A compound with an aggregation-induced emission center, a preparation method thereof, and an application thereof

Abstract

The present invention discloses a compound having an aggregation-induced emission center, a preparation method thereof and an application thereof. The structural formula of the compound is shown as formula (I), formula (II) or formula (III): The compound having an aggregation-induced emission center of the present invention not only has excellent anti-tumor activity, but also has fluorescence characteristics, and can be applied to the preparation of candidate compounds for anti-tumor drugs and aggregation-induced fluorescence imaging of cancer cells.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis, and more specifically to a compound having an aggregation-induced emission center and a preparation method and application thereof. Background Art

[0002] At present, cancer is still one of the most devastating diseases to the human body. Scientists have never stopped their research on cancer and have developed many therapies, which are of great help in treating early-stage cancer patients and can extend the patient's life by months or even years. However, any therapy cannot be separated from the assistance of anti-cancer drugs. Among the many anti-cancer drugs, natural drug active small molecule anti-cancer drugs have been widely studied, such as pentacyclic triterpenes with broad biological activity.

[0003] Pentacyclic triterpenes (PT) are natural secondary metabolites from plants, fungi, marine invertebrates and many other organisms. Natural and semisynthetic pentacyclic triterpenes usually have cytotoxic or antitumor, antiviral, antiparasitic, antibacterial, anti-inflammatory, hepatoprotective, renal protective, neuroprotective, cardioprotective, anti-ulcer, antifeedant, antidiabetic, etc. However, although many pentacyclic triterpenoids such as boswellic acid, oleanolic acid, betulin, betulin, etc. have a wide range of physiological and pharmacological activities and have good performance in antitumor (Molecule, 2015, 20, 1610-1625; Cancer Letters, 2012, 320, 158–170), these pentacyclic triterpenoid derivatives do not have aggregation fluorescence properties, and it is impossible to observe their aggregation degree and distribution in cells, tissues and animals, making it difficult to study their anticancer mechanism. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects and shortcomings of existing pentacyclic triterpenoid compounds that they do not have fluorescent properties, and to provide a compound with an aggregation-induced emission center. On the premise that the anti-tumor activity is equivalent to or further improved on that of the pentacyclic triterpenoid compounds, through structural modification and optimization, the carboxylic acid on the pentacyclic triterpenoid is replaced by a brominated hydrocarbon structure to generate an ester bond reaction to connect the fluorescent group, thereby giving the pentacyclic triterpenoid compounds fluorescent properties.

[0005] Another object of the present invention is to provide a method for preparing the above compound having aggregation-induced emission centers.

[0006] Another object of the present invention is to provide the use of the above compound having aggregation-induced emission center in the preparation of anti-tumor drugs.

[0007] Another object of the present invention is to use the above compound having aggregation-induced emission centers in fluorescence imaging of cancer cells.

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

[0009] The present invention protects a compound having an aggregation-induced emission center, and the structural formula of the compound is shown in Formula (I), Formula (II) or Formula (III):

[0010]

[0011] The compound with an aggregation-induced emission center of the present invention not only retains the inhibitory effect of the parent pentacyclic triterpene structure on cancer cells, but also has a quaternary ammonium salt structure connected to the parent, making it have good water solubility, which is conducive to the absorption and utilization of drugs containing the compound in the body. At the same time, a fluorescent group is connected through an ester bond reaction by substituting the carboxylic acid on the pentacyclic triterpene with a bromohydrocarbon structure. The co-localization degree of the derivative and mitochondria is observed and calculated using a confocal fluorescence microscope. Finally, the aggregation position of the pentacyclic triterpene aggregation-induced fluorescence derivative in cells is obtained, enabling the visualization of pentacyclic triterpene compounds in living cells, thereby quantifying the uptake and distribution of substances in cells, especially realizing the co-localization degree of pentacyclic triterpene compounds and mitochondria in cells, and thus realizing the research on the anti-cancer mechanism of pentacyclic triterpene compounds.

