Sesquiterpene lactone derivative, preparation thereof and application of sesquiterpene lactone derivative in hemodialysis blood vessel protection

Abstract

The invention discloses a sesquiterpene lactone derivative as well as preparation and application thereof in dialysis blood vessel protection. The sesquiterpene lactone derivative disclosed by the invention has a structure as shown in a formula (I), is mainly prepared from sesquiterpene lactone and amino acid through reaction, and can be used for preparing an internal arteriovenous fistula protection medicine for renal dialysis. Expression of PCNA and TLR4 in VSMCs can be effectively inhibited, proliferation of VSMCs in an AVF model is further inhibited, venous stenosis and thrombosis caused by neointimal hyperplasia are reduced, the incidence rate of AVF dysfunction is reduced, and the application prospect in preparation of internal arteriovenous fistula protection drugs for renal dialysis is good. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a sesquiterpene lactone derivative, its preparation and application, and particularly to a sesquiterpene lactone-water-soluble amino acid derivative, its preparation and application in the protection of dialysis blood vessels. Background Art

[0002] Hemodialysis is one of the effective methods for the replacement therapy of patients with end-stage renal disease. The vascular access is the "lifeline" of patients with end-stage renal disease. The autologous arteriovenous fistula (AVF) is the preferred form of vascular access for hemodialysis because of its high patency rate, few complications, convenient surgical operation and low medical cost, but it is prone to frequent failures. According to the analysis of the pooled data provided by the Dialysis Outcomes and Practice Patterns Study (DOPPS) Committee, the primary patency rate of arteriovenous fistulas after 1 year is 85%, and the patency rate after 2 years excluding primary failure is 75%. The primary failure rate in some centers can be as high as 50%. The main reasons for AVF dysfunction are venous stenosis and thrombosis caused by neointimal hyperplasia. Therefore, how to increase the service life of AVF and reduce AVF dysfunction remains a major and urgent clinical problem. Summary of the Invention

[0003] Object of the Invention: The object of the present invention is to provide a sesquiterpene lactone derivative represented by formula (I) or a pharmaceutically acceptable salt thereof, to solve the problem of how to reduce AVF dysfunction caused by neointimal hyperplasia. Another object of the present invention is to provide a method for preparing a sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof, to solve the problem of how to prepare a sesquiterpene lactone derivative. The third object of the present invention is to provide the application of a sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for protecting arteriovenous fistulas for renal dialysis, to solve the problem of how to obtain a drug for protecting arteriovenous fistulas for renal dialysis.

[0004] Technical Solution: A sesquiterpene lactone derivative represented by formula (I) or a pharmaceutically acceptable salt thereof according to the present invention:

[0005]

[0006] Wherein, R is selected from a substituted C 1-6 alkyl group, and the substituent group is selected from one of a mercapto group, a hydroxyl group, an amide group, and a phenyl group substituted with a hydroxyl group.

[0007] Preferably, the alkyl group is a straight-chain alkyl group, for example, a methyl group, an ethyl group, a n-propyl group, a n-butyl group, a n-pentyl group, etc.

[0008] Preferably, the sesquiterpene lactone derivative includes at least one of the following compounds:

[0009]

[0010] The second aspect of the present invention discloses a preparation method of the above sesquiterpene lactone derivative or its pharmaceutically acceptable salt, which comprises the following steps:

[0011]

[0012] Among them, R is selected from one of mercaptomethyl, hydroxymethyl, hydroxyethyl, amidoethyl, amidomethyl, and methylphenol.

[0013] Preferably, the solvent is selected from at least one of fatty acid thioester, sodium ethoxide, tetrahydrofuran, and sodium acetate, and the molar ratio of compound III to compound II is 0.2 - 2:0.5 - 1.

[0014] Preferably, the reaction conditions are as follows: Compound II is added to the solvent, and compound III is slowly dropped into the mixture of compound II and the solvent under stirring conditions not exceeding 40°C. After reacting for 12 - 36 h, compound I is obtained.

[0015] Preferably, the preparation method of compound III is as follows:

[0016] In a dry flask, add an aqueous solution of parthenolide and n-butanol, then add concentrated sulfuric acid and mix well. Heat the mixture under reflux. When the unreacted substrate disappears, stop heating; then transfer the mixture to a separatory funnel, wash it with saturated brine, take the organic phase, dry it and concentrate it. Heat-treat the concentrated product to obtain compound III.

[0017] Preferably, the concentration of the aqueous solution of parthenolide is 0.5 - 2 mol / L, the final concentration of n-butanol is 1 - 2 mg / mL, and the final concentration of concentrated sulfuric acid is 0.1 - 0.2 M.

[0018] Preferably, the heat-treatment conditions of the concentrated product are heating in a water bath at 85 - 95°C for 2 - 3 h.

