A pentacyclic triterpene aggregation-induced fluorescence derivative, its preparation method and application

Abstract

The present invention discloses a pentacyclic triterpenoid aggregation-induced fluorescence derivative, a preparation method thereof and an application thereof. The structural formula of the pentacyclic triterpenoid aggregation-induced fluorescence derivative of the present invention is shown in Formula (I), Formula (II) or Formula (III): #imgabs0# wherein R is #imgabs1# The pentacyclic triterpenoid aggregation-induced fluorescence derivative of the present invention not only has excellent anti-tumor activity, but also is a salt, has better water solubility, can significantly improve pharmacokinetic parameters and improve bioavailability; moreover, it can show aggregation-induced fluorescence characteristics in cells, and further better analyze the mechanism of action of the compound in cells, and can be widely used in 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 pentacyclic triterpene aggregation-induced fluorescence derivative, a preparation method thereof, and an application thereof. Background Art

[0002] As is well known, mitochondria are organelles that provide energy in cells and are called the "energy factories" of cells. The growth of cancer cells not only requires mitochondria to obtain energy, but also requires mitochondria to support various structures required for their wild proliferation. Research shows that the anti-tumor mechanism of pentacyclic triterpenoids is closely related to the mitochondrial apoptosis pathway. For example, betulinic acid can continuously activate caspases 3, caspases 7, and caspases 9, and clear poly(ADP-ribose) polymerase, thereby triggering the release of cytochrome c and Smac proteins on the inner mitochondrial membrane, depolarization of the mitochondrial membrane potential, etc., and ultimately inhibiting the proliferation of cancer cells.

[0003] For example, the prior art discloses a class of anti-tumor pentacyclic triterpene derivatives targeting mitochondria, a preparation method thereof, and an application thereof. This class of pentacyclic triterpene derivatives can target the mitochondria of cancer cells by introducing a mitochondrial targeting vector, enhancing their anti-tumor activity. However, the anti-tumor activity of this pentacyclic triterpene derivative at low nanomolar concentrations needs to be further improved; moreover, since this pentacyclic triterpene derivative does not have aggregation fluorescence characteristics, it is impossible to observe its aggregation degree and distribution in cells, tissues, and animals, and it is difficult to study its anti-cancer mechanism. Summary of the Invention

[0004] The object of the present invention is to overcome the defects and deficiencies that the existing anti-tumor pentacyclic triterpene drugs targeting mitochondria have poor anti-tumor activity at low nanomolar concentrations and do not have aggregation fluorescence characteristics, and to provide a pentacyclic triterpene aggregation-induced fluorescence derivative.

[0005] Another object of the present invention is to provide a preparation method of the above pentacyclic triterpene aggregation-induced fluorescence derivative.

[0006] Another object of the present invention is to provide an application of the above pentacyclic triterpene aggregation-induced fluorescence derivative in the preparation of anti-tumor drugs.

[0007] Another object of the present invention is to provide an application of the above pentacyclic triterpene aggregation-induced fluorescence derivative in fluorescence imaging of cancer cells.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] The present invention protects a pentacyclic triterpene aggregation-induced fluorescence derivative, and its structural formula is shown as formula (I), formula (II), or formula (III):

[0010]

[0011] Among them, R is

[0012] The pentacyclic triterpenoid aggregation-induced fluorescence derivative of the present invention introduces a cation by quaternization reaction of a bromohydrocarbon structure and a pyridine structure to generate pyridinium, thereby increasing its mitochondrial targeting effect in cells, enabling it to achieve selective cytotoxicity at low nanomolar concentrations, and thus significantly improving its anti-tumor activity. At the same time, while ensuring that the pentacyclic triterpenoid aggregation-induced fluorescence derivative has excellent anti-tumor activity, an ester bond reaction is used to connect a fluorescent group by substituting the carboxylic acid on the pentacyclic triterpenoid with a bromohydrocarbon structure. A confocal fluorescence microscope is used to observe and calculate the degree of co-localization of the derivative with mitochondria, and finally the aggregation position of the pentacyclic triterpenoid aggregation-induced fluorescence derivative in cells is obtained, enabling the visualization of pentacyclic triterpenoid compounds in living cells, thereby being able to quantify the uptake and distribution of intracellular substances, especially the degree of co-localization of pentacyclic triterpenoid compounds with mitochondria in cells, so as to study the anti-cancer mechanism of pentacyclic triterpenoid compounds.

[0013] The present invention also protects a preparation method of a pentacyclic triterpenoid aggregation-induced fluorescence derivative, comprising the following steps:

[0014] 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 the pentacyclic triterpenoid aggregation-induced fluorescence derivative;

[0015] Among them, the compound A is

[0016] The compound B is

[0017]

[0018] Any one of them.

[0019] In the specific implementation manner, the molar ratio of the compound A to the compound B in the present invention is 1:1.