[0012] The present invention also protects a preparation method of the above compound, including the following steps:

[0013] Dissolve Compound A and Compound B in an organic solvent, react at 90-110 °C for 10-12 h, and separate and purify to obtain a compound having an aggregation-induced emission center;

[0014] Among them, Compound A is Compound B is

[0015] Any one of them.

[0016] Specifically, the molar ratio of Compound A to Compound B is 1:1.

[0017] Specifically, Compound A can be prepared by the following preparation method:

[0018] S1. Under the protection of nitrogen, dissolve 4,7-dibromobenzothiadiazole, 4-borotriphenylamine, potassium carbonate and tetrakis(triphenylphosphine)palladium in a mixed solution of tetrahydrofuran and water, and heat the reaction system to 65-75 °C, react for 7-9 h, and separate and purify to obtain Intermediate 1;

[0019] S2. Dissolve Intermediate 1, pyridine-4-boronic acid, potassium carbonate and tetrakis(triphenylphosphine)palladium in a mixed solution of tetrahydrofuran and water, react at 65-75 °C for 7-9 h, and separate and purify to obtain Compound A.

[0020] Among them, the CAS number of the above-mentioned 4,7-dibromobenzothiadiazole is 15155-41-6, the CAS number of 4-(triphenylamino)phenylboronic acid is 201802-67-7; the CAS number of pyridine-4-boronic acid is 1692-15-5.

[0021] Specifically, in the mixed solution of tetrahydrofuran and water described in the above steps S1 and S2, the volume ratio of tetrahydrofuran to water is 10:1.

[0022] Specifically, in the above step S1, the molar ratio of 4,7-dibromobenzothiadiazole: 4-(triphenylamino)phenylboronic acid: tetrakis(triphenylphosphine)palladium: potassium carbonate is 0.15:0.15:0.0070:1.00; in the above step S1, the molar ratio of 4,7-dibromobenzothiadiazole: pyridine-4-boronic acid: tetrakis(triphenylphosphine)palladium: potassium carbonate is 0.15:0.15:0.0070:1.00.

[0023] Specifically, the compound B can be prepared by the following preparation method:

[0024] Dissolve the reactant a and 1,4-dibromobutane in acetone, add potassium carbonate at the same time, and raise the temperature of the reaction system to 60-70 °C, and react for 3-5 h to obtain the compound B;

[0025] Among them, the reactant a is any one of betulinic acid, oleanolic acid and ursolic acid.

[0026] The above-mentioned CAS number of betulinic acid is 472-15-1, the CAS number of oleanolic acid is 508-02-1, and the CAS number of ursolic acid is 2955-27-3.

[0027] Specifically, the molar ratio of the reactant a: 1,4-dibromobutane: potassium carbonate is 1:(2-4):1.

[0028] The application of the above-mentioned compound with an aggregation-induced emission center in the preparation of anti-tumor drugs is also within the protection scope of the present invention.

[0029] The present invention also protects the application of the above-mentioned compound with an aggregation-induced emission center in the fluorescence imaging of cancer cells.

[0030] Compared with the prior art, the present invention has the following beneficial technical effects:

[0031] In the present invention, a quaternary ammonium salt is formed by the quaternization reaction of a bromohydrocarbon structure and a pyridine structure to introduce a cation, thereby increasing the mitochondrial targeting effect of the derivative in cells, and further improving the anti-tumor activity of the compound with an aggregation-induced emission center.

[0032] In the present invention, an ester bond is formed by substituting a carboxylic acid on pentacyclic triterpenoids with a bromohydrocarbon structure to connect a fluorescent group. The degree of co-localization between the derivative and mitochondria is observed and calculated using a confocal fluorescence microscope, and finally the aggregation position of pentacyclic triterpenoid compounds in cells is obtained, enabling the visualization of pentacyclic triterpenoid compounds in living cells, thereby quantifying the uptake and distribution of substances in cells. In particular, the degree of co-localization between pentacyclic triterpenoid compounds and intracellular mitochondria can be achieved, thus realizing the research on the anti-cancer mechanism of pentacyclic triterpenoid compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a synthetic route diagram of compound A of the present invention.

