Carrasane sesquiterpene lactone derivative and medical application thereof

Abstract

The invention discloses a carrasane sesquiterpene lactone derivative and a medical application of the carrasane sesquiterpene lactone derivative. Relates to the field of small-molecule drug discovery. The chemical structural formula of the compound is as shown in formula (I): # imgabs0. The carrasane type sesquiterpene lactone derivative disclosed by the invention can be used for effectively improving liver injury, hepatic glucose metabolism disorder, lipid metabolism disorder, liver inflammation and fibrosis degree of NASH mice, and has potential medicinal value.

Description

Technical Field

[0001] The present invention relates to the field of small molecule drug discovery, and in particular to a class of calaburane sesquiterpene lactone derivatives or pharmaceutically acceptable salts thereof and use thereof in preparing non-alcoholic fatty hepatitis drugs. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases in the world, and nonalcoholic steatohepatitis (NASH, also known as metabolic-associated fatty liver disease, MASH) is the most serious histological manifestation of NAFLD. About 20% of patients with NAFLD will progress to NASH, and if NASH is not intervened, it will further progress to malignant diseases such as cirrhosis and liver cancer. At present, the treatments for NAFLD mainly include diet control and exercise, but drug intervention is required after progression to NASH. Since NASH patients usually have diabetes or hyperlipidemia, hypoglycemic and lipid-lowering drugs are often used clinically to control the progression of NASH, such as metformin, statins, and fibrates, but these drugs cannot fundamentally reverse NASH, and inevitably have significant side effects and clinical contraindications. Although the development of new drugs for NASH has been carried out for decades, most clinical trials have suffered heavy losses. Currently, only one new drug, Resmetirom, has just been approved by the FDA for marketing. Therefore, NASH treatment drugs are quite scarce, there is a huge unmet clinical need in the market, and there is an urgent need to discover some safe and effective new NASH drugs. Summary of the invention

[0003] In view of the above problems, the present invention provides a use of a calaburane-type sesquiterpene lactone derivative as shown in formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating non-alcoholic fatty hepatitis.

[0004] The technical solution of the present invention is: the present invention discloses a class of calaburane sesquiterpene lactone derivatives or pharmaceutically acceptable salts thereof, the chemical structural formula of the derivative is shown in formula (I):

[0005]

[0006] Where: Selected from = or -, Formation of C=O, C-OH or

[0007] X is an O or N atom;

[0008] R 1 Alkyl, aryl, Where R 2 and R 3 may be the same or different, and are hydrogen, alkyl, cycloalkyl, or aryl; R 2 , R 3 The N atom and the alkyl group can form a 3-9 membered ring structure, which can be substituted by one or more substituents, including hydrogen, alkyl, ester, aryl, alkylaryl, arylalkyl, arylalkenyl, arylalkynyl or heterocyclic group.

[0009] Furthermore, the derivative is selected from the following compounds:

[0010]

[0011] Another object of the present invention is to provide a pharmaceutical composition comprising one or more of the calaburane-type sesquiterpene lactone derivatives described in Formula I as active ingredients, and pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and / or diluents.

[0012] Another object of the present invention is to provide the use of the calaburane-type sesquiterpene lactone derivative or pharmaceutically acceptable salt, and the pharmaceutical composition thereof in the preparation of a drug for the prevention and treatment of non-alcoholic fatty hepatitis.

[0013] The beneficial effect of the present invention is that the calaburane-type sesquiterpene lactone derivatives of the present invention can effectively improve liver damage, liver glucose metabolism disorder, lipid metabolism disorder, liver inflammation and fibrosis degree of NASH mice, and have potential medicinal value. DETAILED DESCRIPTION

[0014] In order to more clearly illustrate the technical solution of the present invention, the technical solution of the present invention is further described in detail below: Example 1: Preparation of Compound 1