[0020] Specifically, the (A1) can be prepared by the following preparation method:

[0021] Under the protection of nitrogen, dissolve 4-borotriphenylamine and 4-bromopyridine hydrochloride in a mixed solution of tetrahydrofuran and water, add potassium carbonate and tetrakis(triphenylphosphine)palladium at the same time, and heat the reaction system to 60 - 80 °C, and react for 7 - 14 h to obtain compound A1. Specifically, the temperature can be 70 °C and the reaction time can be 8 h.

[0022] Specifically, the volume ratio of tetrahydrofuran to water in the mixed solution of tetrahydrofuran and water is (8-12):(0.5-1.5).

[0023] Specifically, the molar ratio of 4-borotriphenylamine: 4-bromopyridine hydrochloride: potassium carbonate: tetrakis(triphenylphosphine)palladium is 0.15:0.15:0.007:1.00.

[0024] Specifically, the (A2) is prepared by the following preparation method:

[0025] Potassium tert-butoxide is dissolved in N,N-dimethylformamide, then 4-methylpyridine is added. After stirring at room temperature for 30 min, [4-(diphenylamino)phenyl]-1-carbaldehyde is added, and the mixture is heated to 80-100 °C and reacted overnight. After separation and purification, compound A2 is obtained.

[0026] In the specific embodiment, the molar ratio of [4-(diphenylamino)phenyl]-1-carbaldehyde: 4-methylpyridine: potassium tert-butoxide is 1:1:(1.2-1.4).

[0027] Specifically, the compound B can be prepared by the following preparation method:

[0028] Reactant a and 1,4-dibromobutane are dissolved in acetone, potassium carbonate is added simultaneously, and the reaction system is heated to 65 °C and reacted for 4 h to obtain compound B;

[0029] Wherein, the reactant a is any one of betulinic acid, oleanolic acid and ursolic acid.

[0030] The CAS number of the above-mentioned 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.

[0031] Specifically, the molar ratio of reactant a: 1,4-dibromobutane: potassium carbonate is 1:4:1.

[0032] The application of the above-mentioned pentacyclic triterpenoid aggregation-induced fluorescence derivative in the preparation of anti-tumor drugs is also within the protection scope of the present invention.

[0033] The present invention also protects the application of the above-mentioned pentacyclic triterpenoid aggregation-induced fluorescence derivative in cancer cell aggregation-induced fluorescence imaging.

[0034] Compared with the prior art, the present invention has the following beneficial technical effects:

[0035] The pentacyclic triterpene aggregation-induced fluorescence derivative of the present invention has excellent anti-tumor activity. By targeting the pentacyclic triterpene parent compound to mitochondria, it can be better applied to the preparation of anti-tumor drugs. Moreover, this pentacyclic triterpene aggregation-induced fluorescence derivative is a salt. Compared with the pentacyclic triterpene parent structure, its water solubility increases, significantly improving the pharmacokinetic parameters and enhancing the bioavailability. In addition, this pentacyclic triterpene aggregation-induced fluorescence derivative can exhibit aggregation-induced fluorescence properties within cells, enabling the observation of the aggregation position and degree of the compound in cells under a fluorescence microscope, and can be better applied to analyzing the mechanism of action of the compound within cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a synthetic route diagram of compound A1 of the present invention.

[0037] Figure 2 It is a synthetic route diagram of compound A2 of the present invention.

[0038] Figure 3 It is a synthetic route diagram of compound B of the present invention.

[0039] Figure 4 It is an aggregation-induced confocal fluorescence microscopy imaging diagram of compound I in HeLa cells in Example 1.

[0040] Figure 5 It is an aggregation-induced confocal fluorescence microscopy imaging diagram of compound IV in HeLa cells in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0041] 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 raw material reagents purchased conventionally.

[0042] Compound A1

[0043] Compound A1 can be prepared by the following preparation method (the synthetic route is as Figure 1 shown):

[0044] 4-Borotriphenylamine (433.7 mg, 1.50 mmol) is mixed with 4-bromopyridine hydrochloride (289.4 mg, 1.50 mmol), potassium carbonate (1382.1 mg, 10.00 mmol), and tetrakis(triphenylphosphine)palladium (80.9 mg, 0.070 mmol), and then placed in a 100 mL round-bottom flask. 50 mL of THF and 5 mL of water are added, and the mixture is heated under reflux at 70 °C for 8 h. The progress of the reaction is detected by thin-layer chromatography TLC (the developing agent is V 石油醚 : V 乙酸乙酯= 12:1), cooled, concentrated under reduced pressure, extracted with ethyl acetate (50 mL × 3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to silica gel column chromatography (eluent V 石油醚 :V 乙酸乙酯 = 10:1) to obtain compound A1 as an orange-yellow powder (225.0 mg) with a yield of 46.6%.

