Piperine derivative as well as preparation method and application thereof

Abstract

The invention discloses a piperine derivative as well as a preparation method and application thereof, and relates to the technical field of medical chemistry. The invention provides a specific structural formula of the piperine derivative, and also provides a preparation method of the piperine derivative. The piperine derivative shows remarkable anti-proliferative activity and antifeedant activity, can be applied to development of natural botanical insecticides, and shows potential application value in the field of preparation of novel environment-friendly pesticides.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to a piperine derivative and a preparation method and application thereof. Background Art

[0002] In recent years, structural modification of botanical pesticides has become an important approach to enhance their bioactivity. For example, insecticides such as pyrethroids, rotenone, and matrine have significantly improved their insecticidal activity and bioselectivity through structural optimization.

[0003] Piperine, a natural plant secondary metabolite, possesses a wide range of biological activities, including insecticidal, antibacterial, anti-inflammatory, and antioxidant properties. Its natural origin and environmental friendliness make it an ideal candidate for the development of new green pesticides. In recent years, the structural modification of piperine has seen rapid development. However, it is important to note that existing studies have limited the target insect species of piperine, which significantly restricts its widespread application in pest control. Furthermore, in-depth research on the antiproliferative activity of piperine is relatively scarce, resulting in a lack of understanding of its mechanisms of inhibiting pest growth and reproduction at the cellular level. Therefore, structural modification of piperine to broaden its insecticidal spectrum has become an important and pressing issue in this field.

[0004] Diamide insecticides are a new class of highly effective insecticides with a broad spectrum of insecticide activity and strong insecticidal activity. They have novel targets, making it difficult for pests to develop resistance, thus giving them significant advantages. At the same time, they also possess unique cytotoxic effects, acting precisely on pest cells and effectively killing them. A deeper look into their chemical structure reveals that the core structure of diamide insecticides is a benzene ring and two amide bonds connected to it, namely a phenylenediamine structure. Phenylenediamine includes three isomers: paraphenylenediamine, metaphenylenediamine, and o-phenylenediamine. These can provide multiple reaction sites during chemical structure modification, providing opportunities for chemical structure modification and optimization. In contrast, although piperine has certain insecticidal activity, studies have found that its range of action is relatively narrow and it is only effective against a few pests. In view of this, this study proposes to combine piperine with a phenylenediamine structure to form a piperine-phenylenediamine derivative with synergistic effects. Through this structural modification, the insecticidal activity and cytotoxicity of piperine against other pests are enhanced, thereby effectively expanding its insecticidal spectrum. It is expected to solve the limitations of piperine in agricultural applications, and to promote the development and application of botanical insecticides. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a piperine derivative and its preparation method and application. The piperine derivative exhibits significant antiproliferative activity and antifeedant activity, can be applied to the development of natural plant-derived insecticides, and shows potential application value in the field of preparing new environmentally friendly pesticides.

[0006] The present invention solves the above-mentioned technical problems with the following technical solutions: providing a piperine derivative, which is prepared by combining piperine with para-phenylenediamine or meta-phenylenediamine through an amidation reaction, and simultaneously introducing a fatty chain or a substituted benzene ring by changing the side chain group, wherein the piperine derivative has one of the following structural formulas: 、 、 .

[0007] The present invention also provides a method for preparing the above-mentioned piperine derivative, comprising the following steps: (1) Hydrolyze piperine in an ethanol aqueous solution to obtain piperic acid (compound a); The reaction process is as follows: ; (2) Piperonic acid reacts with oxalyl chloride in the presence of dimethylformamide to obtain compound b; The reaction process is as follows: ; (3) Compound b is subjected to a condensation reaction with p-phenylenediamine under alkaline conditions of triethylamine to obtain compound c; The reaction process is as follows: ; (4) Compound c is reacted with acryloyl chloride and cinnamoyl chloride to obtain piperine derivatives (compounds 1 and 2).

[0008] The reaction process is as follows: ; Alternatively, compound b is reacted with m-aminobenzamide to obtain a piperine derivative (compound 3); The reaction process is as follows: .