[0019] The third aspect of the present invention discloses the application of the above sesquiterpene lactone derivative or its pharmaceutically acceptable salt in the preparation of a protective drug for arteriovenous fistulas for renal dialysis.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0021] The present invention can effectively inhibit the expression of PCNA and TLR4 in VSMCs, thereby inhibiting the proliferation of VSMCs in the AVF model, reducing venous stenosis and thrombosis caused by neointimal hyperplasia, and reducing the incidence of AVF dysfunction, and has good application prospects in the preparation of protective drugs for arteriovenous fistulas for renal dialysis. Brief Description of the Drawings

[0022] Figure 1Expression of PCNA and TLR4 in vascular tissues of rabbits in each group;

[0023] Figure 2 Statistical results of relative expression levels of TLR4 mRNA in vascular tissues of rabbits in each group;

[0024] Figure 3 Statistical results of relative expression levels of PCNA mRNA in vascular tissues of rabbits in each group;

[0025] Figure 4 Effect of compound 2 at different concentrations on the expression of PCNA and TLR4 mRNA in VSMCs under the action of high-concentration fetal bovine serum;

[0026] Figure 5 Statistical results of relative expression levels of PCNA mRNA in VSMCs under the action of high-concentration fetal bovine serum with compound 2 at different concentrations;

[0027] Figure 6 Statistical results of relative expression levels of TLR4 mRNA in VSMCs under the action of high-concentration fetal bovine serum with compound 2 at different concentrations. Detailed implementation manners

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0029] Example 1: The chemical structural formula of a sesquiterpene lactone derivative is as follows:

[0030]

[0031] The preparation method of the above compound is as follows:

[0032] (1) Add an aqueous solution of 2 mol / L parthenolide (CAS: 20554-84-1) and n-butanol with a final concentration of 1 mg / mL to a dry flask. Add concentrated sulfuric acid with a final concentration of 0.1 M as a catalyst, and gently stir evenly with a stirrer. Heat the mixture to the reflux temperature and maintain reflux for about two hours to promote the condensation reaction. Monitor the progress by thin-layer chromatography and stop heating when the unreacted substrate disappears. After completion, cool to room temperature, transfer the mixture to a separatory funnel, and wash with saturated brine to remove the unreacted substrate and impurities. Dry the organic phase with anhydrous magnesium sulfate, and then concentrate the product with a rotary evaporator. Heat the concentrated product in a water bath at 90 °C for 2 - 3 h to obtain compound III, whose structural formula is as follows:

[0033]

[0034] (2) The reaction formula for preparing the sesquiterpene lactone derivative using compound III as a raw material is as follows:

[0035]

[0036] Place 25 mL of tetrahydrofuran (THF) and 5 mmol of sodium ethoxide in a reactor, and stir the solvent at 100 - 150 rpm while maintaining the temperature of the reactor at 20 °C. Quickly place 2.5 mmol of cysteine in the reactor, and slowly add 3 mmol of Compound III to the reactor within 1.5 hours. During the addition process, control the internal temperature of the reactor not to exceed 40 °C. After the addition is completed, control the internal temperature of the reactor at 20 °C and react for 24 h. After the reaction is completed, filter the reaction product to obtain the solid using a 3 μm filter paper, and wash the solid with THF. Dry the solid in a vacuum oven at 60 °C to obtain Compound 1.

[0037] (3) Place Compound 1 and tetrahydrofuran in a reactor according to a solid-liquid ratio of 1 g:5 mL, and stir the mixture at 150 - 180 rpm while maintaining the temperature of the reactor at 20 °C. Stir the mixture under the above conditions for 5 hours, filter and wash it using a 10 μm filter paper, and concentrate and distill the filtrate under reduced pressure to obtain Compound 2.

[0038] Characterize Compound 2 by 1H NMR, 13C NMR, two-dimensional NMR spectra, and high-resolution mass spectrometry. The characterization data are shown in Table 1.

[0039] Table 1 of Compound 2 1 1H NMR (600 MHz), 13 13C NMR spectrum (150 MHz) (δ, ppm, TMS, CDCl3)

[0040]

[0041]

[0042] The structure of Compound 2 is as follows:

[0043]

[0044] Example 2: The chemical structural formula of a sesquiterpene lactone derivative is as follows:

[0045]

[0046] The method for this compound is as follows:

[0047] Add an aqueous solution of 0.5 mol / L parthenolide (CAS: 20554-84-1) and n-butanol with a final concentration of 1.5 mg / mL to a dry flask. Add concentrated sulfuric acid with a final concentration of 0.1 M as a catalyst and gently stir evenly with a stirrer. Heat the mixture to the reflux temperature and maintain reflux for about two hours to promote the condensation reaction. Monitor the progress by thin-layer chromatography and stop heating when the unreacted substrate disappears. After completion, cool to room temperature, transfer the mixture to a separatory funnel, and wash with saturated brine to remove the unreacted substrate and impurities. Dry the organic phase with anhydrous magnesium sulfate and then concentrate the product using a rotary evaporator. Heat the concentrated product in a water bath at 90 °C for 2 - 3 h to obtain Compound III.