[0045] Compound A2

[0046] Compound A2 can be prepared by the following preparation method (synthesis route as Figure 2 shown), specifically including the following steps:

[0047] Potassium tert-butoxide (134.6 mg, 1.20 mmol) was dissolved in N,N-dimethylformamide (20 mL), and then 4-methylpyridine (93.0 mg, 1.00 mmol) was added. The mixture was stirred at room temperature for 30 minutes, and [4-(diphenylamino)phenyl]-1-carbaldehyde (273.1 mg, 1.00 mmol) was added. The reaction was heated to reflux at 80 °C overnight; thin-layer chromatography (TLC) was used to detect the reaction (developing agent V 石油醚 :V 乙酸乙酯 = 5:1); cooled, saturated sodium chloride solution was added, extracted with ethyl acetate (50 mL × 3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to silica gel column chromatography (eluent V 石油醚 :V 乙酸乙酯 = 3:1) to obtain compound A2 as a yellow powder (298.0 mg) with a yield of 85.6%.

[0048] Compound B1

[0049] Compound B1 can be prepared by the following preparation method (synthesis route as Figure 3 shown):

[0050] Betulinic 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. The mixture was heated to reflux at 65 °C for 4 h, and TLC was used to detect the progress of the reaction (developing agent V 石油醚 :V 乙酸乙酯 = 3:1), cooled, concentrated under reduced pressure, and subjected to silica gel column chromatography (eluent 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%.

[0051] Compound B2

[0052] Compound B2 can be prepared by the following preparation method (synthesis route as Figure 3 shown):

[0053] Oleanolic acid (456.7 mg, 1.00 mmol) was mixed with 1,4-dibromobutane (855.6 mg, 4.00 mmol) and potassium carbonate (138.2 mg, 1.00 mmol) in 20 mL of acetone, and the mixture was heated under reflux at 65 °C for 4 h. The progress of the reaction was monitored by TLC (the developing solvent was V 石油醚 :V 乙酸乙酯 = 3:1). After cooling, the mixture was concentrated under reduced pressure, and silica gel column chromatography (the eluent was V 石油醚 :V 乙酸乙酯 = 2:1) was used to obtain compound B2 as a white powder (512.5 mg) with a yield of 86.8%.

[0054] Compound B3

[0055] Compound B3 can be prepared by the following method (the synthetic route is as shown in Figure 3 ):

[0056] Ursolic acid (456.7 mg, 1.00 mmol) was mixed with 1,4-dibromobutane (855.6 mg, 4.00 mmol) and potassium carbonate (138.2 mg, 1.00 mmol) in 20 mL of acetone, and the mixture was heated under reflux at 65 °C for 4 h. The progress of the reaction was monitored by TLC (the developing solvent was V 石油醚 :V 乙酸乙酯 = 3:1). After cooling, the mixture was concentrated under reduced pressure, and silica gel column chromatography (the eluent was V 石油醚 :V 乙酸乙酯 = 2:1) was used to obtain compound B3 as a white powder (522.6 mg) with a yield of 88.5%.

[0057] Example 1

[0058] A pentacyclic triterpenoid aggregation-induced fluorescence derivative (denoted as compound I) has the following structural formula:

[0059]

[0060] The above pentacyclic triterpenoid aggregation-induced fluorescence derivative can be prepared by the following method:

[0061] Compound A1 (161.1 mg, 0.50 mmol) and compound B1 (295.2 mg, 0.50 mmol) were mixed in 10 mL of acetonitrile, and the mixture was heated under reflux at 90 °C for 12 h. After cooling, the mixture was concentrated under reduced pressure, and silica gel column chromatography (the eluent was V 二氯甲烷 :V 甲醇 = 40:1) was used to obtain the pentacyclic triterpenoid aggregation-induced fluorescence derivative (compound I) as a yellow powder (250.1 mg) with a yield of 54.8%.

[0062] The above-mentioned Compound I was subjected to nuclear magnetic resonance detection, and the results are as follows:

[0063] DaDa DoDⅠ: mp: 169 - 178 °C;

[0064] 1 H NMR (400 MHz, ChloDoDoDm-d) δ 9.26 (d, J = 6.7 Hz, 2H), 8.07 (d, J = 6.6 Hz, 2H), 7.69–7.63 (m, 2H), 7.36 (D, J = 7.8 Hz, 4H), 7.24–7.14 (m, 6H), 7.10–7.03 (m, 2H), 4.96 (D, J = 7.5 Hz, 2H), 4.70 (d, J = 2.3 Hz, 1H), 4.59 (D, J = 1.9 Hz, 1H), 4.22–4.06 (m, 3H), 3.17 (dd, J = 11.4, 4.6 Hz, 1H), 2.95 (Dd, J = 10.8, 4.4 Hz, 1H), 2.25–2.16 (m, 1H), 2.20–2.09 (m, 2H), 2.08 (d, J = 8.6 Hz, 1H), 1.81 (q, J = 6.6 Hz, 4H), 1.66 (s, 3H), 1.71–1.50 (m, 3H), 1.50–1.18 (m, 7H), 1.14 (Dd, J = 12.8, 4.0 Hz, 2H), 1.07–0.85 (m, 8H), 0.81 (s, 3H), 0.73 (d, J = 10.0 Hz, 6H), 0.69–0.61 (m, 1H).