[0009] Furthermore, the specific process of step (1) is as follows: dissolving piperine in an ethanol aqueous solution, then adding a 20 wt% potassium hydroxide aqueous solution, reflux at 90°C for 24 h, removing ethanol by rotary evaporation, diluting the residue with water and adjusting the pH to 2-3 with dilute hydrochloric acid for precipitation, and sequentially extracting, drying and vacuum concentrating to obtain piperic acid.

[0010] Furthermore, the molar volume ratio of piperine, ethanol aqueous solution, and potassium hydroxide aqueous solution is 7.00 mmol:10 mL:15 mL.

[0011] Furthermore, the specific process of step (2) is as follows: dissolving piperic acid in ultra-dry dichloromethane, then adding oxalyl chloride and dimethylformamide dropwise, stirring at room temperature for 2 h, and concentrating in vacuo to obtain compound b.

[0012] Furthermore, the molar volume ratio of piperic acid, oxalyl chloride, dimethylformamide, and ultra-dry dichloromethane is 2.00 mmol:2.20 mmol:0.16 mmol:8 mL.

[0013] Furthermore, the specific process of step (3) is as follows: under anhydrous and oxygen-free conditions, p-phenylenediamine is dissolved in ultra-dry dichloromethane, triethylamine is added dropwise under ice bath conditions, and then compound b is added dropwise, and the mixture is stirred in an ice bath for 2 h and then at room temperature for 4 h. After removing the solvent by rotary evaporation, the residue is dissolved in ethyl acetate, and the mixture is washed, dried, vacuum concentrated and purified in sequence to obtain compound c.

[0014] Furthermore, the molar volume ratio of p-phenylenediamine, triethylamine, compound b and ultra-dry dichloromethane is 2.00 mmol:3.00 mmol:2.00 mmol:8 mL.

[0015] Furthermore, the specific process of step (4) is as follows: under anhydrous and oxygen-free conditions, compound c is dissolved in ultra-dry dichloromethane, triethylamine is added dropwise in an ice bath, and then acid chloride is added dropwise, the mixture is stirred in an ice bath for 2 h and then stirred at room temperature overnight, the solvent is removed by rotary evaporation, and the residue is dissolved in ethyl acetate, and the mixture is washed, separated, dried, vacuum concentrated, and purified in sequence to obtain a piperine derivative; Alternatively, under anhydrous and oxygen-free conditions, m-aminobenzamide is dissolved in ultra-dry dichloromethane, and triethylamine is added dropwise in an ice bath, followed by compound b. The mixture is stirred in an ice bath for 2 h and then at room temperature overnight. The solvent is removed by rotary evaporation, and the residue is dissolved in ethyl acetate. The mixture is washed, separated, dried, concentrated in vacuo, and purified to obtain a piperine derivative.

[0016] Furthermore, the molar volume ratio of compound c, triethylamine, acyl chloride and ultra-dry dichloromethane is 0.20 mmol:0.30 mmol:0.24 mmol:8 mL.

[0017] Furthermore, the acid chloride is acryloyl chloride or cinnamoyl chloride.

[0018] Furthermore, the molar volume ratio of m-aminobenzamide, triethylamine, compound b and ultra-dry dichloromethane is 0.20 mmol:0.30 mmol:0.24 mmol:8 mL.

[0019] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is the above-mentioned piperine derivative, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0020] The present invention also provides the use of the piperine derivative, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a drug having anti-proliferative activity and / or antifeedant activity.

[0021] Furthermore, the anti-proliferation activity and / or antifeedant activity is Sf9 cell anti-proliferation activity and / or Lepidoptera insect antifeedant activity.

[0022] Furthermore, the drug is an environmentally friendly natural plant-derived pesticide.

[0023] The present invention has the following beneficial effects: 1. The piperine derivatives of the present invention have both anti-proliferation activity and antifeedant activity, and can be developed as candidates for new botanical pesticides. They have broad application prospects in the preparation of new environmentally friendly natural botanical pesticides.