[0048]

[0049] Place 25 mL of fatty acid thioester (LAT) and 5 mmol of sodium ethoxide in a reactor as a solvent, and stir the solvent at 100 - 150 rpm while maintaining the temperature of the reactor at 20 °C. Quickly place 2.5 mmol of glutamine in the reactor, and slowly add 3 mmol of Compound III to the reactor within 1.5 hours. During the addition process, control the internal temperature of the reactor not to exceed 40 °C. After the addition is completed, control the internal temperature of the reactor at 20 °C and react for 24 h. After the reaction is completed, filter the reaction product to obtain a solid using a 3 μm filter paper, and wash the solid with THF. Dry the solid in a vacuum oven at 60 °C to obtain Intermediate 3.

[0050] Place Intermediate 3 and tetrahydrofuran (THF) in a reactor according to a solid-liquid ratio of 1 g:10 mL, and stir the mixture at 150 - 180 rpm while maintaining the temperature of the reactor at 20 °C. Stir the mixture under the above conditions for 5 hours, filter and wash it using a 10 μm filter paper, and concentrate and distill the filtrate under reduced pressure to obtain Compound 3.

[0051] Characterize Compound 3 by 1H NMR, 13C NMR, two-dimensional NMR spectroscopy, and high-resolution mass spectrometry. The characterization data are shown in Table 2.

[0052] Table 2 of the compound 1 1H NMR (600 MHz), 13 13C NMR spectrum (150 MHz) (δ, ppm, TMS, CDCl3)

[0053] Position δ(H) δ(C) 1 6.33 (dd, J=11.6, 5.1 Hz, 1H) 141.6 (CH) 2 2.11 - 2.08 (m, 1H) 29.0 (CH) 3.07 - 3.09 (m, 1H) 3 5.43 (t, J=8.1 Hz, 1H) 70.3 (CH) 4 4.07 - 4.09 (m, 2H) <![CDATA[123.2(CH2)]]> 5 6.11 (d, J=9.1 Hz, 1H) <![CDATA[125.5(CH2)]]> 6 5.47 - 5.50 (m, 1H) 70.4 (CH) 7 2.58 - 2.66 (br.s, 1H) 46.9 (CH) 8 5.72 - 5.54 (m, 2H) <![CDATA[71.6(CH2)]]> 9 3.10 - 3.15 (m, 2H) <![CDATA[30.2(CH2)]]> 10 143 11 2.77 - 2.86 (br.s, 1H) 48.9 (CH) 12 165.6 13 7.32 (d, J=2.1 Hz, 2H) <![CDATA[136.0(CH2)]]> 5.56 (d, J=2.1 Hz, 1H) <![CDATA[R1 , > 5.47 (t, J=8.2 Hz, 1H) 69.8 (CH) <![CDATA[R2 , > 2.21 (s, 2H) <![CDATA[24.2(CH2)]]> <![CDATA[R3 , > 181.3 <![CDATA[R4 , > 4.24 (s, 2H) <![CDATA[22.1(CH2)]]> <![CDATA[R5 , > 109.1

[0054] The structure of Compound 3 is as follows:

[0055]

[0056] Example 3: The chemical structural formula of a sesquiterpene lactone derivative is as follows:

[0057]

[0058] The method for preparing this compound is as follows:

[0059] Add an aqueous solution of 2 mol / L parthenolide (CAS: 20554 - 84 - 1) and n-butanol with a final concentration of 1 mg / mL to a dry flask. Add concentrated sulfuric acid with a final concentration of 0.1 M as a catalyst and gently stir evenly with a stirrer. Heat the mixture to the reflux temperature and maintain reflux for about two hours to promote the condensation reaction. Monitor the progress by thin-layer chromatography and stop heating when the unreacted substrate disappears. After completion, cool to room temperature, transfer the mixture to a separatory funnel, and wash with saturated brine to remove the unreacted substrate and impurities. Dry the organic phase with anhydrous magnesium sulfate and then concentrate the product using a rotary evaporator. Heat the concentrated product in a water bath at 90 °C for 2 - 3 h to obtain Compound III.

[0060]

[0061] Place 25 mL of fatty acid thioester (LAT) and 3 mmol of sodium acetate (NaOAc) as solvents in a reactor, and stir the solvents at 100 - 150 rpm while maintaining the temperature of the reactor at 20 °C. Quickly place 2.5 mmol of tyrosine in the reactor, and slowly add 3 mmol of Compound III to the reactor within 1.5 hours. During the addition process, control the internal temperature of the reactor not to exceed 40 °C. After the addition is completed, control the internal temperature of the reactor at 20 °C and react for 36 h. After the reaction is completed, filter the reaction product to obtain a solid using a 3 - μm filter paper, and wash the solid with THF. Dry the solid in a vacuum oven at 60 °C to obtain Intermediate 4.

[0062] Place Intermediate 4 and tetrahydrofuran (THF) in a reactor according to a solid-liquid ratio of 1 g:5 mL, and stir the mixture at 150 - 180 rpm while maintaining the temperature of the reactor at 20 °C. Stir the mixture under the above conditions for 5 hours, filter and wash using a 10 - μm filter paper, and concentrate and distill the filtrate under reduced pressure to obtain Compound 4.

