A paeonol derivative, and a preparation method and application thereof

Abstract

A kudzuvine flower terpine derivative, a preparation method and application thereof, a structure formula is shown as follows: wherein R1, R2 are H, CH3, CH2F, CHF2, CF3, t-Bu, MOM, EE, THP, a straight chain or branched alkane with carbon atom number being 1-5, an ester group, a silyl ether, a trifluoro or polyfluorinated alkane straight chain terminal alkyne alkane n=1, 2, 3, 4 or 5, m=1, 2, 3, 4, 5 or 6, when one group is a terminal alkyne, the other two groups are alkane or fluorinated alkane. The kudzuvine flower terpine derivative can inhibit the proliferation growth of liver cancer cells, induce liver cancer cell apoptosis and G2 / M cycle arrest, inhibit the migration and invasion of liver cancer cells, achieve the effect of resisting tumors, and provide a new leading drug for clinical treatment of hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention belongs to the application field of puerarin derivatives of compounds, and particularly relates to a puerarin derivative, a preparation method thereof, and an application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. In China, due to the large population and high incidence rate, the number of HCC cases ranks first in the Asian and African regions. Most HCC patients are infected with hepatitis B virus and further develop into liver cirrhosis. Most patients have entered the middle and late stages when they seek medical treatment, with severe conditions, rapid progression, poor prognosis, and extremely low five-year survival rate. Currently, advanced HCC patients develop drug resistance to traditional radiotherapy and chemotherapy drugs, which greatly limits the use of anti-tumor drugs available for patients. Therefore, there is an urgent need to develop new and effective anti-cancer treatment drugs.

[0003] In recent years, chemotherapy and prevention using the chemical diversity, structural complexity, and inherent biological activity characteristics of plant-derived compounds have achieved satisfactory results due to their high efficiency, effectiveness, and low side effects, and thus have become a very attractive anti-cancer strategy. A variety of plant-derived natural products with anti-tumor activity have been identified, such as flavonoids, terpenoids, alkaloids, and phenols, which can inhibit tumor cell proliferation, induce apoptosis, reduce telomerase activity, inhibit angiogenesis, improve immune function, reverse multidrug resistance, etc. Therefore, using advanced and innovative research techniques to explore the characteristics of plant-derived compounds, explore the mechanism of action, and apply them clinically will be the main direction for the prevention and treatment of liver cancer in the future.

[0004] Pueraria lobata, as a traditional Chinese medicine in China, has various pharmacological activities such as relieving hangover, protecting the liver, anti-inflammatory, and anti-tumor. Puerarin is an active ingredient isolated and extracted from fresh flowers of Pueraria lobata earlier, belonging to the isoflavone natural product category. The puerarin derivative prepared in the present invention is a brand-new drug, and the synthesis scheme mainly involves introducing competitive groups at the phenolic hydroxyl positions of puerarin. There has been no report on the modification of the same drug before. Later phenotypic experiments found that this compound has a certain inhibitory effect on liver cancer cells, can inhibit cell proliferation and migration, induce apoptosis and cell cycle arrest, and its use is disclosed for the first time. Summary of the Invention

[0005] Technical Problem to be Solved: In order to develop more new anti-cancer drugs that can effectively inhibit the growth activity of liver cancer cells, the present invention provides a puerarin derivative, a preparation method thereof, and an application thereof, in order to help with the clinical treatment of hepatocellular carcinoma.

[0006] Technical Solution: The puerarin derivative has the following structural formula: R1 and R2 are both straight-chain or branched alkanes, esters, silyl ethers, trifluoro or polyfluoroalkanes with 1-5 carbon atoms, including H, CH3, CH2F, CHF2, CF3, t-Bu, MOM, EE, THP, and carbon atoms 1-5. Straight-chain terminal alkynes n = 1, 2, 3, 4 or 5, m = 1, 2, 3, 4, 5 or 6, when one group is a terminal alkyne, the other two groups are alkanes or fluoroalkanes.

[0007] The ester groups mentioned above are Ac, t-BuCO, PhCO, Ts, Ms, Tf, PO(OR`)2, where R` = alkyl or aryl.