[0034] Figure 2 It is a synthetic route diagram of compound B of the present invention.

[0035] Figure 3 It is a confocal fluorescence microscopy imaging diagram of compound 1 in HeLa cells in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention will be further described below in conjunction with the specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the raw material reagents used in the embodiments of the present invention are conventionally purchased raw material reagents.

[0037] Compound A

[0038] Compound A can be prepared by the following preparation method (the synthetic route is as Figure 1 shown), and specifically includes the following steps:

[0039] S1. Mix 4,7-dibromobenzothiadiazole (437.7 mg, 1.50 mmol) with 4-borotriphenylamine (433.7 mg, 1.50 mmol), potassium carbonate (1382.1 mg, 10.00 mmol), and tetrakis(triphenylphosphine)palladium (80.9 mg, 0.070 mmol), place them in a 100 mL round-bottom flask, add 50 mL of THF and 5 mL of water, heat and reflux at 70 °C for 8 h, and detect the progress of the reaction by thin-layer chromatography TLC (the developing agent is V 石油醚 : V 乙酸乙酯 = 20:1), cool, concentrate under reduced pressure, extract with ethyl acetate (50 mL × 3), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and perform silica gel column chromatography (the eluent is V 石油醚 : V 乙酸乙酯 = 10:1) to obtain intermediate 1, which is an orange-yellow powder (236.9 mg) with a yield of 34.6%;

[0040] S2. Mix the intermediate 1 (685.5 mg, 1.50 mmol) described in S1 with pyridine-4-boronic acid (184.6 mg, 1.50 mmol), potassium carbonate (1382.1 mg, 10.00 mmol), and tetrakis(triphenylphosphine)palladium (80.9 mg, 0.070 mmol) in a 100 mL round-bottom flask. Add 50 mL of THF and 5 mL of water, and heat under reflux at 70 °C for 8 h. Monitor the progress of the reaction by thin-layer chromatography TLC (the developing solvent is V 石油醚 : V 乙酸乙酯 = 2:1). Cool, concentrate under reduced pressure, extract with ethyl acetate (50 mL × 3), dry over anhydrous sodium sulfate, concentrate under reduced pressure, and perform silica gel column chromatography (the eluent is V 石油醚 : V 乙酸乙酯 = 1:1) to obtain compound A as an orange-yellow powder (278.8 mg) with a yield of 40.8%.

[0041] Compound B1

[0042] Compound B1 can be prepared by the following method (the synthetic route is as shown in Figure 2 ), and specifically includes the following steps:

[0043] Mix betulinic acid (456.7 mg, 1.00 mmol) with 1,4-dibromobutane (855.6 mg, 4.00 mmol) and potassium carbonate (138.2 mg, 1.00 mmol) in 20 mL of acetone, heat under reflux at 65 °C for 4 h, and monitor the progress of the reaction by TLC (the developing solvent is V 石油醚 : V 乙酸乙酯 = 3:1). Cool, concentrate under reduced pressure, and perform silica gel column chromatography (the eluent is V 石油醚 : V 乙酸乙酯 = 2:1) to obtain compound B1 as a white powder (500.0 mg) with a yield of 84.7%.

[0044] Compound B2

[0045] Compound B2 can be prepared by the following method (the synthetic route is as shown in Figure 2 ), and specifically includes the following steps:

[0046] Mix oleanolic acid (456.7 mg, 1.00 mmol) with 1,4-dibromobutane (855.6 mg, 4.00 mmol) and potassium carbonate (138.2 mg, 1.00 mmol) in 20 mL of acetone, heat under reflux at 65 °C for 4 h, and monitor the progress of the reaction by TLC (the developing solvent is V 石油醚 : V 乙酸乙酯 = 3:1). Cool, concentrate under reduced pressure, and perform silica gel column chromatography (the eluent is V 石油醚 : V 乙酸乙酯= 2:1) Compound B2 was obtained as a white powder (512.5 mg) with a yield of 86.8%.