[0065] 13 C NMR (101 MHz, ChloDoDoDm-d) δ 176.21, 155.08, 152.35, 150.41, 145.73, 144.21, 129.87, 129.02, 126.34, 125.50, 123.72, 122.63, 120.17, 109.70, 78.90, 62.52, 60.39, 59.70, 56.56, 55.31, 50.51, 49.38, 46.99, 42.40, 40.71, 38.84, 38.70, 38.25, 37.16, 37.03, 34.32, 32.07, 30.61, 29.69, 28.50, 27.99, 27.38, 25.50, 21.03, 20.91, 19.32, 18.27, 16.13, 16.09, 15.39, 14.67, 14.19.

[0066] MS (m / z): 833.5 [M - BD]+.

[0067] Example 2

[0068] A pentacyclic triterpenoid aggregation-induced fluorescence derivative (denoted as Compound II), whose structural formula is as follows:

[0069]

[0070] The above pentacyclic triterpenoid aggregation-induced fluorescence derivative can be prepared by the following preparation method:

[0071] Mix Compound A1 (161.1 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 the pentacyclic triterpenoid aggregation-induced fluorescence derivative (Compound II), which is a yellow powder with a yield of 65.8%.

[0072] Perform nuclear magnetic resonance detection on the above Compound II, and the results are as follows:

[0073] DaDa DoDⅡ: Mp: 220 - 227 °C;

[0074] 11H NMR (400 MHz, ChloDoDoDm-d) δ 9.23 (d, J = 6.8 Hz, 2H), 8.10–8.03 (m, 2H), 7.70–7.62 (m, 2H), 7.36 (dd, J = 8.3, 7.4 Hz, 4H), 7.25–7.14 (m, 6H), 7.11–7.02 (m, 2H), 5.23 (D, J = 3.6 Hz, 1H), 4.92 (hepD, J = 7.0, 6.6 Hz, 2H), 4.17–4.02 (m, 3H), 3.19 (dd, J = 11.3, 4.7 Hz, 1H), 2.82 (dd, J = 13.9, 4.5 Hz, 1H), 2.17–2.07 (m, 1H), 2.06 (d, J = 12.3 Hz, 2H), 1.95 (Dd, J = 13.5, 4.0 Hz, 1H), 1.86–1.69 (m, 7H), 1.62 (DD, J = 14.4, 4.9 Hz, 2H), 1.54–1.47 (m, 2H), 1.51–1.39 (m, 1H), 1.43–1.28 (m, 1H), 1.26 (D, J = 7.1 Hz, 2H), 1.24–1.10 (m, 2H), 1.11 (s, 3H), 1.04 (dD, J = 13.6, 3.3 Hz, 1H), 0.97 (s, 3H), 0.94 (d, J = 10.3 Hz, 1H), 0.90 (d, J = 6.7 Hz, 6H), 0.80 (s, 3H), 0.74 (s, 3H), 0.72–0.65 (m, 1H), 0.63 (s, 3H);

[0075] 13 13C NMR (101 MHz, ChloDoDoDm-d) δ 177.92, 155.04, 152.37, 145.72, 144.19, 143.84, 129.88, 129.00, 126.35, 125.52, 123.67, 122.59, 122.29, 120.17, 78.91, 62.91, 60.39, 59.76, 55.15, 47.52, 46.80, 45.83, 41.73, 41.34, 39.32, 38.72, 38.39, 36.97, 33.82, 33.04, 32.72, 32.58, 30.68, 28.55, 28.10, 27.66, 27.17, 25.81, 25.35, 23.68, 23.38, 23.02, 21.03, 18.30, 17.13, 15.60, 15.32, 14.19;

[0076] MS (m / z): 833.5 [M - BD] + 。

[0077] Example 3

[0078] A pentacyclic triterpenoid aggregation-induced fluorescence derivative (denoted as Compound III), whose structural formula is as follows:

[0079]

[0080] The above pentacyclic triterpenoid aggregation-induced fluorescence derivative can be prepared by the following preparation method:

[0081] Mix Compound A1 (161.1 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), to obtain the pentacyclic triterpenoid aggregation-induced fluorescence derivative (Compound III), which is a yellow powder with a yield of 45.5%.

[0082] Perform nuclear magnetic resonance detection on the above Compound III, and the results are as follows:

[0083] DaDa DoDⅢ: mp: 170 - 179 °C;

[0084] 1 1H NMR (400 MHz, ChloDoDoDm-d) δ 9.24 (d, J = 6.6 Hz, 2H), 8.08 (d, J = 6.7 Hz, 2H), 7.71–7.62 (m, 2H), 7.36 (D, J = 7.8 Hz, 4H), 7.24–7.15 (m, 6H), 7.10–7.02 (m, 2H), 5.18 (D, J = 3.6 Hz, 1H), 4.94 (hepD, J = 6.6, 6.1 Hz, 2H), 4.87 (s, 0H), 4.12 (q, J = 7.1 Hz, 1H), 4.04 (D, J = 6.3 Hz, 2H), 3.18 (s, 0H), 2.19 (d, J = 11.3 Hz, 1H), 2.16–1.92 (m, 3H), 1.90–1.60 (m, 5H), 1.60–1.39 (m, 6H), 1.37–1.21 (m, 6H), 1.05 (s, 3H), 0.99–0.88 (m, 7H), 0.87–0.79 (m, 6H), 0.74 (s, 3H), 0.65 (s, 3H);