[0063] Characterize Compound 4 by 1H NMR, 13C NMR, two-dimensional NMR spectra, and high-resolution mass spectrometry. The characterization data are shown in Table 3.

[0064] Table 3 of the compound's 1 1H NMR (600 MHz), 13 13C NMR spectrum (150 MHz) (δ, ppm, TMS, CDCl3)

[0065] Position δ(H) δ(C) 1 6.33 (dd, J=11.6, 5.1 Hz, 1H) 141.6 (CH) 2 2.11 - 2.08 (m, 1H) 29.0 (CH) 3.07 - 3.09 (m, 1H) 3 5.43 (t, J=8.1 Hz, 1H) 70.3 (CH) 4 4.07 - 4.09 (m, 2H) <![CDATA[123.2(CH2)]]> 5 6.11 (d, J=9.1 Hz, 1H) <![CDATA[125.5(CH2)]]> 6 5.47 - 5.50 (m, 1H) 70.4 (CH) 7 2.58 - 2.66 (br.s, 1H) 46.9 (CH) 8 5.72 - 5.54 (m, 2H) <![CDATA[71.6(CH2)]]> 9 3.10 - 3.15 (m, 2H) <![CDATA[30.2(CH2)]]> 10 143 11 2.77 - 2.86 (br.s, 1H) 48.9 (CH) 12 165.6 13 7.32 (d, J=2.1 Hz, 2H) <![CDATA[136.0(CH2)]]> 5.56 (d, J=2.1 Hz, 1H) <![CDATA[R1 , > 5.47 (t, J=8.2 Hz, 1H) 69.8 (CH) <![CDATA[R2 , > 2.21 (s, 2H) <![CDATA[24.2(CH2)]]> <![CDATA[R3 , > 181.3 <![CDATA[R4 , > 6.58 - 7.66 (br.s, 1H) 121.6 (CH) <![CDATA[R5 , > 6.51 - 7.36 (br.s, 1H) 131.4 (CH) <![CDATA[R6 , > 121.4 <![CDATA[R7 , > 5.58 - 6.66 (br.s, 1H) 141.6 (CH) <![CDATA[R8 , > 5.51 - 6.36 (br.s, 1H) 143.4 (CH)

[0066] The structure of Compound 4 is as follows:

[0067]

[0068] Example 4: The chemical structural formula of a sesquiterpene lactone derivative is as follows:

[0069]

[0070] The method for preparing this compound is as follows:

[0071] Add an aqueous solution of 1 mol / L parthenolide (CAS: 20554 - 84 - 1) and n-butanol with a final concentration of 2 mg / mL to a dry flask. Add concentrated sulfuric acid with a final concentration of 0.1 M as a catalyst and gently stir evenly with a stirrer. Heat the mixture to the reflux temperature and maintain reflux for about two hours to promote the condensation reaction. Monitor the progress by thin layer chromatography and stop heating when the unreacted substrate disappears. After completion, cool to room temperature, transfer the mixture to a separatory funnel, and wash with saturated brine to remove unreacted substrate and impurities. Dry the organic phase with anhydrous magnesium sulfate, and then concentrate the product with a rotary evaporator. Heat the concentrated product in a water bath at 90 °C for 2 - 3 h to obtain Compound III.

[0072]

[0073] Place 25 ml of tetrahydrofuran (THF) and 2.5 mmol of sodium acetate (NaOAc) as solvents in a reactor, and stir the solvents at 100 - 150 rpm while maintaining the temperature of the reactor at 20 °C. Quickly place 2.5 mmol of serine into the reactor, and slowly add 3 mmol of Compound III to the reactor within 1.5 hours. During the addition process, control the internal temperature of the reactor not to exceed 40 °C. After the addition is completed, control the internal temperature of the reactor at 30 °C and react for 12 h. After the reaction is completed, filter the reaction product to obtain the solid using a 3 μm filter paper, and wash the solid with THF. Dry the solid in a vacuum oven at 60 °C to obtain Intermediate 5.

[0074] Place Intermediate 5 and tetrahydrofuran (THF) in a reactor according to a material-liquid ratio of 1 g:7 mL, and stir the mixture at 150 - 180 rpm while maintaining the temperature of the reactor at 20 °C. Stir the mixture under the above conditions for 5 hours, filter and wash using a 10 μm filter paper, and concentrate and distill the filtrate under reduced pressure to obtain Compound 5.

[0075] Characterize Compound 5 by proton nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum, two-dimensional nuclear magnetic resonance spectrum, and high-resolution mass spectrometry. The characterization data are shown in Table 4.

