 100°C for 10 minutes to fully denature the proteins. After centrifugation, the cells were stored at -20°C. Proteins were separated using a 10% SDS-PAGE gel, with 20 μg of protein sample loaded into each well. Stacking gel electrophoresis was performed at 80V, followed by separating gel electrophoresis at 120V. Wet transfer was performed under a constant current of 300 mA to transfer proteins from the gel onto a PVDF membrane. The PVDF membrane was blocked with 5% BSA at room temperature for 2 hours. The membrane was then incubated overnight at 4°C with antibodies MDM2 (abcam, ab16895), P53 (Proteintech, 10442-1-AP), Bcl-2 (CST, 15071S), Bax (CST, 2772T), and β-actin (abcam, ab8226). The membrane was then washed three times with TBST for 10 minutes each time. Incubation with secondary antibody (Beyotime) at room temperature for 2 hours was performed. ECL chemiluminescence imaging solution (Beyotime, BeyoECL-star) was added, and the gel was detected using a gel imaging system (ClinxChemScope, China).

[0105] The results are as follows Figure 2 As shown, Figure 2The results in A showed that B14 could significantly degrade MDM2 protein at 50 μM. Figure 2 The B-value in the study showed that the degradation ability decreased after the addition of the protease system inhibitor MG-132 (purchased from MCE, catalog number 149688), indicating that the effect of B14 on MDM2 occurs through the proteasome system, further proving that B14 can inhibit the MDM2RING domain.

[0106] (2) Inhibitory activity test of B14 against the MDM2RING domain

[0107] Homogeneous time-resolved fluorescence (HTRF) was performed using the MDM2HTRF binding kit (PerkinElmer), which included GST Eu crystallization antibody, MI-1061red ligand, Standard, MDM2 protein, dilution buffer #9, and PROTACBinding Buffer. The GST Eu crystallization antibody, MI-1061red ligand, and MDM2 protein were diluted 50-fold with PROTACBinding Buffer, and equal volumes of GST Eu crystallization antibody and MI-1061red ligand were mixed as HTRF reagents. The Standard stock solution was diluted with dilution buffer #9 according to the recommended procedure to obtain a standard curve, with final concentrations of 2000, 400, 80, 16, 3.2, 0.64, 0.128, and 0 nM. The test samples were also diluted with dilution buffer #9 to concentrations of 250, 50, 10, 2, 0.4, 0.08, and 0.016 μM. Following the kit instructions, use a white 384-well plate. Add 5 μL of dilution buffer #9 and 5 μL of PROTAC binding buffer to each well in the negative control group. Add 5 μL of the corresponding sample and 5 μL of MDM2 protein to each well in the standard control and sample groups. Then add 10 μL of premixed HTRF and GST Eu antibody working solution to all wells, with 5 auxiliary wells for each sample. Seal and incubate at room temperature for 4 hours. Use a SpectraMax iD5 microplate reader (Molecular Devices) at an excitation wavelength of 350 nm to measure the values ​​at 665 nm and 620 nm, respectively, and calculate the ratio: Ratio = Signal 665nm / Signal 620nm × 10⁻¹⁰. 4 The IC50 value was calculated by plotting the logarithm of concentration on the x-axis and Ratio on the x-axis. The MDM2-p53 interaction inhibitor Nutlin-3a (purchased from Sigma-Aldrich, catalog number 444152) was used as a positive control to determine the in vitro competitive inhibitory effect of the derivative. The results are shown in Table 1.

[0108] Table 1. In vitro competitive inhibition of MDM2 by compound B14

[0109]

[0110] The results in Table 1 show that compound B14 has a certain inhibitory effect on the MDM2-p53 interaction in vitro.

[0111] Figure 2 The results in Table 1 show that B14 can inhibit both the MDM2RING domain and the MDM2-p53 binding domain, thus playing a dual-site inhibitory role.

Claims

1. The application of a triterpenoid compound in the preparation of an antitumor drug, characterized in that, The drug is selected from anti-liver cancer drugs, anti-breast cancer drugs, and anti-human osteosarcoma drugs; when the drug is an anti-liver cancer drug, the drug is selected from one or more of B7 or B14; when the drug is an anti-breast cancer drug, the drug is selected from one or more of B1, B2, B5, B11-B14; when the drug is an anti-human osteosarcoma drug, the drug is selected from one or two of B7 or B9. 。 2. The application according to claim 1, characterized in that, The drug dosage form is tablet, capsule, emulsion, suspension, injection or infusion; the drug contains pharmaceutically acceptable excipients.

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