[0024] 2. In terms of antifeedant activity, piperine derivatives showed significant structure-activity relationship (SAR). The experimental results showed that at a concentration of 5.00 mg / mL, the antifeedant rate of compound 1 reached 94.61%, and its EC 50 The value was 0.184 mg / mL, which was lower than that of piperine (0.195 mg / mL), indicating that compound 1 could effectively inhibit the feeding behavior of Spodoptera exigua at low concentrations.

[0025] 3. In terms of cytotoxicity, the experimental results showed that compound 2 and compound 3 had μ The inhibition rate of Sf9 cells at the concentration of M was over 90%, showing a high anti-proliferative activity. 50 The results showed that the IC values ​​of compounds 2 and 3 were 50 The values ​​are 4.137 μ M and 3.726 μ M, much lower than piperine (107.490 μ M), and is close to azadirachtin (3.532 μ M), indicating that these two derivatives have significant advantages in cytotoxicity.

[0026] 4. The preparation method of the conjugate of piperine and phenylenediamine of the present invention is simple and easy, and the raw materials are low in cost and readily available, and are suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the cell morphology observation picture after treatment with compound 2; Figure 2 This is the cell morphology observation picture after treatment with compound 3; Figure 3 This is the fluorescence staining image of cells after treatment with compound 2; Figure 4 This is the fluorescence staining image of cells after treatment with compound 3; Figure 5 The results are analyzed by flow cytometry. DETAILED DESCRIPTION

[0028] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0029] Example 1 A piperine derivative, the structural formula of which is: ; The preparation method of the above-mentioned piperine derivatives, and its preparation route are as follows: ; The specific steps include: (1) Piperine (2 g, 7.00 mmol) was dissolved in 10 mL of ethanol-water solution, and then 15 mL of 20 wt% potassium hydroxide solution was added. The mixture was refluxed at 90 °C for 24 h. The reaction progress was monitored by TLC. After the reaction was completed, ethanol was removed by rotary evaporation. The residue was diluted with water and the pH was adjusted to 2-3 with 0.1 mol / L dilute hydrochloric acid to precipitate the target product. The product was extracted with ethyl acetate several times, dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain piperic acid (compound a, light yellow solid, yield 82%). (2) Dissolve piperic acid (436 mg, 2.00 mmol) in 8 mL of ultra-dry dichloromethane, and then slowly add oxalyl chloride (190 μ L, 2.20 mmol) and dimethylformamide (120 μ L, 0.16 mmol), stirred at room temperature for 2 h until the reaction solution gradually became clear, and concentrated in vacuo to remove the reaction solvent to obtain compound b; (3) Under anhydrous and oxygen-free conditions, p-phenylenediamine (216 mg, 2.00 mmol) was dissolved in 8 mL of ultra-dry dichloromethane, and triethylamine (417 μl, 3.00 mmol) as a base catalyst, then compound b (473 mg, 2.00 mmol) was dissolved in a small amount of ultra-dry dichloromethane and slowly added dropwise to the reaction solution. The mixture was stirred in an ice bath for 2 h, then the ice bath was removed and stirred at room temperature for 4 h. The reaction progress was monitored by TLC. When TLC showed that the reaction was complete, the solvent was removed by rotary evaporation, and the residue was dissolved in ethyl acetate and washed with saturated sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by thin-layer chromatography to obtain compound c. (4) Under anhydrous and oxygen-free conditions, compound c (60 mg, 0.20 mmol) was dissolved in 8 mL of ultra-dry dichloromethane, and triethylamine (42 μ l, 0.30 mmol), then acryloyl chloride (40 mg, 0.24 mmol) was dissolved in a small amount of ultra-dry dichloromethane and slowly added dropwise to the reaction solution. The mixture was stirred in an ice bath for 2 h and then stirred at room temperature overnight. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporation, and the residue was dissolved in ethyl acetate and washed with saturated sodium bicarbonate solution. The layers were separated, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by thin-layer chromatography to obtain a piperine derivative (compound 1, yellow amorphous powder, yield 69%).

