A ligustilide derivative and its preparation method and application

Abstract

The present invention belongs to the field of medicine and discloses a class of ligustilide derivatives and their application in anti-inflammatory drugs. The ligustilide structural derivative is shown in Formula I, #imgabs0# In Formula I, R 1 The group is a substituent at each position on the benzyl group, selected from any one of the following: 2′-F, 3′-F, 4′-F, 2′-Cl, 3′-Cl, 4′-Cl, 4′-Br, 4′-CN2′-CF 3 , 3′-CF 3 , 4′‑CF 3 , 2′‑OCH 3 , 3′‑OCH 3 , 4′‑OCH 3 , 4′‑OCH 2 CH 3 2′‑Ph, 3′‑Ph, 4′‑Ph, 4′‑OH, 4′‑CH 3 、#imgabs1# Based on ligustilide as the parent nucleus and the introduction of benzyl at the N-1 position, a series of ligustilide derivatives were designed and synthesized, and most of the obtained compounds have excellent anti-inflammatory activity. Compared with the commonly used clinical anti-inflammatory drugs indomethacin and dexamethasone, the anti-inflammatory activity of the lead compound 4f is better than that of the commonly used clinical drugs. The modified 4f structure is efficient and stable, and has the potential to be developed into a new anti-inflammatory drug.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to a class of ligustilide derivatives and a preparation method thereof and their application in anti-inflammatory drugs. Background Art

[0002] Inflammation is a protective response of the body, but persistent inflammatory responses can damage tissues and even lead to loss of function, tumors, and death. Unfortunately, the anti-inflammatory drugs currently used in clinical practice generally have poor selectivity and obvious side effects in clinical use, which limits the clinical application of traditional anti-inflammatory drugs and requires the development of new anti-inflammatory drugs. Ligustilide is a traditional Chinese medicine extract with strong anti-inflammatory and analgesic properties obtained from the traditional Umbelliferae Chinese medicine Angelica sinensis. However, due to the unstable chemical properties of ligustilide, the butenyl group connected to its C-3 position is easily oxidized or degraded by light, heat, and enzymes in the environment, and its drugability is poor. Therefore, a series of structurally stable ligustilide compounds with anti-inflammatory inhibitory activity were designed. As potential anti-inflammatory drugs, they have good application scenarios and help improve the effect of clinical anti-inflammatory treatment. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a ligustilide compound with anti-inflammatory activity. The derivative can inhibit NF- κ B and MAPK signaling pathways exert their anti-inflammatory effects, and the effect is significant.

[0004] The solution of the present invention to solve the above technical problems is as follows:

[0005] A ligustilide compound having anti-inflammatory activity, the chemical structure of the compound is shown in the following formula (I):

[0006]

[0007] Where R 1 The group is one of hydrogen, halogen, 2-8 carbon atom alkyl, cycloalkane, alkoxy, cyano, hydroxyl, amine, aryl, heteroaryl, and isopropyl:

[0008] 2′-F, 3′-F, 4′-F, 2′-C1, 3′-C1, 4′-C1, 4′-Br, 4′-CN

[0009] 2′-CF 3 , 3′-CF 3 , 4′-CF 3 , 2′-OCH 3 , 3′-OCH 3 , 4′-OCH 3 , 4′-OCH 2 CH 3

[0010] 2′-Ph, 3′-Ph, 4′-Ph, 4′-OH, 4′-CH 3

[0011] Preferably, the ligustilide derivatives of the present invention have the chemical structure shown in Table 1:

[0012] Table 1 Structure and nomenclature of compounds

[0013]

[0014]

[0015]

[0016]

[0017]

[0018] A method for preparing the above-mentioned ligustilide derivative specifically comprises the following steps:

[0019] (1.1) Phthalide and a cyclohexane aqueous solution containing n-butyl lithium are reacted in a solvent, and the reaction solution obtained after the reaction is purified to obtain compound 1;

[0020] (1.2) Compound 1, liquid ammonia and Na are reacted in a solvent, and the obtained reaction solution is purified to obtain compound 2;

[0021] (1.3) Compound 2, pyridine and methanesulfonyl chloride are reacted in a solvent, and the obtained reaction solution is purified to obtain compound 3;

[0022] (1.4) Compound 3 is reacted with an amine in a solvent under heating reflux, and the resulting reaction solution is purified to obtain compounds 4a-4v.

[0023] The synthetic route is:

[0024]

[0025] The application of the above-mentioned ligustilide compounds in the preparation of anti-inflammatory drugs.

[0026] The drug refers to at least one of the ligustilide compounds or its pharmaceutically acceptable salts and solvates.

[0027] The medicament further comprises one or more pharmaceutically acceptable carriers or excipients.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0029] (1) The present invention uses ligustilide as the mother core, modifies and transforms it, and designs and synthesizes a series of ligustilide compounds with stable chemical properties, and applies them to the development of anti-inflammatory drugs. Among them, when the group connected to the lactam ring of the modified ligustilide derivative is trifluoromethylbenzylamine (4f), it is better than the positive control compound and can effectively reduce the production of cellular inflammatory factors.

[0030] (2) A new, highly effective and stable ligustilide derivative 4f was developed, which has the potential to be developed into a new anti-inflammatory drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The effect of different concentrations of compound 4f, indomethacin and ligustilide on the expression of ROS in RAW264.7 cells after 24 hours of treatment. DETAILED DESCRIPTION

[0032] Unless otherwise specified, the reagents used in the examples can be purchased from the market.