[0008] The aforementioned silyl ethers are TMS, TES, TBS, TIPS, or TBDPS.

[0009] The preferred compounds of the puerarin derivatives have the following structural formulas:

[0010]

[0011] The preparation method of the kudzu root extract derivative includes the following steps:

[0012]

[0013] The preparation method of the above-mentioned puerarin derivative is characterized by the following steps: First, dissolving the drug: Puerarin is dissolved in N,N-dimethylformamide, and K2CO3 is added and stirred at room temperature for 15 min, wherein the molar ratio of K2CO3 to puerarin is 1.2:1; Second, substitution reaction: hept-6-yn-1-yl ethanesulfonate is added, wherein the molar ratio of hept-6-yn-1-yl ethanesulfonate to puerarin is 1:1, and the mixture is stirred overnight at 50°C. The reaction is monitored for completeness by TLC and LCMS; Third, purification: the mixture is filtered and passed through a reverse flash column to obtain a white solid puerarin derivative.

[0014] The application of the above-mentioned puerarin derivatives in the preparation of drugs that inhibit liver cancer cells SMMC-7721.

[0015] The drug that inhibits liver cancer cells SMMC-7721 contains the above-mentioned puerarin derivative as its active ingredient.

[0016] Beneficial effects: First, the present invention successfully prepared puerarin derivatives with high yield; second, the puerarin derivatives prepared by the present invention have the effect of inhibiting the growth, proliferation, invasion and migration of liver cancer cells, and can induce apoptosis and cell cycle arrest of liver cancer cells, which can effectively alleviate the drug resistance problem in clinical practice and provide a new lead drug for the treatment of hepatocellular carcinoma. Attached Figure Description

[0017] Figure 1 Identification of the HK derivative of kudzu root extract by liquid chromatography-mass spectrometry (LCMS);

[0018] Figure 2 Identification of the kudzu root extract derivative SQYHT-01 by liquid chromatography-mass spectrometry (LCMS);

[0019] Figure 3 Identification of the kudzu root extract derivative SQYHT-02 by liquid chromatography-mass spectrometry (LCMS);

[0020] Figure 4 Identification of the kudzu root extract derivative SQYHT-03 by liquid chromatography-mass spectrometry (LCMS);

[0021] Figure 5 Identification of the puerarin derivative SQYHT-04 by liquid chromatography-mass spectrometry (LCMS);

[0022] Figure 6 NMR identification (H1NMR spectrum) of the kudzu extract derivative: HK: 1 H NMR (500MHz, DMSO-d6) δ9.55(s,1H),8.37(s,1H),7.37-7.45(m,3H),7.21(s,1H),6.77-6.83(m,2H),4.06(t,J=6 .5Hz,2H),3.92(s,3H),2.76(t,J=2.6Hz,1H),2.16-2.23(m,2H),1.74-1.83(m,2H),1.49-1.57(m,4H).MS(ESI)C 23 H 22 O5, Calculated for [M+H] + 379.1540; Found: 378.9;

[0023] Figure 7 NMR identification (H1N1 spectrum) of the kudzu extract derivative SQYHT-01: SQYHT-01: 1 H NMR(500MHz,DMSO-d6)δ9.65(s,1H),8.44(s,1H),7.47(s,1H),7.42(s,1H),7.41-7.36( m,4H),7.14(t,J=7.4Hz,1H),6.99(d,J=7.9Hz,2H),6.80(d,J=8.1Hz,2H),3.93(s,3H);

[0024] Figure 8 NMR identification (H1N1 spectrum) of the kudzu root extract derivative SQYHT-02: SQYHT-02: 1H NMR (500MHz, DMSO-d6) δ9.62(s,1H),8.37(s,1H),7.44(s,1H),7.40(d,J=8.1Hz,2H),7.21(s,1H),6.80(d,J=8.1Hz,2H),4. 42-4.36(m,1H),3.91(s,3H),1.99-1.93(m,2H),1.76-1.69(m,2H),1.57-1.52(m,1H),1.48-1.36(m,4H),1.28-1.22(m,1H);