[0030] 1 H NMR (400 MHz, DMSO- d 6) δ 10.1 (s, 1H), 9.9 (s, 1H), 7.6 – 7.6 (m,4H), 7.3 – 7.3 (m, 2H), 7.1 – 6.9 (m, 4H), 6.8 (m, J = 15.3, 6.9 Hz, 1H), 6.3(d, J = 14.9 Hz, 1H), 6.1 (m, J = 15.1, 1.6 Hz, 1H), 6.1 (s, 2H), 1.9 (dd, J = 6.9,1.7 Hz, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ 163.5, 163.1, 147.9, 147.8, 140.6,139.3, 138.6, 134.7, 134.6, 130.7, 126.0, 125.0, 124.3, 122.8, 119.5, 119.4,108.4, 105.6, 101.2, 17.4. 18.6, 13.7. HRESIMS ( m / z): 377.6735 [M + H]+(calcd for C 22 H 20 N2O4, 377.1501).

[0031] Example 2 A piperine derivative, the structural formula of which is: ; The preparation method of the above piperine derivative is similar to that of Example 1, except that acryloyl chloride in step (4) is replaced by cinnamoyl chloride to obtain a piperine derivative (compound 2, yellow amorphous powder, yield 75%).

[0032] 1 H NMR (400 MHz, DMSO- d 6) δ 10.18 (s, 1H), 10.10 (s, 1H), 7.68 – 7.55(m, 7H), 7.48 – 7.41 (m, 3H), 7.33 – 7.29 (m, 2H), 7.05 – 6.92 (m, 4H), 6.83(dd, J = 15.8, 2.5 Hz, 1H), 6.29 (d, J = 14.9 Hz, 1H), 6.05 (s, 2H). 13 C NMR (100 MHz, DMSO- d 6) δ 163.6, 163.2, 148.0, 147.9, 140.8, 139.9, 138.8, 135.0,134.8, 134.7, 130.8, 129.7, 129.1, 129.0, 127.7, 125.1, 124.4, 122.9, 122.4,119.6, 116.9, 108.5, 105.7, 101.3. HRESIMS ( m / z ): 439.1649 [M + H]+ (calcdfor C 27 H 22 N2O4, 439.1658).

[0033] Example 3 A piperine derivative, the structural formula of which is: ; The preparation method of the piperine derivative is similar to steps (1)-(2) of Example 1, and step (3) is specifically as follows: Under anhydrous and oxygen-free conditions, m-aminobenzamide (60 mg, 0.40 mmol) was dissolved in 8 mL of ultra-dry dichloromethane, and triethylamine (83 μ l, 0.60 mmol), then compound b (104 mg, 0.44 mmol) was dissolved in a small amount of ultra-dry dichloromethane and slowly added dropwise to the reaction solution. The mixture was stirred in an ice bath for 2 h and then stirred at room temperature overnight. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporation, and the residue was dissolved in ethyl acetate and washed with saturated sodium bicarbonate solution. The layers were separated, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by thin-layer chromatography to obtain a piperine derivative (compound 3, off-white amorphous powder, yield 72%).

[0034] 1 H NMR (400 MHz, DMSO- d 6) δ 10.1 (s, 1H), 10.0 (s, 1H), 8.0 (s, 1H), 7.4 (d, J = 7.9 Hz, 1H), 7.4 – 7.2 (m, 3H), 7.2 (t, J = 8.0 Hz, 1H), 7.1 – 6.8(m, 4H), 6.3 (d, J = 14.8 Hz, 1H), 6.1 (s, 2H), 2.0 (s, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ 168.3, 163.9, 148.0, 147.9, 140.9, 139.7, 139.6, 138.9, 130.8,128.8, 125.1, 124.5, 122.9, 114.1, 110.0, 108.5, 105.7, 101.3, 24.0. HRESIMS( m / z ): 351.1325 [M + H]+ (calcd for C20H18N2O4, 351.1345).