[0033] Example 1: Preparation of 3-(1-hydroxybutyl)benzofuranone (Compound 1)

[0034] Dissolve diisopropylamine in tetrahydrofuran, at -78°C, protect with Ar, and keep stirring, then slowly drop a cyclohexane aqueous solution containing n-butyl lithium (containing 0.015 mol of n-butyl lithium). After the solvent is added dropwise, continue the reaction for about 10 minutes to prepare an aqueous solution of diisopropylammonium amide. Cool to -70°C, then slowly drop a phthalide (C 8 H 6 O 2 , 0.01 mol) THF solution (dropping time about 20

[0035] min), a bright yellow precipitate was generated in the reaction solution. After the solution was added dropwise, the reaction was continued for 15 min. Butyraldehyde (0.015 mol) was slowly added dropwise. After the solution was added dropwise, the reaction was continued for 20 min. 3 g of crushed ice was added and the reaction was terminated for 20 min. The temperature was raised to room temperature and 4 mL of H 2 O, vacuum concentrate under Ar protection to remove the residual organic solvent in the reaction system, then adjust the pH to 3 with HCl, extract with 30 mL × 3 ethyl acetate, then combine the organic phases, and add MgSO 4 After being fully dried, the product was filtered and concentrated in vacuo under Ar protection to obtain a yellow viscous oil with a yield of 90%. 1 H NMR (400 MHz, CDCl 3)δ7.83(d,J=7.6Hz,1H),7.60(t,J=7.3Hz,1H),7.53(d,J=7.6Hz,1H),7.47(t,J=7.3Hz,1H),5.33(dd,J=1 1.0,4.1Hz,1H),4.01–3.82(m,1H),1.52(q,J=12.2,10.3Hz,3H),1.41–1.29(m,1H),0.87(t,J=7.0Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ170.60,147.08,134.00,129.41,126.58,125.77,123.18,83.59,72.56,34.50,18.76,13.88.

[0036] Example 2: Preparation of 3-(1-hydroxybutyl)-4,5-dihydroisobenzofuran (Compound 2)

[0037] Place 1 in a three-necked beaker, dissolve it with a little tetrahydrofuran, and then add liquid ammonia. Protect with Ar at -50℃, add isopropanol while stirring, and then slowly add chopped Na (0.15mol). At this time, the solution color turns dark blue, and then keep it at room temperature and continue to react for 6h. Slowly add NH 4 Cl solid until the dark blue color of the solution disappears completely. Then slowly and carefully add H 2 O, the reaction system temperature rises to room temperature and then concentrated in vacuo, and the remaining NH 3 The pH of the aqueous layer was adjusted to 1 with 3 mol / L HCl, and the organic phases were combined after extraction with 30 mL × 3 ether.

[0038] mL×3 saturated NaCl solution was used to wash the solution until the effluent water layer was neutral. 4 After being fully dried, the product was filtered and concentrated in vacuo under Ar protection to obtain a brown-red viscous oily product with a yield of 60% and a Chinese medicine aroma. 1 H NMR (400 MHz, CDCl 3 )δ6.18(dt,J=9.7,2.1Hz,1H),5.94(dt,J=9.3,4.4Hz,1H),4.90(dd,J=19.9,3.6Hz,1H),3.86(dq,J=10.0,5. 6,4.9Hz,1H),2.69–2.50(m,2H),2.43(dtt,J=15.2,7.1,2.8Hz,2H),1.65–1.35(m,5H),0.93(t,J=7.3Hz,3H). 13C NMR (101 MHz, CDCl 3 )δ171.59,159.83,129.44,126.09,117.17,85.89,72.41,34.86,22.74,22.49,21.59,19.28,14.31.

[0039] Example 3: Preparation of ligustilide (Compound 3)

[0040] Compound 2 was treated with CH 2 Cl 2 Dissolve, -15℃, Ar protection, under stirring, add pyridine (0.03 mol), react for 10 min after the addition is completed. Slowly add methanesulfonyl chloride solution (0.013 mol), continue to react for 10 min after the addition is completed, naturally warm to room temperature, Ar protection, vacuum concentrate below 40℃ to remove CH 2 Cl 2 . Add 20 mL of pyridine to the system, protect with Ar, heat under reflux for 40 min, cool to 0 °C, adjust pH to 1 with 3 mol / L HCl, warm to room temperature, extract with 30 mL × 3 ether, combine the organic phases, and then wash with 30 mL × 3 saturated NaCl solution until the outflowing water layer is neutral. Use MgSO 4 After being fully dried, the product was filtered and concentrated in vacuo under Ar protection. Column chromatography was performed with a frozen acetone-petroleum ether solvent under light-proof conditions to obtain a brown-red viscous oil with a yield of 55%. The product had a strong aroma of traditional Chinese medicine. 1 H NMR (400 MHz, CDCl 3 )δ6.22(d,J=9.6Hz,1H),5.93(dt,J=9.1,4.2Hz,1H),5.15(t,J=8.0Hz,1H),2.53(t,J=9.6Hz ,2H),2.44–2.35(m,2H),2.31(q,J=7.6Hz,2H),1.44(h,J=7.4Hz,2H),0.89(t,J=7.4Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ167.67,148.61,147.08,129.90,124.05,117.17,112.94,28.16,22.45,18.57,13.81.

[0041] Example 4: Preparation of 3-butyl-2-(4-chlorobenzyl)-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4a)

[0042] Take the intermediate ligustilide (100 mg, 0.53 mmol) and p-chlorobenzylamine (211 mg, 1.5 mmol) as raw materials, dissolve 0.53 mmol of ligustilide in 5 mL of anhydrous tetrahydrofuran at room temperature, and then add p-chlorobenzylamine (1.5 mmol) dissolved in 2 mL of anhydrous tetrahydrofuran. The mixture is reacted at 60°C for 12 hours. After the reaction is completed, the mixture is concentrated under vacuum to a residue, and 50 mL of petroleum ether is added to mix to form a homogeneous solution. Place for 12 hours.

[0043] After 3 h, the product crystals were filtered and the crude product was recrystallized from a mixed solvent of petroleum ether and acetone (5:1) to obtain 4a as a white solid with a yield of 65%. 1 H NMR (400 MHz, CDCl 3 )δ7.39–7.31(m,2H),7.27(dd,J=7.9,5.9Hz,2H),6.26(dt,J=9.5,2.0Hz,1H),5.92(dt ,J=9.7,4.0Hz,1H),4.54(d,J=15.3Hz,1H),4.33(d,J=15.2Hz,1H),3.16(s,1H),2.63–2 .47(m,1H),2.43(dddd,J=11.5,7.8,3.8,1.9Hz,2H),2.35–2.24(m,1H),1.78–1.62(m, 3H),1.02(dddd,J=13.3,9.0,7.5,6.1Hz,1H),0.90–0.68(m,2H),0.65(t,J=7.3Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ168.79,137.25,133.12,130.08,129.03,128.83,128.46,117.25,92.17,41.09,33.41,25.20,22.78,22.22,18.61,13.65.HRMS(ESI)of compound 4a:calcd.for C 19 H 22 ClNO 2 [M+H] + =332.1339, found[M+H] + =332.1421.