[0025] Figure 9 NMR identification (H1N1 spectrum) of the kudzu extract derivative SQYHT-03: SQYHT-03: 1 H NMR(500MHz,DMSO-d6)δ9.63(s,1H),8.41-8.37(m,1H),7.46(s,1H),7.43-7.37(m,2H),7.25(s,1H),6.8 4-6.78(m,2H),4.86-4.81(m,1H),4.76-4.71(m,1H),4.39-4.34(m,1H),4.33-4.28(m,1H),3.94(s,3H);

[0026] Figure 10 NMR identification (H1N1 spectrum) of the kudzu root extract derivative SQYHT-04: SQYHT-04: 1 H NMR(500MHz,DMSO-d6)δ9.61(s,1H),8.42-8.38(m,1H),7.54-7.50(m,1H),7.43-7.37(m,2H), 7.29-7.25(m,1H),6.84-6.79(m,2H),6.56-6.31(m,1H),4.48-4.38(m,2H),3.96-3.92(m,3H);

[0027] Figure 11 To investigate the effect of different concentrations of the puerarin derivative HK on the proliferation of hepatocellular carcinoma cells SMMC-7721 by the MTT assay;

[0028] Figure 12 To evaluate the effect of the puerarin derivative HK on the proliferation of SMMC-7721 hepatocellular carcinoma cells using a clonogenic assay;

[0029] Figure 13 The effect of different concentrations of the puerarin derivative HK on apoptosis of SMMC-7721 liver cancer cells at 24h and 48h;

[0030] Figure 14The effect of different concentrations of the puerarin derivative HK on cell cycle arrest in SMMC-7721 hepatocellular carcinoma cells over 24 hours;

[0031] Figure 15 To investigate the effect of different concentrations of the puerarin derivative HK on the invasive ability of SMMC-7721 hepatocellular carcinoma cells in a Transwell assay over 24 hours;

[0032] Figure 16 To investigate the effect of different concentrations of the puerarin derivative HK on the migration ability of hepatocellular carcinoma cells SMMC-7721 using a scratch assay. Detailed Implementation

[0033] Example 1

[0034] 1. Materials

[0035] 1.1 Sample: The puerarin derivative HK was prepared using the following method: Step 1, drug dissolution: Puerarin was dissolved in dimethylformamide, and K2CO3 was added and stirred at room temperature for 15 min. The molar ratio of K2CO3 to puerarin was 1.2:1. Step 2, substitution reaction: Hepten-6-yn-1-yl ethanesulfonate was added. The molar ratio of hepten-6-yn-1-yl ethanesulfonate to puerarin was 1:1. The mixture was stirred overnight at 50 °C, and the reaction was monitored for completeness by TLC and LCMS. Step 3, purification: The mixture was filtered and passed through a reverse flash column (H2O / 0.05% FA / MeCN: 20%-80%, 20 min) to obtain a white solid puerarin derivative. The preparation and detection results are as follows: Figure 1-10 As shown.

[0036] 1.2 Cells: SMMC-7721 liver cancer cells were provided by the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0037] 1.3 Main culture media and reagents: RPMI 1640 medium, standard fetal bovine serum, trypsin, crystal violet staining solution, 4% paraformaldehyde, MTT reagent, 100% dimethyl sulfoxide (DMSO), PBS solution, 70% ethanol solution, etc.

[0038] 1.4 Main instruments: cell culture incubator, centrifuge, ELISA reader, flow cytometer, pipette, microscope, etc.

[0039] 1.5 Consumables: EP tubes, tip heads, 96-well cell culture plates, 6-well cell culture plates, 24-well cell culture plates, centrifuge tubes, disposable cell embedding dishes, etc.

[0040] 2. Methods and Results

[0041] 2.1 MTT assay for the inhibitory effect of puerarin derivatives on hepatocellular carcinoma cells SMMC-7721

[0042] (1) Cell suspension preparation: SMMC-7721 cells in the logarithmic growth phase were prepared into a single-cell suspension, and the cell number was adjusted to 2×10⁻⁶. 4 / mL.

[0043] (2) Cell seeding: The cell suspension was seeded into 96-well cell plates at a volume of 100 μL / well and cultured in a humid incubator at 37°C and 5% CO2 for 24 h.