[0035] Test Example 1 Study on Antifeedant Activity 1. Experimental methods To evaluate the antifeedant properties of piperine derivatives against the beet armyworm (Spodoptera exigua), a leaf disc assay was conducted. Beet armyworm larvae were reared on cabbage leaves under a 16-hour light / 8-hour dark photoperiod, a temperature of 24 ± 1°C, and a relative humidity of 70%. Synthetic piperine derivative 1 was selected, and commercially available azadirachtin was used as a positive control. Samples were first dissolved in 10% acetone and then diluted to the desired concentration (5.00 mg / mL) with deionized water containing 0.2% Tween-80.

[0036] The experimental steps are as follows: Cut fresh cabbage leaves into leaf discs with a diameter of 5 cm, soak them in the prepared solution for 20 minutes, take them out and air-dry them. Place two treated leaf discs in a 15 cm diameter culture dish, and place 8 healthy third-instar larvae that have been starved for 6 hours in each culture dish. The experiment was carried out in a growth chamber with the same breeding conditions (light cycle 16:8, temperature 24±1 ℃, relative humidity 70%), and each treatment was repeated 3 times. After 24 hours, the feeding area of ​​the larvae was determined by the coordinate paper method. For compounds with a feeding refusal rate greater than 90% in the dose-response experiment, their half-lethal concentration (EC50) was further determined. 50 ) value. EC was calculated using Origin2019 software. 50 The values ​​and SD values ​​were obtained. The feed rejection rate (FR) was calculated using the following formula: FR=(CK-T) / CK×100%; Wherein, CK is the feeding area of ​​leaves in the negative control; T is the feeding area of ​​leaves in the treatment group.

[0037] 2. Experimental results The above experimental results are shown in Table 1.

[0038] Table 1 Antifeedant rate and EC of the tested compounds 50 value

[0039] As shown in Table 1, at a concentration of 5.00 mg / mL, the antifeedant activity of compound 1 was 94.61%, which was higher than that of piperine (93.80%). To further evaluate the antifeedant activity of compound 1, azadirachtin was used as a positive control and experiments were conducted at different doses (0.313, 0.625, 1.250, 2.500, and 5.000 mg / mL), and the EC values ​​were calculated. 50 The results showed that the EC 50 The value was 0.184 mg / mL, which was lower than that of piperine (0.195 mg / mL), indicating that compound 1 had stronger antifeedant activity than the parent piperine at the same concentration.

[0040] Experimental Example 2: Study on the cytotoxicity of insect Sf9 cells 1. Experimental methods In order to evaluate the toxic effects of drugs on Sf9 cells, the MTT colorimetric assay was used to detect cell viability. In the experiment, Sf9 cells in the logarithmic growth phase were collected and the cell density was adjusted to 8×10 4 Then, take 100 μ L cell suspension was added to a 96-well cell culture plate, and 100 L cell suspension was added to each well. μ The cells were treated with 0.1% DMSO solution (treated group) or 0.1% DMSO solution (control group) for 48 hours. The experiment was repeated 6 times to ensure the reliability of the data. Cell viability was detected by MTT colorimetry. 20 μL of drug solution (treated group) or 0.1% DMSO solution (control group) were added to each well. μ After incubation in a cell culture incubator for 4 hours, the medium was removed and DMSO was added to dissolve the formazan. The absorbance (OD value) was measured at 492 nm using a microplate reader. The experimental data were analyzed by linear regression using GraphPad Prism 8 software to calculate the half-lethal concentration (IC50) of the drug. 50 The cell lethality rate was calculated as follows: Inhibition rate (IR%) = (OD control group - OD treatment group) / (OD control group - OD blank group) × 100%.

[0041] 2. Experimental results The above experimental results are shown in Table 2.