[0044] Example 5: Preparation of 2-(4-bromobenzyl)-3-butyl-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4b)

[0045] Using the intermediate ligustilide (100 mg, 0.53 mmol) and p-bromobenzylamine (279 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4b as a white solid with a yield of 58%. 1 H NMR (400 MHz, CDCl 3 )δ7.44–7.38(m,2H),7.29(d,J=8.4Hz,2H),6.25(dt,J=9.6,2.0Hz,1H),5.92(dt,J= 9.6,4.1Hz,1H),4.52(d,J=15.3Hz,1H),4.30(d,J=15.3Hz,1H),3.24(s,1H),2.64–2. 49(m,1H),2.43(dddd,J=11.6,7.8,3.9,1.8Hz,2H),2.37–2.22(m,1H),1.79–1.63(m ,3H),1.07–0.93(m,1H),0.90–0.68(m,2H),0.65(t,J=7.2Hz,3H),0.57–0.43(m,1H). 13 C NMR (101 MHz, CDCl 3 )δ168.81,152.33,137.76,131.42,130.44,128.99,128.82,121.20,117. 24,92.17,41.14,33.43,25.20,22.78,22.23,18.62,13.66.HRMS(ESI)of compound 4b:calcd.for C 19 H 22 BrNO 2 [M+H] + =375.0834, found [M+H] + =376.0913.

[0046] Example 6: Preparation of 3-butyl-2-(4-fluorobenzyl)-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4c)

[0047] Using the intermediate ligustilide (100 mg, 0.53 mmol) and p-fluorobenzylamine (187 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4c as a white solid with a yield of 63%. 1 H NMR (400 MHz, CDCl 3)δ7.43–7.37(m,2H),7.02–6.94(m,2H),6.27(dt,J=9.4,1.9Hz,1H),5.93(dt,J=9.8,4.0Hz,1H),4 .55(d,J=15.2Hz,1H),4.36(d,J=15.2Hz,1H),2.97(s,1H),2.62–2.50(m,1H),2.43(dddd,J=9.8,8 .2,4.0,2.2Hz,2H),2.36–2.24(m,1H),1.76–1.70(m,2H),1.02(ddt,J=13.3,8.9,6.8Hz,1H),0.92 –0.79(m,1H),0.80–0.69(m,1H),0.65(t,J=7.3Hz,3H),0.52(dddd,J=22.2,13.0,10.0,5.9Hz,1H). 13 C NMR (101 MHz, CDCl 3 )δ168.68,152.04,134.56,130.40,130.32,129.17,128.78,117.28,115.24, 115.02,92.16,41.03,33.40,25.21,22.79,22.21,18.58,13.65.HRMS(ESI)of compound 4c:calcd.for C 19 H 22 FNO 2 [M+H] + =316.3884, found[M+H] + =316.1707.

[0048] Example 7: Preparation of 3-butyl-3-hydroxy-2-(4-methoxybenzyl)-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4d)

[0049] Using the intermediate ligustilide (100 mg, 0.53 mmol) and p-methoxybenzylamine (205 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4d as a white solid with a yield of 52%. 1 H NMR (400 MHz, CDCl 3)δ7.32(d,J=8.2Hz,2H),6.80(dd,J=7.0,4.4Hz,2H),6.27–6.18(m,1H),5.88(dt,J=9.4,4.1H z,1H),4.57(d,J=15.1Hz,1H),4.19(d,J=15.1Hz,1H),4.06(s,1H),3.76(s,3H),2.61–2.46(m ,1H),2.45–2.35(m,2H),2.34–2.17(m,1H),1.68(dd,J=9.4,7.2Hz,2H),0.93(ddd,J=13.5,10 .5,6.2Hz,1H),0.78–0.62(m,2H),0.58(t,J=7.1Hz,3H),0.43(ddt,J=19.4,13.6,6.9Hz,1H). 13 C NMR (101 MHz, CDCl 3 )δ168.94,158.79,152.57,130.94,130.00,128.77,128.49,117.39,113.67, 92.22,55.32,41.06,33.58,25.14,22.77,22.21,18.61,13.67.HRMS(ESI)of compound 4d:calcd.for C 20 H 25 NO 3 [M+H] + =327.1834, found [M + H] + =328.1919.

[0050] Example 8: Preparation of 4-(3-butyl-3-hydroxy-1-oxo-1,3,4,5-tetrahydro-2H-isoindol-2-yl)benzonitrile (Compound 4e)

[0051] Using the intermediate ligustilide (100 mg, 0.53 mmol) and p-cyanobenzylamine (198 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4e as a white solid with a yield of 61%. 1 H NMR (400 MHz, CDCl 3)δ7.65–7.55(m,3H),7.55–7.47(m,2H),6.24(dt,J=9.6,1.9Hz,1H),5.93(dt,J=9.5, 4.0Hz,1H),4.55(d,J=15.5Hz,1H),4.42(d,J=15.6Hz,1H),3.27–3.21(m,1H),2.61–2. 24(m,4H),1.71(dddd,J=38.7,14.0,11.9,4.6Hz,2H),1.34–1.23(m,0H),1.11–0.93( m,1H),0.93–0.69(m,2H),0.66(d,J=7.1Hz,2H),0.66–0.56(m,1H),0.59–0.45(m,1H). 13 C NMR (101 MHz, CDCl 3 )δ168.84,152.36,144.19,132.21,129.35,129.25,129.09,118.72,117.08, 111.10,92.08,41.48,33.41,25.22,22.75,22.20,18.66,13.68.HRMS(ESI)of compound 4e:calcd.forC 20 H 22 N 2 O 2 [M+H] + =323.1681, found[M+H] + =323.1807.