[0085] 1313C NMR (101 MHz, ChloDoDoDm-d) δ 177.71, 155.01, 152.35, 145.72, 144.21, 138.34, 129.87, 129.01, 126.35, 125.50, 125.39, 123.68, 122.61, 120.17, 78.93, 62.92, 59.78, 55.15, 52.83, 48.17, 47.46, 42.06, 39.53, 39.07, 38.82, 38.72, 38.57, 36.90, 36.84, 33.00, 30.61, 28.60, 28.14, 27.99, 27.20, 25.32, 24.20, 23.50, 23.27, 21.14, 18.28, 17.18, 17.00, 15.65, 15.46;

[0086] MS (m / z): 833.5 [M - BD] + 。

[0087] Example 4

[0088] A pentacyclic triterpenoid quaternary ammonium salt fluorescent derivative (denoted as Compound Ⅳ), whose structural formula is as follows:

[0089]

[0090] The above pentacyclic triterpenoid quaternary ammonium salt fluorescent derivative can be prepared by the following preparation method:

[0091] Mix the above Compound A2 (174.1 mg, 0.50 mmol) and Compound B1 (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 to obtain Compound Ⅳ as a red powder (320.0 mg) with a yield of 68.2%.

[0092] Perform nuclear magnetic resonance detection on the above Compound Ⅳ, and the results are as follows:

[0093] DaDa DoDⅣ: mp: 182 - 191 °C;

[0094] 11H NMR (400 MHz, ChloDoDoDm-d) δ 9.13 (d, J = 6.6 Hz, 2H), 7.92 (d, J = 6.5 Hz, 2H), 7.64 (d, J = 16.0 Hz, 1H), 7.50–7.43 (m, 2H), 7.32 (D, J = 7.8 Hz, 4H), 7.14 (dD, J = 6.9, 2.9 Hz, 6H), 7.02–6.97 (m, 3H), 6.94 (s, 1H), 4.89 (D, J = 7.4 Hz, 2H), 4.71 (d, J = 2.3 Hz, 1H), 4.59 (D, J = 2.0 Hz, 1H), 4.21–4.05 (m, 3H), 3.17 (dd, J = 11.3, 4.7 Hz, 1H), 2.94 (Dd, J = 10.8, 4.4 Hz, 1H), 2.24–2.10 (m, 2H), 2.08 (q, J = 5.8 Hz, 2H), 1.89–1.74 (m, 4H), 1.66 (s, 4H), 1.71–1.43 (m, 5H), 1.43–1.28 (m, 6H), 1.26 (D, J = 7.1 Hz, 2H), 1.25–1.08 (m, 2H), 1.01 (Dd, J = 12.7, 4.4 Hz, 1H), 0.94 (d, J = 2.0 Hz, 6H), 0.90–0.83 (m, 1H), 0.81 (s, 3H), 0.76 (s, 3H), 0.71 (s, 3H), 0.65 (d, J = 10.5 Hz, 1H);

[0095] 13 13C NMR (101 MHz, ChloDoDoDm-d) δ 176.17, 153.97, 150.87, 150.41, 146.37, 143.93, 142.23, 129.93, 129.65, 126.92, 125.86, 124.72, 123.27, 120.87, 118.82, 109.70, 78.91, 62.51, 60.39, 59.80, 56.55, 55.32, 50.52, 49.38, 46.98, 42.40, 40.72, 38.83, 38.70, 38.24, 37.17, 37.03, 34.33, 32.08, 30.60, 29.68, 28.46, 27.97, 27.38, 25.50, 21.04, 20.91, 19.32, 18.30, 16.17, 16.09, 15.37, 14.67, 14.19;

[0096] MS (m / z): 859.5 [M - BD] + 。

[0097] Example 5

[0098] A pentacyclic triterpenoid quaternary ammonium salt fluorescent derivative (denoted as Compound V), and its structural formula is as follows:

[0099]

[0100] The above pentacyclic triterpenoid quaternary ammonium salt fluorescent derivative can be prepared by the following preparation method:

[0101] Mix the above Compound A2 (174.1 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 to obtain Compound V as a red powder (340.5 mg) with a yield of 72.5%.