[0047] Compound B3

[0048] Compound B3 can be prepared by the following preparation method (the synthetic route is as Figure 2 shown), and specifically includes the following steps:

[0049] Ursolic acid (456.7 mg, 1.00 mmol), 1,4-dibromobutane (855.6 mg, 4.00 mmol), and potassium carbonate (138.2 mg, 1.00 mmol) were mixed and placed in 20 mL of acetone, and heated under reflux at 65 °C for 4 h. The progress of the reaction was detected by TLC (the developing agent was V 石油醚 :V 乙酸乙酯 = 3:1). After cooling, it was concentrated under reduced pressure, and silica gel column chromatography (the eluent was V 石油醚 :V 乙酸乙酯 = 2:1) gave Compound B3 as a white powder (522.6 mg) with a yield of 88.5%.

[0050] Example 1

[0051] A compound with an aggregation-induced emission center (denoted as Compound I), and its structural formula is as follows:

[0052]

[0053] The above compound with an aggregation-induced emission center can be prepared by the following preparation method:

[0054] The above Compound A (228.1 mg, 0.50 mmol) and Compound B1 (295.2 mg, 0.50 mmol) were mixed and placed in 10 mL of acetonitrile, and heated under reflux at 90 °C for 12 h. After cooling, it was concentrated under reduced pressure, and silica gel column chromatography (the eluent was V 二氯甲烷 :V 甲醇 = 40:1) gave Compound I as a black-purple powder (281.0 mg) with a yield of 53.7%.

[0055] The above Compound I was subjected to nuclear magnetic resonance detection, and the results were as follows:

[0056] Data for Ⅰ: mp: 235 - 242 °C;

[0057] 11H NMR (400 MHz, Chloroform-d) δ 9.52–9.46 (m, 2H), 8.96–8.89 (m, 2H), 8.31 (d, J = 7.7 Hz, 1H), 7.95–7.83 (m, 3H), 7.32 (dd, J = 8.5, 7.3 Hz, 4H), 7.24–7.08 (m, 8H), 5.13 (t, J = 7.5 Hz, 2H), 4.71 (d, J = 2.3 Hz, 1H), 4.59 (t, J = 1.9 Hz, 1H), 4.27–4.07 (m, 2H), 3.14 (dd, J = 11.5, 4.5 Hz, 1H), 2.95 (td, J = 10.8, 4.5 Hz, 1H), 2.25–2.17 (m, 1H), 2.21–2.13 (m, 1H), 2.19–2.00 (m, 1H), 1.85 (h, J = 6.3, 5.9 Hz, 3H), 1.66 (s, 3H), 1.62–1.51 (m, 1H), 1.48–1.18 (m, 6H), 1.17–1.09 (m, 1H), 1.06–0.85 (m, 7H), 0.87–0.80 (m, 1H), 0.78 (s, 3H), 0.65 (d, J = 7.6 Hz, 6H);

[0058] 13 13C NMR (101 MHz, Chloroform-d) δ 176.26, 153.98, 153.02, 152.64, 150.38, 149.53, 146.92, 144.48, 138.47, 132.26, 130.49, 129.55, 128.46, 126.41, 126.34, 125.55, 124.13, 122.99, 121.64, 109.74, 78.88, 62.32, 60.42, 56.58, 55.25, 50.48, 49.39, 47.00, 42.40, 40.70, 38.79, 38.66, 38.25, 37.12, 34.30, 30.61, 29.70, 28.63, 27.93, 27.34, 25.60, 25.49, 20.89, 19.32, 18.20, 16.09, 16.04, 15.31, 14.66;