[0044] (3) Add puerarin derivatives: working concentrations of 2, 4, 8, 16, 32 and 64 μg / mL, with each concentration group repeated 3 times. Puerarin and cisplatin were set as control groups. The mixture was incubated at 37℃ and 5% CO2 for 24 h.

[0045] (4) Result detection: Add 10 μL MTT (5 mg / mL) to each well, mix well, and incubate in the dark for 4 h. Then, aspirate the culture medium, wash with PBS, add 100 μL DMSO solution to each well, and shake to mix for 10 min. Measure the absorbance at 490 nm using a microplate reader. Calculate the cell viability using the formula: Cell viability = [(As-Ab)] / [(Ac-Ab)]×100%, where As represents wells containing cell culture medium and drug, Ac represents wells containing cell culture medium but no drug, and Ab represents wells containing only culture medium and no cells or drug.

[0046] (5) Five compounds, namely, the derivatives of kudzu root extract HK, SQYHT-01, SQYHT-02, SQYHT-03, and SQYHT-04, all showed good inhibitory effects on SMMC-7721 liver cancer cells. The results are shown in Table 1 and... Figure 11 As shown, HK exhibited the most significant inhibitory activity against SMMC-7721 liver cancer cells among the five puerarin derivatives, with an IC50 concentration of [missing value]. 50 With a concentration of 2.5 μM, it increased the anticancer activity by nearly 30 times compared with pufatin, and its effect was better than that of cisplatin.

[0047] Table 1. Effects of cisplatin, bufotin, and bufotin derivatives on cellular IC50. 50 (μM)

[0048]

[0049] 2.2 Colony formation assay to evaluate the effect of puerarin derivative HK on the proliferation of hepatocellular carcinoma cells SMMC-7721.

[0050] (1) Cell suspension preparation and inoculation: SMMC-7721 cells were prepared into a cell suspension and inoculated into 6-well cell plates at a density of 700 cells / well. 2 mL of RPMI 1640 complete culture medium was added and the cells were incubated statically at 37°C, 5% CO2 and saturated humidity for 24 h.

[0051] (2) Add 0.25, 1 and 4 μM concentrations of puerarin derivatives and continue culturing for 14 days. During this period, the corresponding drug-containing culture medium for each group is changed every two to three days, and the cell status is observed. The cisplatin group is set as a positive control.

[0052] (3) Cell fixation and staining: Add 1 mL of 4% paraformaldehyde to each well for 30-60 min, wash three times with PBS, add 1 mL of 0.1% crystal violet solution to each well for 10-20 min, and wash several times with PBS solution until the background is clean.

[0053] (4) Observe and photograph the cells in each well and dry them. Observe the number of cell colonies in each well under a microscope and take photos to record the results.

[0054] (5) Results of the effect of the puerarin derivative HK on the proliferation of SMMC-7721 cells are as follows: Figure 12 Plate cloning experiments showed that as the concentration of puerarin derivatives increased, colony formation in SMMC-7721 cells decreased significantly, and the effect of puerarin derivatives in inhibiting cell proliferation was better than that in the Control group.

[0055] 2.3 Detection of the apoptosis-inducing effect of the puerarin derivative HK on SMMC-7721 hepatocellular carcinoma cells.

[0056] (1) Cell suspension preparation and seeding: SMMC-7721 cells with a confluence of 80%-90% were prepared into a cell suspension and seeded at 1×10⁻⁶ cells per cell. 5 Cells were seeded at a density of 10 cells / well in 6-well cell culture plates, and 2 mL of RPMI 1640 complete medium was added. The cells were then cultured at 37°C, 5% CO2 and saturated humidity for 24 h.

[0057] (2) Add the working concentrations of the kudzu root extract derivatives at 0.25, 1 and 4 μM respectively, and incubate in an incubator for 24 h and 48 h respectively.

[0058] (3) Digest cells: Collect the original culture medium in the 6-well plate and set aside. Wash the cells in the wells 1-2 times with PBS solution, then add 1 mL of trypsin (preferably EDTA-free) to each well to digest the adherent cells. Stop digestion with the collected culture medium, centrifuge the cell suspension at 1000g for 5 min, discard the supernatant, and collect the cells.