[0102] Perform nuclear magnetic resonance detection on the above Compound V, and the results are as follows:

[0103] DaDa DoDⅤ: Mp: 193 - 199 °C;

[0104] 1 H NMR (400 MHz, ChloDoDoDm-d) δ 9.10 (d, J = 6.7 Hz, 2H), 7.91 (d, J = 6.7 Hz, 2H), 7.63 (d, J = 16.0 Hz, 1H), 7.50–7.43 (m, 2H), 7.32 (dd, J = 8.5, 7.2 Hz, 4H), 7.18–7.07 (m, 6H), 7.04–6.96 (m, 2H), 6.95 (d, J = 16.0 Hz, 1H), 5.23 (D, J = 3.6 Hz, 1H), 4.86 (Dd, J = 7.3, 4.8 Hz, 2H), 4.17–4.02 (m, 3H), 3.19 (dd, J = 11.2, 4.6 Hz, 1H), 2.82 (dd, J = 13.7, 4.5 Hz, 1H), 2.07 (s, 2H), 2.06 (d, J = 15.7 Hz, 2H), 1.95 (Dd, J = 13.4, 3.9 Hz, 1H), 1.88–1.69 (m, 4H), 1.69–1.52 (m, 1H), 1.57 (s, 3H), 1.55–1.44 (m, 5H), 1.44–1.10 (m, 7H), 1.11 (s, 3H), 1.09–1.01 (m, 1H), 0.96 (s, 4H), 0.90 (d, J = 7.5 Hz, 7H), 0.83 (s, 3H), 0.74 (s, 3H), 0.72–0.65 (m, 1H), 0.63 (s, 3H);

[0105] 1313C NMR (101 MHz, ChloDoDoDm-d) δ 177.89, 153.96, 150.91, 146.37, 143.91, 143.81, 142.25, 129.92, 129.66, 126.88, 125.88, 124.74, 123.25, 122.31, 120.85, 118.77, 78.93, 62.86, 59.85, 55.17, 47.54, 46.80, 45.84, 41.74, 41.35, 39.33, 38.72, 38.42, 37.00, 33.83, 33.04, 32.73, 32.59, 30.68, 28.44, 28.09, 27.67, 27.15, 25.82, 25.36, 23.67, 23.40, 23.03, 18.32, 17.13, 15.59, 15.36;

[0106] MS (m / z): 859.5 [M - BD] + 。

[0107] Example 6

[0108] A pentacyclic triterpenoid quaternary ammonium salt fluorescent derivative (denoted as Compound VI), whose structural formula is as follows:

[0109]

[0110] The above pentacyclic triterpenoid quaternary ammonium salt fluorescent derivative can be prepared by the following method:

[0111] Mix the above Compound A2 (174.1 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 to obtain Compound VI, which is a red powder (300.1 mg) with a yield of 63.9%.

[0112] Perform nuclear magnetic resonance detection on the above Compound VI, and the results are as follows:

[0113] DaDa DoD VI: Mp: 172 - 180 °C;

[0114] 11H NMR (400 MHz, ChloDoDoDm-d) δ 9.11 (d, J = 6.7 Hz, 2H), 7.93 (d, J = 6.5 Hz, 2H), 7.64 (d, J = 16.1 Hz, 1H), 7.50–7.43 (m, 2H), 7.32 (dd, J = 8.5, 7.2 Hz, 4H), 7.18–7.10 (m, 6H), 7.02–6.97 (m, 3H), 6.95 (s, 1H), 5.18 (D, J = 3.5 Hz, 1H), 4.86 (DD, J = 7.5, 4.3 Hz, 2H), 4.12 (q, J = 7.2 Hz, 1H), 4.03 (D, J = 6.3 Hz, 2H), 3.19 (dd, J = 11.3, 4.4 Hz, 1H), 2.18 (d, J = 11.2 Hz, 1H), 2.04 (s, 1H), 2.11–1.92 (m, 3H), 1.92–1.80 (m, 1H), 1.80–1.67 (m, 2H), 1.70–1.61 (m, 1H), 1.64–1.53 (m, 1H), 1.54 (d, J = 10.9 Hz, 1H), 1.51–1.41 (m, 4H), 1.37–1.29 (m, 1H), 1.30 (s, 1H), 1.27 (dD, J = 14.3, 5.9 Hz, 4H), 1.05 (s, 4H), 0.95 (q, J = 8.4, 5.9 Hz, 8H), 0.84 (D, J = 3.2 Hz, 6H), 0.74 (s, 3H), 0.65 (s, 3H);

[0115] 13 13C NMR (101 MHz, ChloDoDoDm-d) δ 177.68, 153.94, 150.86, 146.37, 143.93, 142.21, 138.30, 129.93, 129.65, 126.93, 125.86, 125.43, 124.71, 123.29, 120.86, 118.83, 78.94, 62.86, 60.39, 59.87, 55.16, 52.86, 48.17, 47.48, 42.07, 39.53, 39.07, 38.82, 38.72, 38.61, 36.93, 36.84, 33.02, 30.62, 28.48, 28.13, 28.00, 27.19, 25.33, 24.21, 23.50, 23.29, 21.15, 21.04, 18.30, 17.18, 17.00, 15.64, 15.50, 14.19;

[0116] MS (m / z): 859.5 [M - BD] + 。

[0117] Performance Test

[0118] 1. Cytotoxicity Control Test on Cancer Cells and Normal Cells

[0119] (1) Experimental Materials

[0120] 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.