[0059] MS (m / z): 967.5 [M - Br] + 。

[0060] Example 2

[0061] A compound with an aggregation-induced emission center (denoted as Compound II), whose structural formula is as follows:

[0062]

[0063] The above-mentioned compound with an aggregation-induced emission center can be prepared by the following preparation method:

[0064] Mix the above-mentioned compound A (228.1 mg, 0.50 mmol) and compound B2 (295.2 mg, 0.50 mmol), place them in 10 mL of acetonitrile, heat under reflux at 90 °C for 12 h, cool, concentrate under reduced pressure, and perform silica gel column chromatography (the eluent is V 二氯甲烷 :V 甲醇 = 40:1), to obtain compound II, which is a black-purple powder (297.2 mg), and the yield is 56.8%.

[0065] Perform nuclear magnetic resonance detection on the above-mentioned compound II, and the results are as follows:

[0066] Data DorⅡ: mp: 214 - 219 °C;

[0067] 1 1H NMR (400 MHz, ChloroDorm-d) δ 9.47 (d, J = 6.7 Hz, 2H), 8.93 (d, J = 6.7 Hz, 2H), 8.33 (dd, J = 7.6, 1.9 Hz, 1H), 7.90 (dd, J = 8.9, 2.4 Hz, 2H), 7.86 (dd, J = 7.8, 2.3 Hz, 1H), 7.32 (t, J = 7.7 Hz, 4H), 7.19 (t, J = 8.4 Hz, 6H), 7.12 (t, J = 7.3 Hz, 2H), 5.23 (d, J = 3.8 Hz, 1H), 5.11 (dp, J = 12.6, 6.0 Hz, 2H), 4.17–4.07 (m, 2H), 3.15 (dd, J = 11.2, 4.6 Hz, 1H), 2.83 (dd, J = 14.1, 4.4 Hz, 1H), 2.17 (dp, J = 14.2, 7.0 Hz, 2H), 2.06 (d, J = 16.2 Hz, 1H), 1.95 (td, J = 13.5, 3.8 Hz, 1H), 1.88–1.79 (m, 4H), 1.74–1.63 (m, 1H), 1.60 (d, J = 13.7 Hz, 2H), 1.54–1.44 (m, 4H), 1.43 (s, 2H), 1.41–1.30 (m, 1H), 1.30–1.21 (m, 2H), 1.19 (d, J = 4.3 Hz, 1H), 1.17–1.10 (m, 1H), 1.10 (s, 3H), 1.04 (d, J = 14.0 Hz, 1H), 1.00–0.82 (m, 10H), 0.74 (s, 3H), 0.67 (s, 3H), 0.64 (s, 0H), 0.62 (s, 3H);

[0068] 13 C NMR(101MHz, Chloroform - d) δ 177.94, 153.96, 153.00, 152.55, 149.52, 146.92, 144.48, 143.80, 138.55, 132.32, 130.48, 129.55, 128.45, 126.38, 125.55, 124.12, 122.31, 121.63, 78.87, 62.86, 60.49, 55.10, 47.49, 46.82, 45.83, 41.73, 41.34, 39.31, 38.67, 38.35, 36.92, 33.82, 33.04, 32.70, 32.60, 30.68, 28.67, 28.06, 27.67, 27.10, 25.80, 25.45, 23.68, 23.38, 23.04, 18.26, 17.14, 15.53, 15.27;

[0069] MS(m / z): 967.5[M - Br] + 。

[0070] Example 3

[0071] A compound with an aggregation - induced emission center (denoted as Compound III), whose structural formula is as follows:

[0072]

[0073] The above - mentioned compound with an aggregation - induced emission center can be prepared by the following method:

[0074] Mix the above - mentioned Compound A (228.1 mg, 0.50 mmol) and Compound B3 (295.2 mg, 0.50 mmol), place them in 10 mL of acetonitrile, heat under reflux at 90 °C for 12 h, cool, concentrate under reduced pressure, and perform silica gel column chromatography (the eluent is V 二氯甲烷 : V 甲醇 = 40:1), then Compound III is obtained as a black - purple powder (260.6 mg), and the yield is 49.8%.