[0059] (4) Cell counting: Gently resuspend cells in PBS solution and count them. Take 5 × 10⁶ cells.4 -1×10 5 Centrifuge the resuspended cells again at 1000g for 5 minutes and discard the supernatant.

[0060] (5) For flow cytometry analysis, first add 195 μL of Annexin V-FITC binding solution, gently resuspend the cells, then add 5 μL of Annexin V-FITC, gently mix, and finally add 10 μL of PI staining solution, gently mix. Incubate at room temperature (20-25℃) in the dark for 10-20 min, then place in an ice bath and perform the analysis within 1 hour.

[0061] (6) Results: After treating SMMC-7721 cells with 0.25, 1, and 4 μM of the puerarin derivative HK for 24 and 48 h, respectively, the puerarin derivative HK induced apoptosis in SMMC-7721 hepatocellular carcinoma cells. Figure 13 As shown, compared with the Control group, the number of cells undergoing early apoptosis, late apoptosis, and necrosis increased with increasing concentration. These concentrations of the puerarin derivative HK promoted apoptosis in a dose- and time-dependent manner.

[0062] 2.4 Detection of the cell cycle arrest effect of the puerarin derivative HK on hepatocellular carcinoma cells SMMC-7721

[0063] (1) Cell suspension preparation and seeding: SMMC-7721 cells with a confluence of 80%-90% were prepared into a cell suspension and seeded at 1×10⁻⁶ cells per cell. 5 Cells were seeded at a density of 10 cells / well in 6-well cell culture plates, and 2 mL of RPMI 1640 complete medium was added. The cells were then cultured at 37°C, 5% CO2 and saturated humidity for 24 h.

[0064] (2) Add the kudzu root extract derivatives at working concentrations of 0.25, 1 and 4 μM respectively, and incubate in an incubator for 24 h.

[0065] (3) Digest the cells. Carefully collect the original culture medium in the 6-well plate for later use. Add 1 mL of trypsin to each well to digest the adherent cells. Stop the digestion with the original culture medium. Collect the cell suspension in a centrifuge tube and centrifuge at 1000g for 5 min. Discard the supernatant and precipitate the cells.

[0066] (4) Resuspend the cells gently with pre-cooled PBS solution, centrifuge again at 1000g for 5min, discard the supernatant, and gently tap the bottom of the centrifuge tube to disperse the cells appropriately and avoid cell clumping.

[0067] (5) Cell fixation: Add 1 mL of pre-cooled 70% ethanol to each well, gently pipette to mix, and fix at 4°C for at least 2 hours. Centrifuge at 1000g for 5 minutes, discard the supernatant, add 1 mL of pre-cooled PBS solution, resuspend the cells, centrifuge again to precipitate the cells, and gently tap the bottom of the centrifuge tube to disperse the cells appropriately and avoid cell clumping.

[0068] (6) Cell staining: Prepare propidium iodide staining solution according to the instructions. Add 0.5 mL of propidium iodide staining solution to each tube of cell sample, slowly and fully resuspend the cells, incubate at 37°C in the dark for 30 min, and then store in an ice bath in the dark.

[0069] (7) Flow cytometry detection: Flow cytometry detection was completed within 24 hours after staining.

[0070] (8) The results are as follows Figure 14 As shown, after 24 hours of treatment of hepatocellular carcinoma SMMC-7721 cells with the puerarin derivative HK, the proportion of cells in the G2 / M phase was significantly increased and the distribution of cells in the G0 / G1 phase was reduced. It is believed that the puerarin derivative mainly inhibits the growth of hepatocellular carcinoma SMMC-7721 cells by inducing partial G2 / M cell cycle arrest in a dose-dependent manner, thereby achieving an anti-tumor effect.

[0071] 2.5 Transwell assay to detect the inhibitory effect of puerarin derivative HK on the invasion of hepatocellular carcinoma cells SMMC-7721.

[0072] (1) Cell culture: First, SMMC-7721 cells were starved for 12-24 hours after serum withdrawal. Then, the cells were resuspended in serum-free RPMI 1640 medium, counted, and the cell concentration was adjusted to 5×10⁶. 5 indivual.