[0042] Table 2 Inhibition rate and IC of the tested compounds 50 value

[0043] From Table 2, we can see that the concentration is 100 μ M, the anti-proliferative effect of compound 2 and compound 3 on Sf9 cells can reach more than 90%, and they can be used as the most potential anti-proliferative active compounds. IC 50 4.137 respectively μ M and 3.726 μ M, with piperine (IC 50 = 107.490 mM) showed a significant improvement, among which compound 3 (IC 50 = 3.726 μ M) had the strongest inhibitory effect on Sf9 insect cells, and was comparable to azadirachtin (IC 50 = 3.532 μ M) approach.

[0044] Experimental Example 3 Cell morphology observation 1. Experimental methods In order to evaluate the effect of the test drug on the morphology of Sf9 cells, high-power microscopy was used to analyze the cell morphology. In the experiment, Sf9 cells in the logarithmic growth phase were first collected and the cell density was adjusted to 1×10 5 cells / mL. Subsequently, 4 mL of the cell suspension was added to a 6-well cell culture plate, and 4 mL of the test drug solution or azadirachtin solution (as a positive control) was added to each well. The cells were treated for 6, 12, 24, and 36 hours. At each time point, changes in cell morphology were observed and recorded using a high-power microscope to assess potential effects of the drugs on cell structure and integrity.

[0045] 2. Experimental results The above experimental results are as follows Figure 1-Figure 2 shown.

[0046] Depend on Figure 1 and Figure 2 As can be seen, untreated cells in the control group were round, well-adhered, and proliferated normally. Treatment with the positive control, azadirachtin, resulted in morphological changes. Compared to normal cells, a small number of floating cells were observed after 6 hours of treatment with compounds 2 and 3. After 12, 24, and 36 hours of induction, the number of adherent cells decreased and apoptotic bodies increased. These results suggest that compounds 2 and 3 may inhibit cell proliferation by inducing apoptosis.

[0047] Experimental Example 4 Cell Fluorescence Staining 1. Experimental methods Sf9 cells were seeded on coverslips and cultured in 24-well plates for 24 hours, followed by co-incubation with compounds 2 and 3 for 24 hours. Apoptosis was then detected using the Annexin V-FITC apoptosis detection kit for adherent cells: a. Aspirate the cell culture medium into a suitable centrifuge tube, wash the adherent cells once with PBS, and add an appropriate amount of trypsin digestion solution (optionally containing EDTA) to digest the cells. Incubate at room temperature until the adherent cells can be dislodged by gentle pipetting, then remove the trypsin digestion solution. Avoid over-digestion with trypsin. Note: The trypsin digestion step is critical for adherent cells. If the trypsin digestion time is too short, the cells will require vigorous pipetting to dislodge, which can damage the cell membrane and lead to false positives for cell necrosis. If the digestion time is too long, this can also damage the cell membrane and result in false positives for cell necrosis. It can also affect the binding of phosphatidylserine on the cell membrane to Annexin V-FITC, thereby interfering with the detection of cell apoptosis. Furthermore, the trypsin digestion solution should be EDTA-free, as EDTA may interfere with the binding of Annexin V to phosphatidylserine.

[0048] b. Add the cell culture medium collected in step a. Gently pipette the cells to dislodge them, transfer them to a centrifuge tube, and centrifuge at 1000g for 5 minutes. Discard the supernatant, collect the cells, gently resuspend them in PBS, and count them. Note: Adding the cell culture medium from step a is very important. Firstly, it allows the collection of apoptotic or necrotic cells that have already been suspended. Second, the serum in the cell culture medium can effectively inhibit or neutralize residual trypsin. Residual trypsin will digest and degrade the subsequently added Annexin V-FITC, resulting in staining failure.

[0049] c. Take 50,000 to 100,000 resuspended cells, centrifuge at 1000 g for 5 minutes, discard the supernatant, and add 195 μ Gently resuspend the cells in Annexin V-FITC conjugate solution.

[0050] d. Add 5 μ l Annexin V-FITC, mix gently.

[0051] e. Add 10 μ l Propidium iodide staining solution, mix gently.