[0052] Example 9: Preparation of 3-butyl-3-hydroxy-2-(4-trifluoromethylbenzyl)-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4f)

[0053] Using the intermediate ligustilide (100 mg, 0.53 mmol) and p-trifluoromethylbenzylamine (262 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4f as a white solid with a yield of 60%. 1 H NMR (400 MHz, CDCl 3)δ7.54(d,J=2.7Hz,4H),6.27(d,J=9.7Hz,1H),5.94(dt,J=9.0,4.2Hz,1H),4.63(d,J=1 5.3Hz,1H),4.41(d,J=15.3Hz,1H),3.17(d,J=4.9Hz,1H),2.58–2.50(m,1H),2.44(td,J= 9.9,9.5,3.6Hz,2H),2.36–2.25(m,1H),1.91–1.51(m,4H),0.99(h,J=8.2,7.2Hz,1H),0. 74(dh,J=10.6,6.1,4.9Hz,2H),0.61(t,J=7.0Hz,3H),0.47(dh,J=17.6,6.8,5.4Hz,1H). 13 C NMR (101 MHz, CDCl 3 )δ168.86,152.37,142.77,129.03,128.96,128.94,125.37,125.33,125.29,125. 26,117.19,92.18,41.34,33.42,25.21,22.76,22.17,18.62,13.56.HRMS(ESI)of compound 4f:calcd.for C 20 H 22 F 3 NO 2 [M+H]=366.1605,found[M+H] + =366.1660.

[0054] Example 10: Preparation of 3-butyl-3-hydroxy-2-(2-trifluoromethylbenzyl)-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4g)

[0055] Using the intermediate ligustilide (100 mg, 0.53 mmol) and o-trifluoromethylbenzylamine (262 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4 g of white solid with a yield of 52%. 1 H NMR (400 MHz, CDCl 3)δ7.64(d,J=7.9Hz,1H),7.56–7.44(m,2H),7.34(t,J=7.6Hz,1H),6.34(dd,J=9.7,2.1 Hz,1H),5.98(dt,J=9.4,4.3Hz,1H),4.85(d,J=16.7Hz,1H),4.62(d,J=16.6Hz,1H),2. 62–2.43(m,3H),2.35(p,J=5.5,4.7Hz,2H),1.74(td,J=8.2,7.2,3.1Hz,2H),1.39–1.2 4(m,1H),1.08(dq,J=13.8,7.0Hz,1H),0.98–0.73(m,3H),0.69(td,J=7.2,1.9Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ156.53,151.59,131.00,129.32,128.58,128.41,126.56,121.06,117.47,110 .53,91.78,55.54,35.67,33.72,25.53,22.83,22.39,18.66,13.81.HRMS(ESI)of compound4g:calcd.for C 20 H 22 F 3 NO 2 [M+H] + =366.1605, found [M + H] + =366.1688.

[0056] Example 11: Preparation of 3-butyl-3-hydroxy-2-(3-trifluoromethylbenzyl)-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4h)

[0057] Using the intermediate ligustilide (100 mg, 0.53 mmol) and m-trifluoromethylbenzylamine (262 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4h as a white solid with a yield of 56%. 1 H NMR (400 MHz, CDCl 3)δ7.65(d,J=7.4Hz,2H),7.53(d,J=7.8Hz,1H),7.43(t,J=7.8Hz,1H),6.30(dd,J=9.8,2.2Hz,1H),5.95 (dt,J=9.5,4.4Hz,1H),4.65(d,J=15.3Hz,1H),4.44(dd,J=15.4,1.8Hz,1H),2.82(d,J=1.7Hz,1H),2.6 0–2.51(m,1H),2.50–2.39(m,2H),2.38–2.24(m,1H),1.80–1.68(m,2H),1.01(dp,J=13.9,7.0Hz,1H),0 .79(ttd,J=16.6,11.3,9.8,4.5Hz,2H),0.63(dd,J=8.0,6.2Hz,3H),0.51(td,J=13.9,13.0,6.6Hz,1H). 13 C NMR (101 MHz, CDCl 3 )δ168.76,152.11,139.74,132.21,129.18,128.97,128.91,125.26,125.22,124.17, 124.13,117.24,92.13,41.39,33.40,25.25,22.78,22.13,18.60,13.57.HRMS(ESI)of compound 4h:calcd.for C 20 H 22 F 3 NO 2 [M+H] + =366.1605, found [M + H] + =366.1674.

[0058] Example 12: Preparation of 2-(1,1'-biphenyl]-4-ylmethyl)-3-butyl-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4i)

[0059] Using the intermediate ligustilide (100 mg, 0.53 mmol) and p-phenylbenzylamine (274 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4i as a white solid with a yield of 41%. 1 H NMR (400 MHz, CDCl 3)δ7.54(dd,J=21.1,6.0Hz,6H),7.44(t,J=7.5Hz,2H),7.35(t,J=7.3Hz,1H),6.35–6.27(m,1 H),5.94(dt,J=9.2,4.2Hz,1H),4.65(d,J=15.2Hz,1H),4.41(d,J=15.1Hz,1H),3.20(s,1H),2 .65–2.51(m,1H),2.45(td,J=10.6,4.2Hz,2H),2.39–2.24(m,1H),1.76(t,J=8.3Hz,2H),1.0 8–0.87(m,1H),0.75(ddt,J=23.1,15.1,7.8Hz,2H),0.61(t,J=7.1Hz,3H),0.58–0.51(m,1H). 13 C NMR (101 MHz, CDCl 3 )δ168.81,152.25,140.96,140.32,137.77,129.16,129.08,128.76,128.71,127.24,127. 14,127.08,117.39,92.27,41.45,33.47,25.26,22.82,22.22,18.62,13.71.HRMS(ESI)of compound 4i:calcd.For C 25 H 27 NO 2 [M+H] + =374.2042, found [M + H] + =374.2119.