[0073] (2) Cell seeding: Using sterile forceps, place the Transwell chamber into a 24-well cell plate. Add 600 μL of RPMI 1640 complete culture medium containing 10% fetal bovine serum to each well in the lower chamber, and add 200 μL of cell suspension to each well in the upper chamber.

[0074] (3) Add the working concentrations of the kudzu root extract derivatives at 0.25, 1 and 4 μM respectively, and culture in a 37℃, 5% CO2 cell incubator for 24 h.

[0075] (4) Cell fixation and staining: Remove the Transwell chambers, aspirate the culture medium, add 1 mL of 4% paraformaldehyde to a clean well in a 24-well plate, place the chambers in and fix for 10-15 min, wash with PBS solution, stain with 0.1% crystal violet solution for 5-10 min, rinse with PBS solution 3 times, and gently wipe the upper side of the chambers with a cotton swab to remove the dye that is not specifically bound to the upper surface of the chambers for subsequent microscopic examination.

[0076] (5) Cell counting: Observe and count the positive cells that turn purple under an inverted microscope, take pictures and count the results.

[0077] (6) The results are as follows Figure 15 As shown, compared with the Control group, after SMMC-7721 cells were treated with different concentrations of the puerarin derivative HK for 24 h, the number of cells in the lower chamber decreased with the increase of the puerarin derivative concentration, and the cell migration and invasion ability were significantly inhibited.

[0078] 2.6 Scratch assay to detect the inhibitory effect of puerarin derivative HK on the migration of hepatocellular carcinoma cells SMMC-7721.

[0079] (1) Cell suspension preparation and seeding: SMMC-7721 cells in the logarithmic growth phase were prepared into a cell suspension and seeded at 5 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 10 cells / well in 6-well cell culture plates, and 2 mL of RPMI 1640 complete medium was added. The cells were then cultured at 37°C, 5% CO2 and saturated humidity for 24 h.

[0080] (2) After adding the puerarin derivative and the cells covered the bottom wall, use the tip of a 20 μL pipette to streak the bottom of the 6-well plate evenly. The drug concentrations were 0.25, 1 and 4 μM. The experimental group cells were treated with RPMI 1640 complete medium containing 2% fetal bovine serum. Cisplatin group was set as positive control.

[0081] (3) Observation and photography: The cells were incubated in a constant temperature incubator for 0h, 6h, 12h, 24h, 48h and 72h, and photographed under an inverted microscope to record the results. The inhibitory effect of the pueraria lobata derivative on the migration of SMMC-7721 cells was analyzed.

[0082] (4) Results of the scratch test are as follows Figure 16 As shown, compared with the cisplatin control group, treatment of SMMC-7721 cells with the puerarin derivative HK began to inhibit cell migration after 6 h, and significantly reduced the scratch healing rate at 24 h, 48 h and 72 h in a time- and dose-dependent manner.

Claims

1. A derivative of kudzu root extract, characterized in that, The structural formula is as follows:

2. The method for preparing the puerarin derivative according to claim 1, characterized in that, The steps are as follows:

3. The method for preparing the puerarin derivative according to claim 2, characterized in that, Step 1, drug dissolution: Dissolve puerarin in N,N-dimethylformamide, add K2CO3 and stir at room temperature for 15 min, the molar ratio of K2CO3 to puerarin is 1.2:1; Step 2, substitution reaction: Add hept-6-yn-1-ylethanesulfonate, the molar ratio of hept-6-yn-1-ylethanesulfonate to puerarin is 1:1, stir overnight at 50 °C, and monitor the reaction for completeness by TLC and LCMS; Step 3, purification: Filter and pass through a reverse flash column to obtain a white solid puerarin derivative.

4. The use of the puerarin derivative of claim 1 in the preparation of a drug for treating liver cancer by inhibiting SMMC-7721 liver cancer cells.

5. A drug for treating liver cancer by inhibiting SMMC-7721 liver cancer cells, characterized in that, The active ingredient contains the kudzu root extract derivative as described in claim 1.

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