[0076] Table 4 of the compound's 1 1H NMR (600 MHz), 13 13C NMR spectrum (150 MHz) (δ, ppm, TMS, CDCl3)

[0077] Position δ(H) δ(C) 1 6.47 (dd, J=11.6, 5.1 Hz, 1H) 141.6 (CH) 2 2.12 - 2.15 (m, 1H) 30.0 (CH) 3.10 - 3.15 (m, 1H) 3 5.51 (t, J=8.2 Hz, 1H) 72.8 (CH) 4 4.07 - 4.09 (m, 2H) <![CDATA[123.2(CH2)]]> 5 6.18 (d, J=9.4 Hz, 1H) <![CDATA[128.2(CH2)]]> 6 5.47 - 5.50 (m, 1H) 71.4 (CH) 7 2.58 - 2.66 (br.s, 1H) 47.9 (CH) 8 5.72 - 5.54 (m, 2H) <![CDATA[72.2(CH2)]]> 9 3.10 - 3.15 (m, 2H) <![CDATA[31.8(CH2)]]> 10 148 11 2.32 - 2.35 (br.s, 1H) 43.1 (CH) 12 177.2 13 7.32 (d, J = 2.1 Hz, 2H) <![CDATA[141.0(CH2)]]> 5.99 (d, J = 2.1 Hz, 2H) <![CDATA[R1 , > 5.43 (t, J = 8.2 Hz, 1H) 66.8 (CH) <![CDATA[R2 , > 2.32 (s, 2H) <![CDATA[23.1(CH2)]]> <![CDATA[R3 , > 178.3

[0078] The structure of Compound 5 is as follows:

[0079]

[0080] Example 5: The chemical structural formula of a sesquiterpene lactone derivative is as follows:

[0081]

[0082] The method for preparing this compound is as follows:

[0083] Add an aqueous solution of 1.5 mol / L parthenolide (CAS: 20554-84-1) and n-butanol with a final concentration of 1.5 mg / mL to a dry flask. Add concentrated sulfuric acid with a final concentration of 0.1 M as a catalyst and gently stir evenly with a stirrer. Heat the mixture to the reflux temperature and maintain reflux for about two hours to promote the condensation reaction. Monitor the progress by thin-layer chromatography and stop heating when the unreacted substrate disappears. After completion, cool to room temperature, transfer the mixture to a separatory funnel, and wash with saturated brine to remove the unreacted substrate and impurities. Dry the organic phase with anhydrous magnesium sulfate and then concentrate the product using a rotary evaporator. Heat the concentrated product in a water bath at 85 °C for 2 - 3 h to obtain Compound III.

[0084]

[0085] Place 25 ml of fatty acid thioester (LAT) and 5 mmol of sodium ethoxide in a reactor as solvents, and stir the solvents at 100 - 150 rpm while maintaining the temperature of the reactor at 20 °C. Quickly place 3 mmol of threonine in the reactor, and slowly add 2.5 mmol of Compound III to the reactor within 1.5 hours. During the addition, control the internal temperature of the reactor not to exceed 40 °C. After the addition is complete, control the internal temperature of the reactor at 20 °C and react for 24 h. After the reaction is complete, filter the reaction product to obtain a solid using a 3-μm filter paper, and wash the solid with THF. Dry the solid in a vacuum oven at 60 °C to obtain Intermediate 6.

[0086] The intermediate 6 and tetrahydrofuran (THF) were placed in a reactor according to a material-liquid ratio of 1 g: 10 mL, and the mixture was stirred at 150 - 180 rpm while maintaining the temperature of the reactor at 20 °C. The mixture was stirred under the said conditions for 5 hours, filtered and washed using a 10-μm filter paper, and the filtrate was concentrated and distilled under reduced pressure to obtain Compound 6.

[0087] Compound 6 was characterized by 1H NMR, 13C NMR, two-dimensional NMR spectra, and high-resolution mass spectrometry. The characterization data are shown in Table 5.

[0088] Table 5 of the compound 1 1H NMR (600 MHz), 13 13C NMR spectrum (150 MHz) (δ, ppm, TMS, CDCl3)

[0089]

[0090]

[0091] The structure of Compound 6 is as follows:

[0092]

[0093] Example 6: The chemical structural formula of a sesquiterpene lactone derivative is as follows:

[0094]

[0095] The method for preparing this compound is as follows:

[0096] An aqueous solution of 1 mol / L parthenolide (CAS: 20554-84-1) and n-butanol with a final concentration of 2 mg / mL were added to a dry flask. Concentrated sulfuric acid with a final concentration of 0.2 M was added as a catalyst, and the mixture was gently stirred evenly with a stirrer. The mixture was heated to the reflux temperature and maintained at reflux for about two hours to promote the condensation reaction. The progress was monitored by thin-layer chromatography, and heating was stopped when the unreacted substrate disappeared. After completion, it was cooled to room temperature, and the mixture was transferred to a separatory funnel and washed with saturated brine to remove the unreacted substrate and impurities. The organic phase was dried with anhydrous magnesium sulfate, and then the product was concentrated using a rotary evaporator. The concentrated product was heated in a water bath at 95 °C for 2 - 3 h to obtain Compound III.