[0060] Example 13: Preparation of 3-butyl-3-hydroxy-2-(4-hydroxybenzyl)-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4j)

[0061] Using the intermediate ligustilide (100 mg, 0.53 mmol) and p-hydroxybenzylamine (184 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4j as a white solid with a yield of 68%. 1 H NMR (400 MHz, CDCl 3)δ7.48(d,J=7.6Hz,1H),7.31–7.18(m,1H),6.99–6.83(m,2H),6.29(dd,J=9.8,2.0Hz,1H),5.91(dt,J= 9.2,4.3Hz,1H),4.68(d,J=15.3Hz,1H),4.44(d,J=15.3Hz,1H),3.90(d,J=1.6Hz,3H),3.17(s,1H),2.62 –2.50(m,1H),2.49–2.39(m,2H),2.38–2.25(m,1H),1.95–1.84(m,1H),1.77(td,J=13.3,12.6,5.4Hz,1 H),1.06(ddq,J=35.5,13.8,7.0Hz,2H),0.85–0.77(m,1H),0.74(t,J=7.3Hz,3H),0.67(t,J=7.2Hz,1H). 13 C NMR (101 MHz, CDCl 3 )δ156.53,151.59,131.00,129.32,128.58,128.41,126.56,121.06,117.47,110 .53,91.78,55.54,35.67,33.72,25.53,22.83,22.39,18.66,13.81.HRMS(ESI)of compound 4j:calcd.for C 19 H 23 NO 3 [M+H] + =314.1678, found [M + H] + =314.1758.

[0062] Example 14: Preparation of 3-butyl-3-hydroxy-2-(4-methylbenzyl)-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4k)

[0063] Using the intermediate ligustilide (100 mg, 0.53 mmol) and p-methylbenzylamine (181 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4k as a white solid with a yield of 59%. 1H NMR(400MHz,Chloroform-d)δ7.35–7.27(m,2H),7.10(d,J=7.7Hz,2H),6.28(dt,J=9.5,1.9Hz,1H), 5.91(dt,J=9.7,4.1Hz,1H),4.54(d,J=15.1Hz,1H),4.36(d,J=15.1Hz,1H),2.60–2.49(m,1H),2.43 (ddt,J=13.2,6.3,2.0Hz,2H),2.32(s,3H),1.73(ddd,J=10.3,5.6,3.8Hz,2H),1.44–1.16(m,1H),1 .00(dddd,J=12.9,8.8,7.4,5.9Hz,1H),0.85–0.66(m,2H),0.63(t,J=7.3Hz,3H),0.59–0.45(m,1H). 13 C NMR(101MHz,Chloroform-d)δ168.69,152.08,136.87,135.67,129.10,128.99,128.64,12 8.60,117.41,92.22,41.48,33.41,25.21,22.81,22.22,21.05,18.58,13.65.HRMS(ESI)of compound 4k:calcd.for C 20 H 25 NO 2 [M+H] + =312.1885, found[M+H] + =312.1964

[0064] Example 15: Preparation of 3-butyl-3-hydroxy-2-(4-isopropylbenzyl)-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 41)

[0065] Using the intermediate ligustilide (100 mg, 0.53 mmol) and p-isopropylbenzylamine (225 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 41 as a white solid with a yield of 67%. 1H NMR(400MHz,Chloroform-d)δ7.38–7.31(m,2H),7.19–7.10(m,2H),6.29(dt,J=9.5,2.0Hz,1H),5.91(dt ,J=9.7,4.1Hz,1H),4.61(d,J=15.0Hz,1H),4.33(d,J=15.1Hz,1H),3.01(s,1H),2.88(p,J=6.9Hz,1H),2 .60–2.49(m,1H),2.43(ddt,J=12.9,6.1,1.9Hz,2H),2.36–2.21(m,1H),1.75–1.69(m,2H),1.23(d,J=6. 9Hz, 6H), 0.95 (tdd, J=11.2, 7.5, 5.4Hz, 1H), 0.76–0.63 (m, 2H), 0.59 (t, J=6.9Hz, 3H), 0.54–0.41 (m, 1H). 13 C NMR(101MHz,Chloroform-d)δ168.65,152.02,148.02,136.07,129.10,128.78,128.59,126.38,1 17.43,92.23,41.44,33.85,33.42,25.26,24.05,23.98,22.81,22.15,18.55,13.75.HRMS(ESI)of compound 4l:calcd.forC 22 H 29 NO 2 [M+H] + =340.2198, found [M + H] + =340.2284

[0066] Example 16: Preparation of 3-butyl-2-(4-dimethylaminobenzyl)-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4m)

[0067] Using the intermediate ligustilide (100 mg, 0.53 mmol) and 4-dimethylaminobenzylamine (231 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4m as a white solid with a yield of 43%. 1H NMR(400MHz,Chloroform-d)δ7.33–7.26(m,2H),6.66(d,J=8.1Hz,2H),6.27(dt,J=9.6,2.0H z,1H),5.90(dt,J=8.8,4.1Hz,1H),4.47–4.28(m,2H),3.06(s,1H),2.90(s,6H),2.61–2.47(m ,1H),2.42(tdd,J=8.9,4.2,1.9Hz,2H),2.33–2.22(m,1H),1.73–1.68(m,2H),1.04(ddd,J=2 1.2,11.2,6.8Hz,1H),0.86(tt,J=13.3,7.0Hz,2H),0.66(t,J=7.3Hz,3H),0.61–0.54(m,1H). 13 C NMR(101MHz,Chloroform-d)δ168.49,151.85,129.72,129.17,128.41,126.82,117.50,112.7 5,92.16,41.22,40.84,33.49,25.25,22.83,22.29,18.55,13.77,1.03.HRMS(ESI)ofcompound 4m:calcd.for C 21 H 28 N 2 O 2 [M+H] + =341.2151, found[M+H] + =341.2230

[0068] Example 17: Preparation of 3-butyl-2-(4-ethoxybenzyl)-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4n)

[0069] Using the intermediate ligustilide (100 mg, 0.53 mmol) and p-ethoxybenzylamine (205 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4n as a white solid with a yield of 54%. 1H NMR(400MHz,Chloroform-d)δ7.37–7.32(m,2H),6.82(dq,J=9.6,3.1,2.5Hz,2H),6.28(dt,J=9.6,1.9Hz,1H ),5.92(dt,J=9.6,4.1Hz,1H),4.43(q,J=15.1Hz,2H),4.01(q,J=7.0Hz,2H),2.59–2.48(m,2H),2.43(ddt,J =11.9,6.4,2.1Hz,2H),2.35–2.23(m,1H),1.81–1.72(m,2H),1.41(t,J=7.0Hz,4H),1.28(s,1H),1.10–0.98 (m,1H),0.90(tt,J=13.7,6.4Hz,1H),0.74(dt,J=13.2,5.7Hz,1H),0.68(t,J=7.3Hz,3H),0.63–0.58(m,1H). 13 C NMR(101MHz,Chloroform-d)δ158.18,151.70,130.79,129.93,129.31,128.64,117.39,11 4.37,92.16,63.49,41.17,33.36,25.25,22.83,22.26,18.55,14.81,13.74.HRMS(ESI)of compound 4n:calcd.forC 21 H 27 NO 3 [M+H] + =342.1991, found[M+H] + =342.2055.