[0121] Experimental Drugs: Three pentacyclic triterpenoid aggregation-induced fluorescence derivatives (Compound I, Compound II, Compound III, Compound Ⅳ, Compound Ⅴ, Compound Ⅵ) prepared by the present invention.

[0122] 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 Pusaisai Life Science Co., Ltd.;

[0123] HSF (human skin fibroblasts) was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.;

[0124] DMEM culture medium, fetal bovine serum, and trypsin were purchased from Gibco, USA;

[0125] Cell CounDing KiD-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazolium monosodium salt) was purchased from MedChemexpDess Biotechnology Co., USA;

[0126] DMSO (dimethyl sulfoxide) was purchased from Sigma, USA;

[0127] PBS (phosphate buffer solution) was purchased from Solarbio Co., Ltd.

[0128] Instruments: BioDek Elx800 microplate reader: BioDek, USA; Pipette: Eppendorf China Co., Ltd.; Carbon dioxide incubator: TheDmo, USA; Autoclave: Panasonic, Japan.

[0129] (2) Experimental Methods

[0130] 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 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin in a 37 °C incubator with 5% CO2 and saturated humidity.

[0131] For the above cells: Logarithmic growth phase cells were collected and inoculated in a 96-well culture plate with DMEM medium containing 10% fetal bovine serum, 7000 cells per well, and 100 μL of culture medium per well. They were cultured in the above incubator for 24 h to allow the cells to adhere completely. After complete adhesion, the solution in each well was replaced with 100 μL of DMEM medium containing 10% fetal bovine serum with different concentrations of drugs, and 3 controls were set for each concentration. After culturing in the dark for 48 h, each well was replaced with 100 μL of basal DMEM medium without fetal bovine serum containing 10% CCK-8. After culturing in the dark for 1 h - 2 h, the absorbance value of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0132] All results were defined by the half-maximal inhibitory concentration (IC 50 value) as the drug concentration when 50% of the tumor cells survived. The cell growth inhibition rate was calculated using the following method as an evaluation index.

[0133] Inhibition rate (%) = [1 - (mean OD value of experimental group - mean OD value of blank group) / (mean OD value of control group - mean OD value of blank group)] × 100%. According to the cell growth inhibition rate, the IC 50 value was calculated using the linear regression method.

[0134] (3) Experimental results

[0135] The results of the positive control experiment are shown in Table 1. Betulinic acid (BA), oleanolic acid (OA), ursolic acid (UA), Compound I, Compound II, Compound III, Compound IV, Compound V, Compound VI, and doxorubicin hydrochloride (DX) toxicity a control study.

[0136] Table 1

[0137]

[0138]

[0139] 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.

[0140] As can be seen from Table 1: The pentacyclic triterpene aggregation-induced fluorescence derivatives prepared in the present invention showed higher proliferation inhibitory effects on Hela, MDA-MB-231, MCF-7, and Hep G2 cell lines compared with betulinic acid, oleanolic acid, and ursolic acid, indicating that the introduction of a fluorescent group and a pyridinium structure would increase the anti-tumor activity of the pentacyclic triterpene nucleus.

[0141] 2. Aggregation-induced confocal fluorescence microscopy imaging test

[0142] (1) Experimental materials

[0143] Experimental drugs: Compound I prepared in Example 1 of the present invention, Compound IV prepared in Example 4;

[0144] Cell culture: The HeLa cell line was purchased from Wuhan Punosai Life Science Co., Ltd.;

[0145] DMEM culture medium, fetal bovine serum, and trypsin were purchased from Gibco Company, USA;

[0146] DMSO (dimethyl sulfoxide) was purchased from Sigma Company, USA;

[0147] PBS (phosphate buffer solution) was purchased from Solarbio Life Sciences Co., Ltd.

[0148] Instruments: LSM 880 conDocal laseD scanning micDoscope: Carl Zeiss AG, Germany; Pipettes: Eppendorf China Co., Ltd.; Carbon dioxide incubator: Thermo Fisher Scientific, USA; Autoclave: Panasonic, Japan.

[0149] Experimental reagents and consumables: 35-mm confocal dishes (801001): Wuxi NEST Biotechnology Co., Ltd. Live cell imaging medium (FluoDoBDiDe TM DMEM): Thermo Fisher Scientific (China) Co., Ltd.

[0150] Mitochondrial deep red fluorescence probe ( Deep Red FM): Yeasen Biotech Co., Ltd.

[0151] (2) Experimental methods

[0152] Hela cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 mg / mL streptomycin in a 37 °C incubator with 5% CO2 and saturated humidity.