[0075] Perform nuclear magnetic resonance detection on the above - mentioned Compound III, and the results are as follows:

[0076] Data for III: mp: 188 - 193 °C;

[0077] 11H NMR (400 MHz, Chloroform-d) δ 9.47 (d, J = 6.5 Hz, 2H), 8.93 (d, J = 6.4 Hz, 2H), 8.33 (d, J = 7.6 Hz, 1H), 7.88 (dd, J = 17.8, 8.0 Hz, 3H), 7.32 (t, J = 7.7 Hz, 4H), 7.15 (dt, J = 25.1, 7.8 Hz, 8H), 5.18 (d, J = 3.8 Hz, 1H), 5.11 (td, J = 7.4, 3.0 Hz, 2H), 4.09 (dt, J = 13.1, 6.7 Hz, 3H), 3.15 (dd, J = 11.5, 4.4 Hz, 1H), 2.17 (dd, J = 15.0, 8.9 Hz, 3H), 2.15–1.92 (m, 3H), 1.74 (ddd, J = 17.3, 14.2, 8.4 Hz, 3H), 1.70–1.61 (m, 3H), 1.60–1.38 (m, 8H), 1.28 (tdd, J = 14.0, 10.4, 5.8 Hz, 6H), 1.04 (s, 3H), 0.92 (d, J = 5.3 Hz, 6H), 0.84 (d, J = 6.3 Hz, 3H), 0.77 (s, 3H), 0.66 (d, J = 14.9 Hz, 7H);

[0078] 13 13C NMR (101 MHz, Chloroform-d) δ 177.72, 153.85, 153.00, 152.54, 149.52, 146.92, 144.49, 138.60, 138.32, 132.31, 130.48, 129.55, 128.44, 126.37, 125.55, 125.42, 124.12, 122.94, 121.62, 78.89, 62.83, 60.52, 55.11, 52.86, 48.20, 47.44, 42.07, 39.53, 39.08, 38.83, 38.67, 38.54, 36.87, 32.99, 30.62, 28.70, 28.10, 28.00, 27.14, 25.42, 24.22, 23.49, 23.28, 21.14, 18.24, 17.20, 16.99, 15.58, 15.43;

[0079] MS (m / z): 967.5 [M - Br] + 。

[0080] Performance Test

[0081] 1. Cytotoxicity Control Test on Cancer Cells and Normal Cells

[0082] (1) Experimental materials

[0083] Control drugs: Betulinic acid, oleanolic acid, and ursolic acid were all purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.; doxorubicin hydrochloride was purchased from Anhui Zesheng Technology Co., Ltd.

[0084] Experimental drugs: Compound I, Compound II, and Compound III prepared in the present invention.

[0085] Cell culture: Hela (human cervical cancer cells), MDA-MB-231 (human breast cancer cells), MCF-7 (human breast cancer cells), Hep G2 (human liver cancer cells), and penicillin-streptomycin solution (100×) were all purchased from Wuhan Punosai Life Science Co., Ltd.;

[0086] HSF (human skin fibroblasts) was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.;

[0087] DMEM culture medium, fetal bovine serum, and trypsin were purchased from Gibco, USA;

[0088] Cell Counting Kit-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) was purchased from MedChemexpress Biotechnology Co., USA;

[0089] DMSO (dimethyl sulfoxide) was purchased from Sigma, USA;

[0090] PBS (phosphate buffer solution) was purchased from Solarbio Co., Ltd.

[0091] Instruments: Biotek Elx800 microplate reader: Biotek, USA; pipette: Eppendorf China Co., Ltd.; carbon dioxide incubator: Thermo, USA; autoclave: Panasonic, Japan.