[0015] The medicinal material of North Heshi (600g) was immersed in 80% ethanol (6L), heated at 95°C for reflux extraction three times, each time for 1h, the filtrate was collected, and concentrated under reduced pressure to obtain a dark brown extract, which was dissolved in 20% ethanol (200ml), and water (200mL) was added, extracted with ethyl acetate (300mL×3), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a yellow oil; it was then purified by silica gel column chromatography, eluted with petroleum ether-ethyl acetate gradient, and the fractions rich in compound 1 were collected in stages, combined and concentrated to obtain white solid compound 1, with a yield of 0.08%. 1H NMR (500MHz, CDCl3) δ (ppm): 6.26 (d, J = 2.9Hz, 1H), 5.57 (d, J = 2.4Hz, 1H), 4.82-4.77 (m, 1H), 3.20-3.14 (m, 1H), 2.55 (t, J = 7.5Hz, 2H), 2. 38-2.32(m,2H),2.18(s,3H),1.66-1.63(m,1H),1.58-1.54(m,1H),1 .08(s,3H),1.00-0.90(m,2H),0.49-0.45(m,1H),0.41-0.37(m,1H). 13 C NMR(125MHz, CDCl3)δ(ppm):208.68,170.47,139.00,122.56,75.60,43.58,37.7 3,37.29,34.23,30.73,30.09,23.35,22.91,18.23,17.22.HRMS(ESI):m / z[M+Na] + Calcd for C 15 H 20 NaO3:271.1310,Found 271.1344.

[0016] Example 2: Preparation of Compound 2

[0017] The extraction operation was the same as compound 1. The extract was purified by silica gel column flash chromatography and eluted with petroleum ether-ethyl acetate gradient. The fractions rich in compound 2 were collected in sections, combined and concentrated to obtain yellow liquid compound 2 with a yield of 1.05%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.26 (d, J = 2.7Hz, 1H), 5.57 (d, J = 2.3Hz, 1H), 4.83-4.78 (m, 1H), 3.84 (q, J = 6.2Hz, 1H), 3.21-3.15 (m, 1H), 2.40-2.32 (m, 2H),1.61-1.52(m,2H),1.45(s,1H),1.42-1.35(m,2H),1.22(d,J=6.1Hz, 3H),1.10(s,3H),1.02-0.92(m,2H),0.50-0.46(m,1H),0.39-0.35(m,1H). 13C NMR (125MHz, CDCl3) δ (ppm): 170.56, 139.12, 122.50, 75.74, 67.90, 39.30, 37.8 1,37.43,35.02,30.87,25.38,23.64,22.93,18.32,17.12.HRMS(ESI):m / z[M+H] + Calcd for C 15 H 23 O3:251.1647,Found:251.1675.

[0018] Example 3: Preparation of Compound 3

[0019]

[0020] Compound 2 (100 mg, 0.4 mmol) was dissolved in dichloromethane (2 mL), and acetyl chloride (0.6 mmol) and DIPEA (153 mg, 1.2 mmol) were added thereto. The mixture was reacted at room temperature (20°C) for 3 h. The reaction of compound 2 was completed by TLC monitoring. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL×3). The organic layers were combined, washed with saturated sodium chloride solution (8 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The product was purified by 200-300 mesh silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 to 5:1) to obtain the product as a white solid with a yield of 80.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 8.01 (d, J = 8.8Hz, 2H), 6.93 (d, J = 8.8Hz, 2H), 6.25 (d, J = 2.8H) z,1H),5.55(d,J=2.4Hz,1H),5.19-5.12(m,1H),4.82-4.77(m,1H),3.88(s,3H),3.19-3. 13(m,1H),2.38-2.31(m,1H),1.89-1.82(m,1H),1.75-1.68(m,1H),1.44-1.39(m,2H),1. 35(d,J=6.2Hz,3H),1.08(s,3H),1.00-0.88(m,2H),0.51-0.47(m,1H),0.38-0.34(m,1H). 13 C NMR (125MHz,CDCl3)δ(ppm): 13CNMR(126MHz, CDCl3)δ(ppm):170.51,165.95,163.27,139.07,131.49,123.25,122.48,113.56,75.68,7 1.00,55.44,37.82,37.42,36.19,34.85,30.84,25.10,22.92,20.14,18.25,17.14.HRMS(ESI):m / z[M+H] + Calcd for C 23 H 29 O5:385.2015,Found:385.2009.