[0070] Example 18: Preparation of 3-butyl-2-(2-chlorobenzyl)-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4o)

[0071] Using the intermediate ligustilide (100 mg, 0.53 mmol) and o-chlorobenzylamine (211 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4o as a white solid with a yield of 57%. 1 H NMR (400 MHz, CDCl 3)δ7.50(ddd,J=18.8,7.4,3.0Hz,1H),7.38–7.27(m,1H),7.19(td,J=8.4,7.2,4.3Hz,2H),6.31(d,J =9.8Hz,1H),5.95(dt,J=9.5,4.0Hz,1H),4.78(d,J=16.0Hz,1H),4.57(d,J=15.9Hz,1H),2.73(s,1H) ,2.62–2.51(m,1H),2.50–2.40(m,2H),2.39–2.26(m,1H),1.86–1.51(m,5H),1.28(s,1H),1.06(dh, J=12.8,6.5,5.8Hz,1H),0.90(dp,J=21.4,6.3,5.6Hz,2H),0.83–0.73(m,1H),0.69(t,J=7.4Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ168.86,152.18,136.03,132.82,130.46,129.35,129.17,128.86,128.49,126. 98,117.30,92.06,38.52,33.32,25.39,22.82,22.30,18.68,13.69.HRMS(ESI)of compound 4o:calcd.for C 19 H 22 ClNO 2 [M+H] + =332.1339, found[M+H] + =332.1421.

[0072] Example 19: Preparation of 3-butyl-2-(2-fluorobenzyl)-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4p)

[0073] Using the intermediate ligustilide (100 mg, 0.53 mmol) and o-fluorobenzylamine (187 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4p as a white solid with a yield of 52%. 1 H NMR (400 MHz, CDCl 3)δ7.64–7.48(m,2H),7.31–7.20(m,2H),7.08(dq,J=17.0,8.9,7.9Hz,3H),6.29(dt,J=9.5,2 .2Hz,1H),5.93(dt,J=8.9,4.1Hz,1H),4.74–4.61(m,1H),4.60–4.49(m,1H),2.62(d,J=2.9Hz ,1H),2.58–2.49(m,1H),2.44(tdd,J=9.3,3.9,2.1Hz,2H),2.37–2.26(m,1H),1.88–1.75(m,2 H),1.43–1.27(m,1H),1.06(dp,J=20.7,7.1Hz,1H),1.00–0.85(m,2H),0.68(t,J=7.3Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ168.71,151.94,131.37,129.05,128.97,128.78,125.59,125.44,124.28,117.31, 115.32,115.10,91.98,34.19,33.12,25.38,22.81,22.28,18.59,13.71.HRMS(ESI)of compound 4p:calcd.for C 19 H 22 FNO 23 [M+H] + =316.3884, found[M+H] + =316.1730.

[0074] Example 20: Preparation of 3-butyl-3-hydroxy-2-(2-methoxybenzyl)-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4q)

[0075] Using the intermediate ligustilide (100 mg, 0.53 mmol) and o-methoxybenzylamine (205 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4q as a white solid with a yield of 63%. 1 H NMR (400 MHz, CDCl 3)δ7.65(d,J=7.4Hz,2H),7.53(d,J=7.8Hz,1H),7.43(t,J=7.8Hz,1H),6.30(dd,J=9.8,2.2Hz,1H),5.95 (dt,J=9.5,4.4Hz,1H),4.65(d,J=15.3Hz,1H),4.44(dd,J=15.4,1.8Hz,1H),2.82(d,J=1.7Hz,1H),2.6 0–2.51(m,1H),2.50–2.39(m,2H),2.38–2.24(m,1H),1.80–1.68(m,2H),1.01(dp,J=13.9,7.0Hz,1H),0 .79(ttd,J=16.6,11.3,9.8,4.5Hz,2H),0.63(dd,J=8.0,6.2Hz,3H),0.51(td,J=13.9,13.0,6.6Hz,1H). 13 C NMR (101 MHz, CDCl 3 )δ168.65,159.65,152.03,140.30,129.40,128.68,120.94,117.39,114.05,112 .92,92.19,55.26,41.74,33.43,25.31,22.81,22.23,18.59,13.70.HRMS(ESI)of compound 4q:calcd.forC 20 H 25 NO 3 [M+H] + =328.1834, found [M+H] + =328.1915.

[0076] Example 21: Preparation of 2-(1,1'-biphenyl]-2-ylmethyl)-3-butyl-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound r)

[0077] Using the intermediate ligustilide (100 mg, 0.53 mmol) and o-phenylbenzylamine (274 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4r as a white solid with a yield of 41%. 1 H NMR (400 MHz, CDCl 3)δ7.39(d,J=4.9Hz,1H),7.32–7.12(m,3H),6.27(dd,J=9.7,2.1Hz,1H),5.93(dt,J=10 .0,4.1Hz,1H),4.56(d,J=15.3Hz,1H),4.32(d,J=15.3Hz,1H),3.22(s,1H),2.62–2.50( m,1H),2.49–2.38(m,2H),2.37–2.22(m,1H),1.79–1.59(m,4H),1.01(tq,J=8.0,5.4,2. 9Hz,1H),0.88–0.68(m,2H),0.65(t,J=7.1Hz,3H),0.53(tdd,J=16.4,13.1,6.3Hz,2H). 13 C NMR (101 MHz, CDCl 3 )δ168.85,152.41,140.73,134.20,129.66,128.97,128.84,128.64,127.45,126. 81,117.27,92.17,41.23,33.44,25.26,22.77,22.20,18.63,13.69.HRMS(ESI)of compound 4r:calcd.for C 25 H 27 NO 2 [M+H] + =374.2042, found [M + H] + =374.2118.