[0052] f. Incubate at room temperature (20-25°C) in the dark for 10-20 minutes, then place on ice. Aluminum foil can be used to protect from light. Resuspend the cells 2-3 times during the incubation period to improve staining.

[0053] For fluorescence microscopy, centrifuge at 1000 g for 5 minutes, collect the cells, and use 50-100 μ Resuspend the cells gently in Annexin V-FITC conjugate solution, smear, and observe under a fluorescence microscope. Note: The cells must be tested as soon as possible after staining, usually within 1 hour. When used for flow cytometry, if too many false-positive cells are found when Annexin V-FITC is stained alone, and the problem cannot be solved by adjusting the relevant settings and parameters, PBS can be used. Dilute Annexin V-FITC 3-10 times before testing. 2. Experimental results 2. Experimental results The above experimental results are as follows Figure 3-Figure 4 shown.

[0054] Depend on Figure 3-Figure 4It can be seen that the treatment with compound 2 and compound 3 had a significant effect on Sf9 cells. In the control group, the cell morphology was intact, the nucleus and cytoplasm were evenly distributed, the V-FITC and PI fluorescent markers were also relatively evenly distributed, and the green and red fluorescence basically did not overlap. However, in the compound 2 and compound 3 treatment groups, the cell morphology changed, the separation of the nucleus and cytoplasm was obvious, and some cells even had cell membrane rupture. The distribution of fluorescent markers became uneven, and the green and red fluorescence overlapped, indicating that the integrity of the cell nucleus was destroyed. These observations show that compound 2 and compound 3 have obvious cytotoxic effects on Sf9 cells, leading to destruction of cell structure and rupture of cell membranes, further supporting the hypothesis that compound 2 and compound 3 have anti-proliferative activity on Spodoptera frugiperda Sf9 cells.

[0055] Test Example 5 Flow Cytometry 1. Experimental methods Instrument Preparation: Turn on the flow cytometer in advance, preheat it, and calibrate it to ensure it is in optimal working condition. Use standard fluorescent microspheres to calibrate the instrument's fluorescence channel and adjust the parameters of the scattered light and fluorescence channels to ensure detection accuracy.

[0056] Cell staining: Wash the treated SF9 cells with PBS and resuspend them in binding buffer. Add Annexin V-FITC and PI staining solutions and incubate at room temperature in the dark for 10 minutes. Then, add an appropriate amount of binding buffer and mix thoroughly before analysis.

[0057] Instrument Settings: Based on the characteristics of Annexin V-FITC (green fluorescence) and PI (red fluorescence), set the excitation wavelength to 488 nm, and detect green and red fluorescence signals in the FL1 and FL2 / FL3 channels, respectively. Adjust the scattered light (FSC and SSC) parameters to ensure a clear distribution of cell populations.

[0058] Data Acquisition: Place the stained cell sample into a flow cytometer tube, set the flow rate to an appropriate level, and acquire at least 10,000 cell events. Observe the scatter plot to ensure that the cell populations are clearly distributed and without significant overlap.

[0059] Data Recording: Use the accompanying software to record experimental data and save it in FCS file format for subsequent analysis. Also record experimental conditions, including cell treatment concentration, staining time, and instrument parameters.

[0060] 2. Experimental results The above experimental results are as follows Figure 5 shown.

[0061] Depend on Figure 5As shown, treatment with Compound 2 and Compound 3 significantly altered the apoptosis rate of Sf9 cells. The control group had a low apoptosis rate, with the proportions of early and late apoptotic cells being 0.14% and 4.84%, respectively. The apoptosis rates of D15 and J1 cells in the treated groups were significantly increased, with D15 having an early apoptosis rate of 26.18% and a late apoptosis rate of 11.25%; and J1 having an early apoptosis rate of 22.70% and a late apoptosis rate of 19.77%. Overall, treatment with D15 and J1 significantly induced apoptosis, with the early apoptosis rate of D15 slightly higher than that of J1, while the late apoptosis rate of J1 was relatively high. Flow cytometric analysis further supported the findings of the fluorescence staining experiments.