[0153] For the above cells: Logarithmic growth phase cells were collected and inoculated in 35 mm confocal dishes with DMEM medium containing 10% fetal bovine serum. The inoculation cell specification was 4100 cells / cm 2 culture area. They were cultured in the above incubator for 24 h to allow the cells to adhere completely. After complete adhesion, serum-free medium containing Compound I was added, and the concentration of Compound I was 1 μmol / L. After culturing in the incubator for 3 - 6 h, they were washed three times with PBS, then serum-free medium containing a mitochondrial deep red fluorescence probe was added, and the concentration of the mitochondrial deep red fluorescence probe was 250 nmol / L. After culturing in the incubator for 0.5 h, they were washed three times with PBS, and then 2 mL of the above FluoDoBDiDe TM DMEM was added. Fluorescence distribution within the cells was photographed under an inverted confocal fluorescence microscope. The experimental results were as Figure 4 shown. Among them, 4a was the imaging result of Compound I (channel 405 nm), 4b was the imaging result of a commercial mitochondrial deep red fluorescence probe (channel 633 nm), and 4c was the superimposed image of channel 405 nm and channel 633 nm. From Figure 4 the experimental results and the principle of aggregation-induced emission, it can be concluded that Compound I can smoothly cross the cell membrane and aggregate in mitochondria under the action of low concentration (1 μmol / L) and long time (3 - 6 h), and then produce a targeted anti-tumor effect.

[0154] Compound IV prepared in Example 4 was detected by the above method. The experimental results were as Figure 5 shown. 5a was the imaging result of Compound IV (channel 488 nm), 5b was the imaging result of a commercial mitochondrial deep red fluorescence probe (channel 633 nm), and 5c was the superimposed image of channel 488 nm and channel 633 nm. It can be found from Figure 5 this that Compound IV can also smoothly cross the cell membrane and aggregate in mitochondria under the action of low concentration (1 μmol / L) and long time (3 - 6 h), indicating that the pyridinium structure has the effect of targeting mitochondria, and can target the pentacyclic triterpene parent structure to mitochondria, exerting the anti-tumor mechanism of the pentacyclic triterpene structure to promote mitochondrial dysfunction.

[0155] Compound II, Compound III, Compound V, Compound VI are similar in properties to Compound I and Compound IV, and can all cross the cell membrane and aggregate in mitochondria to produce a targeted anti-tumor effect.

[0156] In summary, the pentacyclic triterpene aggregation-induced fluorescence derivative prepared by the present invention shows the ability to target mitochondria through the pyridinium structure, and can target and deliver the pentacyclic triterpene parent drug to mitochondria, exerting the anti-tumor mechanism in which pentacyclic triterpenoids are closely related to the mitochondrial apoptosis pathway, and can inhibit the activity of tumor cells under low concentration conditions. Compound II and Compound III, similar in properties to Compound I, can both target and deliver the pentacyclic triterpene parent drug to mitochondria, thus producing targeted anti-tumor effects.

[0157] 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 list all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A pentacyclic triterpenoid aggregation-induced fluorescence derivative, characterized in that, The structural formulas of the pentacyclic triterpene aggregation-induced fluorescence derivatives are shown in Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V) or Formula (VI) as follows: Formula (I); Formula (II); Formula (III); Formula (IV); Formula (V); Formula (VI).

2. A method for preparing the pentacyclic triterpene aggregation-induced fluorescence derivative according to claim 1, characterized in that, It includes the following steps: Dissolve compound A and compound B in an organic solvent, react at 90-110 °C for 10-12 h, and then separate and purify to obtain the pentacyclic triterpene aggregation-induced fluorescence derivative; wherein, the compound A is or ; The compound B is , or any one of them.

3. The preparation method of the pentacyclic triterpene aggregation-induced fluorescence derivative 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 pentacyclic triterpene aggregation-induced fluorescence derivative according to claim 2, characterized in that, The said is prepared by the following preparation method: Under the protection of nitrogen, dissolve 4-borotriphenylamine and 4-bromopyridine hydrochloride in a mixed solution of tetrahydrofuran and water, add potassium carbonate and tetrakis(triphenylphosphine)palladium at the same time, and heat the reaction system to 60-80 °C and react for 7-14 h to obtain the product.

5. The preparation method of the pentacyclic triterpene aggregation-induced fluorescence derivative according to claim 4, characterized in that, In the mixed solution of tetrahydrofuran and water, the volume ratio of tetrahydrofuran to water is (8-12):(0.5-1.5).

6. The preparation method of the pentacyclic triterpene aggregation-induced fluorescence derivative according to claim 2, characterized in that, The is prepared by the following preparation method: Dissolve potassium tert-butoxide in N,N -dimethylformamide, then add 4-methylpyridine. After stirring at room temperature for 30 min, add [4-(diphenylamino)phenyl]-1-carbaldehyde, heat to 80-100 °C, react overnight, and separate and purify to obtain the product.

7. The preparation method of the pentacyclic triterpene aggregation-induced fluorescence derivative according to claim 2, characterized in that, Compound B is prepared by the following 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 and react for 3-5 h to obtain compound B; Among them, the reactant a is any one of betulinic acid, oleanolic acid and ursolic acid.

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

1.

9. Use of the pentacyclic triterpene aggregation-induced fluorescence derivative according to claim 1 in the preparation of an anti-tumor drug.

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