[0078] Example 22: Preparation of 3-butyl-2-(3-chlorobenzyl)-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound s)

[0079] Using the intermediate ligustilide (100 mg, 0.53 mmol) and m-chlorobenzylamine (211 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4s as a white solid with a yield of 68%. 1 H NMR (400 MHz, CDCl 3)δ7.30–7.14(m,1H),7.03–6.89(m,2H),6.84–6.71(m,1H),6.34–6.19(m,1H),5.93(dtt,J=10.8,4. 6,2.2Hz,1H),4.54(d,J=15.2Hz,1H),4.40(dd,J=15.1,7.2Hz,1H),3.79(q,J=2.3Hz,3H),2.91–2.78 (m,1H),2.61–2.48(m,1H),2.49–2.38(m,2H),2.37–2.23(m,1H),1.81–1.70(m,2H),1.02(dq,J=13. 8,6.9Hz,1H),0.84(dq,J=13.2,6.9Hz,1H),0.78–0.68(m,1H),0.69–0.62(m,3H),0.62–0.52(m,1H). 13 C NMR (101 MHz, CDCl 3 )δ168.65,159.65,152.03,140.30,129.40,128.68,120.94,117.39,114.05,112 .92,92.19,55.26,41.74,33.43,25.31,22.81,22.23,18.59,13.70.HRMS(ESI)of compound 4s:calcd.for C 19 H 22 ClNO 2 [M+H] + =332.1339, found[M+H] + =332.1422.

[0080] Example 23: Preparation of 3-butyl-2-(3-fluorobenzyl)-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4t)

[0081] Using the intermediate ligustilide (100 mg, 0.53 mmol) and m-fluorobenzylamine (187 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4t as a white solid with a yield of 56%. 1 H NMR (400 MHz, CDCl 3)δ7.30–7.20(m,2H),7.13–7.01(m,2H),6.25(dt,J=9.5,1.9Hz,1H),5.89(dt,J=9.6,3. 9Hz,1H),3.70(ddd,J=13.8,9.5,5.4Hz,1H),3.33(ddd,J=13.9,9.3,6.6Hz,1H),3.11(d dd,J=13.0,9.4,6.8Hz,1H),2.98(ddd,J=13.9,9.3,5.4Hz,1H),2.55–2.25(m,4H),2.07 (s,1H),1.96–1.78(m,2H),1.35–1.25(m,3H),0.95–0.90(m,1H),0.88(d,J=7.3Hz,3H). 13 C NMR (101 MHz, CDCl 3 )δ168.45,151.50,131.25,131.20,129.39,128.66,128.30,128.22,124.10,117.25,115.47 ,115.25,91.68,38.79,33.00,28.70,25.44,22.81,22.46,18.63,13.90,1.03.HRMS(ESI)of compound 4t:calcd.for C 19 H 22 FNO 2 [M+H] + =316.3884, found[M+H] + =316.1713.

[0082] Example 24: Preparation of 3-butyl-3-hydroxy-2-(3-methoxybenzyl)-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4u)

[0083] Using the intermediate ligustilide (100 mg, 0.53 mmol) and m-methoxybenzylamine (205 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4u as a white solid with a yield of 63%. 1 H NMR (400 MHz, CDCl 3)δ7.65(d,J=7.4Hz,2H),7.53(d,J=7.8Hz,1H),7.43(t,J=7.8Hz,1H),6.30(dd,J=9.8,2.2Hz,1H),5.95 (dt,J=9.5,4.4Hz,1H),4.65(d,J=15.3Hz,1H),4.44(dd,J=15.4,1.8Hz,1H),2.82(d,J=1.7Hz,1H),2.6 0–2.51(m,1H),2.50–2.39(m,2H),2.38–2.24(m,1H),1.80–1.68(m,2H),1.01(dp,J=13.9,7.0Hz,1H),0 .79(ttd,J=16.6,11.3,9.8,4.5Hz,2H),0.63(dd,J=8.0,6.2Hz,3H),0.51(td,J=13.9,13.0,6.6Hz,1H). 13 C NMR (101 MHz, CDCl 3 )δ168.65,159.65,152.03,140.30,129.40,128.68,120.94,117.39,114.05,112 .92,92.19,55.26,41.74,33.43,25.31,22.81,22.23,18.59,13.70.HRMS(ESI)of compound 4u calcd.forC 20 H 25 NO 3 [M+H] + =328.1834, found [M+H] + =328.1911.

[0084] Example 25: Preparation of 2-(1,1'-biphenyl]-3-ylmethyl)-3-butyl-3-hydroxy-2,3,4,5-tetrahydro-1H-isoindol-1-one (Compound 4v)

[0085] Using the intermediate ligustilide (100 mg, 0.53 mmol) and m-phenylbenzylamine (274 mg, 1.5 mmol) as raw materials, the synthesis method was the same as compound 4a to obtain 4v as a white solid with a yield of 43%. 1 H NMR (400 MHz, CDCl 3)δ7.68–7.54(m,3H),7.42(ddt,J=34.5,14.8,7.6Hz,6H),6.31(d,J=9.7Hz,1H),5.93(dt,J =9.1,4.3Hz,1H),4.68(d,J=15.1Hz,1H),4.47(d,J=15.0Hz,1H),2.86(d,J=2.0Hz,1H),2.62 –2.49(m,1H),2.49–2.38(m,2H),2.37–2.25(m,1H),1.75(tq,J=14.0,7.6,4.7Hz,2H),1.29( s,1H),0.99(dt,J=14.7,7.5Hz,1H),0.75(ddd,J=22.9,12.7,6.3Hz,2H),0.63–0.57(m,3H). 13 CNMR (101MHz, CDCl 3 )δ168.69,152.05,141.39,139.28,129.18,128.86,128.75,128.73,127.72,127.47,127.32, 127.20,126.10,117.41,92.25,41.81,33.44,25.30,22.81,22.20,18.59,13.70.HRMS(ESI)of compound 4v:calcd.for C 25 H 27 NO 2 [M+H] + =374.2042, found [M + H] + =374.2123.