[0097]

[0098] 25 ml of fatty acid thioester (LAT) and 5 mmol of sodium ethoxide were placed in a reactor, and the solvent was stirred at 100 - 150 rpm while maintaining the temperature of the reactor at 20 °C. 3 mmol of asparagine was quickly placed in the reactor, and 3 mmol of compound III was slowly added dropwise to the reactor within 1.5 hours. During the dropping process, the internal temperature of the reactor was controlled not to exceed 40 °C. After the dropping was completed, the internal temperature of the reactor was controlled at 20 °C for reaction for 24 h. After the reaction was completed, the reaction product was filtered through a 3 - μm filter paper to obtain the solid, and the solid was washed with THF. The solid was dried in a vacuum oven at 60 °C to obtain intermediate 7.

[0099] Intermediate 7 and tetrahydrofuran (THF) were placed in a reactor according to a solid - liquid ratio of 1 g:10 mL, and the mixture was stirred at 150 - 180 rpm while maintaining the temperature of the reactor at 20 °C. The mixture was stirred under the above conditions for 5 hours, filtered and washed with a 10 - μm filter paper, and the filtrate was concentrated and distilled under reduced pressure to obtain compound 7.

[0100] Compound 7 was characterized by 1H - NMR, 13C - NMR, two - dimensional NMR spectroscopy, and high - resolution mass spectrometry. The characterization data are shown in Table 6.

[0101] Table 6 of the compound 1 1H NMR (600 MHz), 13 13C NMR spectrum (150 MHz) (δ, ppm, TMS, CDCl3)

[0102] Position δ(H) δ(C) 1 6.31 (dd, J = 11.6, 5.1 Hz, 1H) 146.8 (CH) 2 2.14 - 2.16 (m, 1H) 29.1 (CH) 3.11 - 3.16 (m, 1H) 3 5.35 (t, J = 8.2 Hz, 1H) 71.8 (CH) 4 4.12 - 4.16 (m, 2H) <![CDATA[125.1(CH2)]]> 5 6.23 (d, J = 9.4 Hz, 1H) <![CDATA[129.5(CH2)]]> 6 5.51 - 5.62 (m, 1H) 68.9 (CH) 7 2.45 - 2.61 (br.s, 1H) 49.2 (CH) 8 5.81 - 5.87 (m, 2H) <![CDATA[73.4(CH2)]]> 9 3.19 - 3.20 (m, 2H) <![CDATA[31.2(CH2)]]> 10 145 11 2.23 - 2.25 (br.s, 1H) 41.9 (CH) 12 174.2 13 7.21 (d, J = 2.1 Hz, 2H) <![CDATA[140.0(CH2)]]> 603 (d, J = 2.1 Hz, 2H) <![CDATA[R1 , > 5.36 (t, J = 8.2 Hz, 1H) 67.1 (CH) <![CDATA[R2 , > 2.33 (s, 2H) <![CDATA[25.4(CH2)]]> <![CDATA[R3 , > 169.3 <![CDATA[R4 , > 111.2

[0103] The structure of compound 7 is as follows:

[0104]

[0105] The inhibitory effects of compounds 2 - 7 on the intimal hyperplasia of arteriovenous fistulas in rabbits were tested as follows:

[0106] 33 purebred healthy male New Zealand white rabbits, weighing 2.5 - 3.0 kg and aged 4 - 5 months. 60 mg of compounds 2 - 7 were weighed respectively, and each was fully mixed and dissolved with 1 mL of glacial acetic acid (>99.6%) to obtain the experimental drugs, which were stored at room temperature for later use.

[0107] Twenty - four New Zealand white rabbits were randomly divided into: normal control group (group A, n = 3), 4 - week model group (group B, n = 3), 4 - week drug - administered experimental groups (groups C - H, 20 μg / ml, n = 3×6). The model group and the experimental groups were respectively established with arteriovenous fistula models of the common carotid artery - internal jugular vein in rabbits. After AVF in the experimental groups, each experimental drug was applied to the blood vessels at the venous end of AVF and the anastomosis. The operation method is as follows:

[0108] New Zealand white rabbits were adaptively fed for one week. Four hours before the operation, they were fasted and water-deprived. Anesthesia was induced by intravenous injection of 3% sodium pentobarbital into the marginal ear vein of the rabbits, and the anesthesia dose was calculated at 1 ml / kg. During the operation, lidocaine could be appropriately given to reduce bleeding and for local anesthesia. Then, the four limbs of the rabbits were fixed, the skin was prepared, and disinfected with iodophor, and a sterile towel was laid. The skin and subcutaneous tissue were incised at the midline of the neck, and then the sterile towel was fixed to the skin around the incision to fully expose the surgical field. The right internal jugular vein was dissected, its branches were ligated, and the right common carotid artery was dissected along the trachea, with a dissection length of about 1.5 cm. 200 mg / kg of sodium heparin was injected intravenously to achieve semi-heparinization. Then, the distal end of the internal jugular vein was ligated, a non-invasive vascular clamp was applied to the proximal end, and the distal end of the internal jugular vein was obliquely cut, and the cut end was rinsed with heparin saline. Non-invasive vascular clamps were placed at the distal and proximal ends of the common carotid artery, with a distance of about 0.8 - 1 cm between the two vascular clamps. Then, a longitudinal incision was made on the ascending artery, and the lumen was rinsed with heparin saline. The internal jugular vein and the common carotid artery were sutured end-to-side continuously with 8-0 non-invasive sutures. During the anastomosis process, the lumen and the anastomosis site were rinsed with heparin saline to prevent thrombus formation. After the anastomosis was completed, the vascular clamp at the venous end and the vascular clamps at the distal and proximal ends of the common carotid artery were successively opened. It could be seen successfully that arterial blood flowed through the anastomosis site and the vein dilated and filled. At this time, the experimental drug containing compound 2-7 was evenly applied to the venous side of the anastomosis site in the experimental group. Finally, the subcutaneous tissue and skin were sutured layer by layer. The normal control group was not given AVF surgery treatment and experimental drug administration intervention. After the operation, penicillin was intramuscularly injected for 3 days for routine anti-infection treatment. Low molecular weight sodium heparin was used for anticoagulation to prevent thrombus formation. They were fed with ordinary feed.

[0109] The rabbits in the model group and each experimental group were sacrificed at 4 weeks after the operation, and the venous end blood vessels at the AVF anastomosis site, about 2 - 3 cm, were quickly removed. The internal jugular vein tissue of the normal control group was dissected and removed. The removed blood vessel tissue was quickly placed in liquid nitrogen for freezing and stored in a -80 °C low-temperature refrigerator for RT-PCR.

[0110] The expression of PCNA and TLR4 in the rabbit blood vessel tissue was detected by reverse transcription PCR (RT-PCR), and the method was as follows:

[0111] The total RNA of the above-mentioned blood vessel tissue was extracted using a kit; after the purity and concentration of the total RNA were detected to be qualified, it was used for RT-PCR. The primer sequences of GAPDH, TLR4, and PCNA are shown in the following table:

[0112]

[0113] GAPDH was set as the internal reference, and DNA was electrophoresed on agarose gel: Observation and photography were carried out under a Bio-Rad gel imaging system: DNA bands were visible, clear without miscellaneous bands, plump without defects. Then, the Image Lab image analysis software was used to analyze the bands, and the optical density values were obtained. The relative contents of their mRNAs were expressed as the ratios of the optical density values of PCNA, TLR4 to the internal reference GAPDH. The results were as Figures 1 - 3 shown, Figure 1 In lane A was the blank control group, lane B was the model group, and lanes C-H were the administration groups of compounds 2-7 in sequence.

[0114] Figures 1 - 3 The results showed that, compared with the normal control group (group A) of rabbits, after 4 weeks of AVF in rabbits, the mRNA levels of PCNA and TLR4 in their blood vessels were significantly increased (P < 0.05). After treatment with the experimental drugs (groups C-H), the mRNA levels of PCNA and TLR4 in the blood vessels decreased significantly, significantly lower than those in the model group (group B) (P < 0.05).

[0115] The inhibitory effect of compounds 2-7 on the proliferation of rabbit arteriovenous fistula vascular smooth muscle cells was tested as follows:

[0116] 5 mL of double antibodies and 20% FBS were added to the newly purchased 500 mL DMEM / F12 medium to prepare a complete medium, which was stored at 4 °C for later use. The compound 2 was prepared into drug working solutions with concentration gradients of 0, 10, 100, 500, 1000, and 2000 μg / mL using the complete medium.

[0117] Culture and identification of rabbit VSMCs: New Zealand white rabbits with a 1-month mature arteriovenous fistula of the common carotid artery - internal jugular vein were sacrificed by air embolism. The venous end blood vessels of the rabbit AVF were extracted under sterile conditions and placed in PBS containing double antibodies for later use. The blood vessels obtained were rinsed several times in DMEM in a laminar flow hood, and the adventitia was carefully removed. After soaking in double antibodies for several minutes, trypsin was directly added. The tissue blocks were cut into small pieces with ophthalmic scissors in the trypsin-double antibody mixture. After cutting the middle membrane tissue blocks of the blood vessels into pieces of 1 mm 3 size, before transferring them into the culture flask, the tissue blocks were digested with 0.25% trypsin for 1 min, and then the digestion was terminated with DMEM medium containing FBS. Then, the tissue blocks were transferred into the culture flask. The culture flask was placed upright with the mouth facing up and put into the incubator. After 2 h, the flask was turned over. The cells were cultured in DMEM medium containing 20% fetal bovine serum in an incubator at 37 °C and 5% CO2. After 5 days, cells could be seen crawling out from the edge of the tissue blocks, and a dense cell layer appeared after 10 days. The cells were identified by α-SMA immunofluorescence method.