[0062] In summary, the piperine derivatives of the present invention exhibit significant activity in both antifeedant activity and cytotoxicity, providing important theoretical support for piperine derivatives as new botanical insecticides and promising new solutions for green pest control.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A piperine derivative, characterized in that It is prepared by combining piperine with p-phenylenediamine or m-phenylenediamine through amidation reaction, and by changing the side chain groups, introducing aliphatic chains or substituted benzene rings. Its structural formula is one of the following: 、 、 。 2. The method for preparing the piperine derivative according to claim 1, wherein The following steps are involved: (1) Piperine is hydrolyzed in an ethanol aqueous solution to obtain piperic acid; (2) Piperonic acid reacts with oxalyl chloride in the presence of dimethylformamide to obtain compound b; (3) Compound b is subjected to a condensation reaction with p-phenylenediamine under alkaline conditions of triethylamine to obtain compound c; (4) Compound C is reacted with acryloyl chloride and cinnamoyl chloride to obtain a piperine derivative; Alternatively, compound b is reacted with m-aminobenzamide to obtain a piperine derivative.

3. The method for preparing a piperine derivative according to claim 2, wherein: The specific process of step (1) is as follows: dissolving piperine in an ethanol aqueous solution, then adding a 20 wt% potassium hydroxide aqueous solution, reflux at 90°C for 24 hours, removing ethanol by rotary evaporation, diluting the residue with water and adjusting the pH to 2-3 with dilute hydrochloric acid for precipitation, and sequentially extracting, drying and vacuum concentrating to obtain piperic acid.

4. The method for preparing a piperine derivative according to claim 2, wherein: The specific process of step (2) is as follows: dissolve piperic acid in ultra-dry dichloromethane, then dropwise add oxalyl chloride and dimethylformamide, stir at room temperature for 2 h, and concentrate in vacuo to obtain compound b.

5. The method for preparing a piperine derivative according to claim 2, wherein: The specific process of step (3) is as follows: under anhydrous and oxygen-free conditions, p-phenylenediamine is dissolved in ultra-dry dichloromethane, triethylamine is added dropwise in an ice bath, and then compound b is added dropwise. The mixture is stirred in an ice bath for 2 h and then at room temperature for 4 h. After removing the solvent by rotary evaporation, the residue is dissolved in ethyl acetate, washed, dried, vacuum concentrated and purified in sequence to obtain compound c.

6. The method for preparing a piperine derivative according to claim 2, wherein: The specific process of step (4) is as follows: under anhydrous and oxygen-free conditions, compound c is dissolved in ultra-dry dichloromethane, triethylamine is added dropwise in an ice bath, and then acryloyl chloride is added dropwise, the mixture is stirred in an ice bath for 2 hours and then stirred at room temperature overnight, the solvent is removed by rotary evaporation, and the residue is dissolved in ethyl acetate, and the mixture is washed, separated, dried, vacuum concentrated, and purified in sequence to obtain a piperine derivative; Alternatively, under anhydrous and oxygen-free conditions, m-aminobenzamide is dissolved in ultra-dry dichloromethane, and triethylamine is added dropwise in an ice bath, followed by compound b. The mixture is stirred in an ice bath for 2 h and then at room temperature overnight. The solvent is removed by rotary evaporation, and the residue is dissolved in ethyl acetate. The mixture is washed, separated, dried, concentrated in vacuo, and purified to obtain a piperine derivative.

7. A pharmaceutical composition, characterized in that The invention comprises an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is the piperine derivative according to claim 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

8. Use of the piperine derivative according to claim 1, its stereoisomers or pharmaceutically acceptable salts thereof in the preparation of a medicament having anti-proliferative activity and / or antifeedant activity.

9. The use according to claim 8, characterized in that The anti-proliferation activity and / or antifeedant activity is Sf9 cell anti-proliferation activity and / or Lepidoptera insect antifeedant activity.

10. The use according to claim 8, characterized in that The medicine is an environmentally friendly natural plant-derived pesticide.

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