[0086] Example 26: Griess reagent method to detect the inhibitory effect of compounds on LPS-induced NO release

[0087] Experimental method: The RAW264.7 cells used in this experiment need to be in good growth condition and the seeding density is 2.5×10 4 / well in a 96-well plate. After 24 hours, the prepared drug was added at a concentration of 10μM. Six replicate wells were set for each concentration, and the drug addition volume was 200μL. A blank group (only culture medium) and a control group (with lipopolysaccharide LPS) were also set up. LPS (1μg / mL) was added 3 hours later. After 48 hours of LPS action, the cell supernatant was taken to detect the NO content. NO is easily oxidized in water and body fluids to form NO 2- , under acidic conditions NO 2-The diazo compound is then chemically reacted with the Griess reagent to obtain a diazo compound. The diazo compound is then coupled with p-naphthylethylenediamine. There is a linear relationship between the content of the substance generated in this chemical reaction process and the NO content, so it can be quantitatively measured using an enzyme marker at 540nm. Experimental results: The results of the NO generation inhibition activity are shown in Tables 1 and 2.

[0088] Table 1 Effects of target compounds on LPS-induced NO secretion in RAW264.7 cells. RAW264.7 cells were pretreated with 10 μM of the compound for 3 h and then stimulated with LPS (1 μg / mL) for 48 h. Data are the mean ± SD of three experiments (n>3).

[0089]

[0090] Table 2 IC of compounds inhibiting NO secretion by RAW264.7 50 RAW264.7 cells were pretreated with different concentrations of compounds for 3 h and then stimulated with LPS (1 μg / mL) for 48 h. Data are the mean ± SD of three experiments (n>3)

[0091]

[0092] Experimental results: When the lactone ring of artemisinin is changed to a lactam ring, the stability of the compound increases. On the basis of benzyl as an effective group to improve the activity of artemisinin derivatives, when the benzene ring linking group in the N-1 benzyl group is replaced with a trifluoromethyl group, the activity of the obtained compounds 4f and 4g is better than the positive control. When the trifluoromethyl group is connected to the para position of the benzene ring, the compound's NO inhibition activity is significantly improved, so the para-trifluoromethyl group is the optimal linking group. Its IC for inhibiting NO 50 The IC value was 1.61±0.11μM, which was better than the two positive control drugs indomethacin. 50 (2.81±0.06μM) and dexamethasone IC 50 (2.22±0.02μM).

[0093] Example 27: ELISA method to determine the effects of compounds on inflammatory factors IL-1β, TNF-α and IL-6

[0094] Experimental method: The seeding density of RAW264.7 cells was 2.5×10 5 / well in a 24-well plate, with 6 replicate wells for each experimental group, and cultured overnight. On the second day, after 2 hours of drug pretreatment, LPS = 1 μg / mL was added, and a blank group (only cell culture medium) and a control group (cell culture medium and LPS) were set up at the same time. After 24 hours, the cell supernatant was aspirated.

[0095] The lead compound 4f with a high NO inhibition rate was selected for enzyme-linked immunosorbent assay (ELISA) to detect its effects on pro-inflammatory factors IL-1β, TNF-α and IL-6. Dexamethasone was used as a positive control. The ELISA kit was used to determine the contents of inflammatory factors IL-1β, TNF-α and IL-6 in all samples according to the experimental steps, and the IC values ​​of the corresponding compounds for inhibiting the production of inflammatory factors were calculated. 50 , the experimental results are shown in Table 3.

[0096] Table 3 IC values ​​of six compounds for inhibiting LPS-induced secretion of IL-1β, TNF-α and IL-6 in RAW264.7 cells 50

[0097]

[0098] Experimental results: According to the experimental results in Table 3, it can be concluded that the lead compound 4f exhibits anti-inflammatory activity superior to that of the positive control dexamethasone, and it effectively inhibits the secretion of IL-1β inflammatory factor. 50 was 18.22±0.12nM, effectively inhibiting the secretion of TNF-α inflammatory factor, IC 50 was 6.2±0.02μM, and also inhibited the secretion of IL-6 inflammatory factor, IC 50 In addition, we also found that the anti-inflammatory activity of 4r was better than that of the positive control dexamethasone, and the IC 50 The IC for inhibiting TNF-α secretion was 556.76±0.35nM. 50 The IC for inhibiting IL-6 secretion was 10.85±0.14μM 50 It is 3.34±0.12μM. The above experimental results show that this series of derivatives has excellent anti-inflammatory effects.

[0099] Example 28: Detection of the inhibitory effect of compounds on ROS

[0100] Experimental method: RAW264.7 macrophages (2.5×10 5 Cells / well) were inoculated into black 96-well cell culture plates and cultured for 12 h. Cells were pretreated with the test compound for 1 h and then incubated with 1 mg / mL LPS for 24 h. The culture medium was removed and washed with PBS; 100 mL DCFH-DA was added and diluted to a final concentration of 100 mM in a medium without FBS and cultured at 37°C for 30 min. The culture medium was removed, 100 mL PBS was added to each well, and fluorescence was detected using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The experimental results are shown in the attached figure. Figure 1 shown.

[0101] Experimental results: The ROS kit was used to detect the effects of the compounds on the ROS secretion of RAW264.7 cells induced by LPS. The results showed that compound 4f, indomethacin, and ligustilide could inhibit the ROS secretion of RAW264.7 cells induced by LPS. However, compound 4f (IC 50 =1.44μM) had a slightly better inhibitory effect than indomethacin (IC 50 =2.47 μM) and ligustilide (IC 50 =3.44 μM).

Claims

1. A ligustilide derivative, characterized in that Has the chemical structure shown below, 2. Use of the ligustilide derivative according to claim 1 in the preparation of anti-inflammatory inhibitors.

Images