[0118] The intervention experiment of different concentration compound 2 drug working solutions on rabbit VSMCs induced by high-concentration fetal bovine serum was carried out as follows:

[0119] Cultivate rabbit VSMCs with DMEM medium containing 20% fetal bovine serum. When the cells reach 70%-80% confluence, change to DMEM-F12 medium for synchronous culture for 24 h. After synchronization of VSMCs, change to drug working solutions of compound 2 at 0, 10, 100, 500, 1000, and 2000 μg / mL respectively to culture rabbit VSMCs for 48 h. Collect the cells and use RT-PCR method to detect the relative expression levels of TLR4 and PCNA mRNA in rabbit VSMCs. The results are as Figures 4 - 6 shown. It can be seen that when the concentration of fetal bovine serum is 20%, the proliferation of rabbit VSMCs is obvious and the expression level of TLR4 is the highest. While compound 2 can inhibit the expression of TLR4 and PCNA in VSMCs in a dose-dependent manner, and thus can significantly inhibit the proliferation of vascular smooth muscle cells in arteriovenous fistulas.

[0120] To compare the inhibitory effects of different compounds on the proliferation of VSMCs, the method is as follows:

[0121] Prepare drug working solutions of compounds 2-7 and comparative compounds 8-14 at 2000 μg / mL according to the above method. Use drug working solutions containing different compounds to culture rabbit VSMCs for 6 days respectively. The blank control is to culture rabbit VSMCs with DMEM medium containing 20% fetal bovine serum. Use the CCK-8 kit to detect the cell proliferation viability and calculate the proliferation inhibition rate: Proliferation inhibition rate = (absorbance value of blank control group - absorbance value of drug administration group) / absorbance value of blank control group * 100%

[0122] The structures of the comparative compounds are as follows:

[0123] Table 7 Structural formulas of the comparative compounds

[0124]

[0125]

[0126] The experimental results are as follows:

[0127] Table 8 Proliferation inhibition rates of different compounds on VSMCs

[0128]

[0129]

[0130] As can be seen from the results in Table 8, when the substituents and their substitution sites on the large carbon ring of the sesquiterpene lactone change, the inhibitory effect of comparative compound 8 on the proliferation of VSMCs decreases significantly, indicating that the substituents on the large carbon ring have a significant impact on the overall pharmacodynamic effect of the compound. Comparing comparative compounds 9, 12, and 13 with compound 2, it can be seen that the structure or saturation of the large carbon ring also plays a key role in the exertion of the overall pharmacodynamic effect of the compound. Too high or too low saturation of the large carbon ring will lead to a decrease in the inhibitory effect of the compound on the proliferation of VSMCs. In addition, comparing comparative compounds 10 and 11 shows that the carboxyl group and other remaining groups in the amino acid substitution group are also essential for the generation of the pharmacodynamic effect. Only amino substitution will also lead to a significant decrease in the pharmacodynamic effect. Comparing comparative compound 14 shows that the single sesquiterpene lactone does not have a good inhibitory effect on the proliferation of VSMCs, but the sesquiterpene lactone derivative formed by amino acid group substitution has a good inhibitory effect on the proliferation of VSMCs, and the amino acid group and the sesquiterpene lactone group play a synergistic role in inhibiting the proliferation of VSMCs.

Claims

1. A sesquiterpene lactone derivative represented by formula (I) or a pharmaceutically acceptable salt thereof: in, R is selected from substituted C 1-6 The alkyl group is substituted with a group selected from the group consisting of a mercapto group, a hydroxyl group, an amide group, and a phenyl group substituted with a hydroxyl group.

2. The sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The alkyl group is a straight-chain alkyl group.

3. The sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Includes at least one of the following compounds:

4. The method for preparing the sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: The steps include: Wherein, R is selected from one of mercaptomethyl, hydroxymethyl, hydroxyethyl, amidoethyl, amidomethyl, and methylphenol.

5. The method for preparing the sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The solvent is selected from at least one of fatty acid thioester, sodium ethoxide, tetrahydrofuran and sodium acetate, and the molar ratio of compound III to compound II is 0.2-2:0.5-1.

6. The method for preparing the sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The reaction conditions are as follows: Compound II is added to a solvent, and Compound III is slowly dripped into the mixture of Compound II and the solvent under stirring conditions not exceeding 40° C., and Compound I is obtained after reacting for 12-36 hours.

7. The method for preparing the sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The preparation method of the compound III is: Add the parthenolide aqueous solution and n-butanol to a dry flask, then add concentrated sulfuric acid and mix well, heat the mixture to reflux, and stop heating when the unreacted substrate disappears; then transfer the mixture to a separatory funnel, wash with saturated brine, take the organic phase, dry it, and concentrate it, and heat the concentrated product to obtain compound III.

8. The method for preparing the sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof according to claim 7, characterized in that: The concentration of the parthenolide aqueous solution is 0.5-2 mol / L, the final concentration of n-butanol is 1-2 mg / mL, and the final concentration of concentrated sulfuric acid is 0.1-0.2M.

9. The method for preparing the sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof according to claim 7, characterized in that: The concentrated product is heated in a water bath at 85-95° C. for 2-3 hours.

10. Use of the sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a protective drug for arteriovenous fistula for renal dialysis.

Images