[0092] (2) Experimental methods

[0093] Hela (human cervical cancer cells), MDA-MB-231 (human breast cancer cells), MCF-7 (human breast cancer cells), HepG2 (human liver cancer cells), and HSF (human skin fibroblasts) were cultured in DMEM culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin in a 5% CO 2 , saturated humidity incubator at 37 °C.

[0094] For the above cells: Collect cells in the logarithmic growth phase, inoculate them in a 96-well culture plate with DMEM culture medium containing 10% fetal bovine serum, 7000 cells per well, 100 μL of culture medium per well, and culture them in the above incubator for 24 h to allow the cells to adhere completely. After complete adhesion, replace the solution in each well with 100 μL of DMEM culture medium containing 10% fetal bovine serum with different concentrations of the drug, and set 3 controls for each concentration. After culturing in the dark for 48 h, replace the solution in each well with 100 μL of basic DMEM culture medium without fetal bovine serum containing 10% CCK-8. After culturing in the dark for 1 h - 2 h, measure the absorbance value of each well at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0095] All results were defined as the drug concentration when 50% of the tumor cells survived according to the half-maximal inhibitory concentration (IC 50 value). The cell growth inhibition rate was calculated using the following method as an evaluation index.

[0096] Inhibition rate (%) = [1 - (mean OD value of the experimental group - mean OD value of the blank group) / (mean OD value of the control group - mean OD value of the blank group)] × 100%. According to the cell growth inhibition rate, the IC 50 value was calculated by linear regression method.

[0097] (3) Experimental results

[0098] The results of the active control experiment are shown in Table 1. Betulinic acid (BA), oleanolic acid (OA), ursolic acid (UA), compound I, compound II, compound III, doxorubicin hydrochloride (DX) toxicity a control study.

[0099] Table 1

[0100]

[0101]

[0102] a The value represents the IC 50 mean ± standard deviation (SD) from three independent experiments. Compared with BA, b P < 0.0001; compared with BA, D P < 0.001; compared with OA, c P < 0.0001; compared with UA, d P < 0.0001; e DX was used as an anti-tumor positive control drug.

[0103] As can be seen from Table 1, compared with betulinic acid, oleanolic acid and ursolic acid, Compounds I, II and III of the present invention all showed higher inhibitory effects on the proliferation of Hela, MDA-MB-231, MCF-7 and Hep G2 cell lines, among which the inhibitory effect of Compound II was the most obvious.

[0104] 2. Aggregation-induced confocal fluorescence microscopy imaging test

[0105] (1) Experimental materials

[0106] Experimental drug: Compound I prepared in Example 1 of the present invention;

[0107] Cell culture: HeLa cell line was purchased from Wuhan Punosai Life Science Co., Ltd.;

[0108] DMEM culture medium, fetal bovine serum, and trypsin were purchased from Gibco, USA;

[0109] DMSO (dimethyl sulfoxide) was purchased from Sigma, USA;

[0110] PBS (phosphate buffer solution) was purchased from Solarbio Life Sciences Co., Ltd.

[0111] Instruments: LSM 880 conDocal laser scanning microscope: Carl Zeiss, Germany; Pipette: Eppendorf China Co., Ltd.; Carbon dioxide incubator: Thermo, USA; Autoclave: Panasonic, Japan.

[0112] Experimental reagents and consumables: 35-mm confocal dish (801001): Wuxi NEST Biotechnology Co., Ltd. Live cell imaging medium (FluoroBrite TM DMEM): Thermo Fisher Scientific (China) Co., Ltd.

[0113] Mitochondrial deep red fluorescence probe (Mito Deep Red FM): Yeasen Biotech Co., Ltd.

[0114] (2) Experimental method

[0115] The Hela cells were cultured in DMEM culture medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 mg / mL streptomycin in a 5% CO 2 , saturated humidity incubator at 37 °C.

[0116] For the above cells: Collect cells in the logarithmic growth phase, inoculate them in a 35-mm confocal dish with DMEM culture medium containing 10% fetal bovine serum. The inoculation cell specification is 4,100 cells / cm 2 culture area, and culture them in the above incubator for 24 h to allow the cells to adhere completely. After complete adhesion, add serum-free culture medium containing Compound I, with the concentration of Compound I being 1 μmol / L. After culturing in the incubator for 3 - 6 h, wash three times with PBS, add serum-free culture medium containing a mitochondrial deep red fluorescent probe, with the concentration of the mitochondrial deep red fluorescent probe being 250 nmol / L. After culturing in the incubator for 0.5 h, wash three times with PBS, add 2 mL of the above FluoroBrite TM DMEM, and take pictures of the intracellular fluorescence distribution under an inverted confocal fluorescence microscope. The experimental results are as Figure 3 shown, where 3a is the imaging result of Compound I (channel 405 nm), 3b is the imaging result of the commercial mitochondrial deep red fluorescent probe (channel 633 nm), and 3c is the superimposed image of channel 405 nm and channel 633 nm.

[0117] From Figure 3 the experimental results and the principle of aggregation-induced fluorescence group luminescence, it can be concluded that Compound I can smoothly penetrate the cell membrane and show aggregation-induced fluorescence luminescence properties in cells under the action of low concentration (1 μmol / L) and long time (3 - 6 h), which can analyze the aggregation position of pentacyclic triterpenoid fluorescent derivatives. Compounds II and III, similar in properties to Compound I, can both penetrate the cell membrane and aggregate in mitochondria, producing targeted antitumor effects.

[0118] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A compound with an aggregation-induced emission center, characterized in that, the structural formula of the compound is as follows:

2. A preparation method of the compound according to claim 1, characterized in that, it comprises the following steps: Dissolve compound A and compound B in an organic solvent, react at 90 - 110 °C for 10 - 12 h, and separate and purify to obtain a compound with an aggregation-induced emission center; Among them, the compound A is The compound B is 3. The preparation method of the compound according to claim 2, characterized in that, the molar ratio of compound A to compound B is 1:

1.

4. The preparation method of the compound according to claim 2, characterized in that, compound A is prepared by the following preparation method: S1. Under the protection of nitrogen, dissolve 4,7-dibromobenzothiadiazole, 4-boronic acid triphenylamine, potassium carbonate and tetrakis(triphenylphosphine)palladium in a mixed solution of tetrahydrofuran and water, heat the reaction system to 65 - 75 °C, react for 7 - 9 h, and separate and purify to obtain intermediate 1; S2. Dissolve intermediate 1, pyridine-4-boronic acid, potassium carbonate and tetrakis(triphenylphosphine)palladium described in S1 in a mixed solution of tetrahydrofuran and water, react at 65 - 75 °C for 7 - 9 h, and separate and purify to obtain compound A.

5. The preparation method of the compound according to claim 4, characterized in that, the volume ratio of tetrahydrofuran to water in the mixed solution of tetrahydrofuran and water in S1 and S2 is (8 - 12):

1.

6. The preparation method of the compound according to claim 4, characterized in that, the molar ratio of 4,7-dibromobenzothiadiazole:4-boronic acid triphenylamine:tetrakis(triphenylphosphine)palladium:potassium carbonate in S1 is 0.15:0.15:0.0070:(1 - 2).

7. The preparation method of the compound according to claim 2, characterized in that, compound B is prepared by the following preparation method: Dissolve reactant a and 1,4-dibromobutane in acetone, add potassium carbonate at the same time, and heat the reaction system to 60 - 70 °C, react for 3 - 5 h to obtain compound B; wherein, the reactant a is oleanolic acid.

8. The preparation method of the compound according to claim 7, characterized in that, the molar ratio of reactant a:1,4-dibromobutane:potassium carbonate is 1:(2 - 4):

1.

9. The application of the compound with an aggregation-induced emission center according to claim 1 in the preparation of anti-tumor drugs; the tumor is at least one of cervical cancer, breast cancer or liver cancer.

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