[0021] Example 4: Preparation of Compound 4

[0022]

[0023] The preparation method of compound 4 refers to compound 3. White solid, yield is 75.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 8.06 (dd, J=8.9, 5.5Hz, 2H), 7.13 (t, J=8.7Hz, 2H), 6.25 (d ,J=2.8Hz,1H),5.56(d,J=2.4Hz,1H),5.20-5.14(m,1H),4.83-4.77(m,1H),3.20-3.14( m,1H),2.39-2.32(m,2H),1.90-1.83(m,1H),1.76-1.69(m,1H),1.44-1.39(m,1H),1.36 (d,J=6.3Hz,3H),1.08(s,3H),1.00-0.90(m,2H),0.51-0.47(m,1H),0.39-0.35(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.48, 166.70, 165.21, 164.68, 139.05, 132.04, 131.96, 127.03, 122.51, 115.53, 1 15.35,75.64,71.61,37.79,37.39,36.13,34.75,30.81,25.06,22.92,20.05,18.25,17.13.HRMS(ESI):m / z[M+H] + Calcd for C 22 H 26 FO4:373.1815,Found:373.1813.

[0024] Example 5: Preparation of Compound 5

[0025]

[0026] The preparation method of compound 5 refers to compound 3. Colorless liquid, yield is 71.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 7.75 (d, J = 7.6Hz, 1H), 7.44 (t, J = 7.9Hz, 1H), 6.98 (t, J = 8.1Hz, 2H ),6.25(s,1H),5.56(s,1H),5.22-5.16(m,1H),4.82-4.77(m,1H),4.13(q,J=7.0,6.4Hz,2H), 3.19-3.14(m,1H),2.38-2.31(m,2H),1.87-1.81(m,1H),1.74-1.67(m,1H),1.49-1.42(m,1H) ,1.36(d,J=6.2Hz,3H),1.09(s,3H),1.00-0.90(m,2H),0.52-0.48(m,1H),0.39-0.34(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.56, 166.36, 158.25, 139.06, 133.05, 131.22, 122.53, 121.31, 120.03, 113.13, 75.7 3,71.17,64.43,37.82,37.42,36.15,34.89,30.83,25.07,22.85,20.09,18.26,17.13,14.81.HRMS(ESI):m / z[M+H] + Calcd for C 24 H 31 O5:399.2171,Found:399.2171.

[0027] Example 6: Preparation of Compound 6

[0028]

[0029] The preparation method of compound 6 refers to compound 3. Colorless liquid, yield is 82.4%. 1H NMR (500MHz, CDCl3) δ (ppm): 6.26 (d, J = 2.9 Hz, 1H), 5.57 (d, J = 2.4 Hz, 1H), 4.9 5-4.88(m,1H),4.83-4.78(m,1H),3.21-3.14(m,1H),2.4.-2.32(m,2H),2.05( s,3H),1.73-1.68(m,1H),1.62-1.56(m,1H),1.37-1.32(m,2H),1.24(d,J=6.3 Hz,3H),1.09(s,3H),1.21-0.91(m,2H),0.48-0.44(m,1H),0.38-0.34(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.76, 170.54, 139.42, 122.93, 75.69, 70.73, 37.75, 37.3 5,35.92,34.70,30.77,24.92,22.83,21.35,19.89,18.19,17.05.HRMS(ESI):m / z[M+H] + Calcd for C 17 H 25 O4:293.1753,Found:293.1744.

[0030] Example 7: Preparation of Compound 7

[0031]

[0032] The preparation method of compound 7 refers to compound 3. Pale yellow liquid, yield is 79.7%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.40 (dd, J=17.3, 1.5Hz, 1H), 6.26 (d, J=2.8Hz, 1H), 6.12 (dd ,J=17.3,10.4Hz,1H),5.85–5.81(m,1H),5.57(d,J=2.5Hz,1H),5.04-4.98(m,1H),4.83-4 .77(m,1H),3.19-3.15(m,2H),1.80-1.72(m,1H),1.67-1.63(m,1H),1.38-1.33(m,1H),1. 28(d,J=6.2Hz,3H),1.08(s,3H),1.00-0.89(m,2H),0.49-0.45(m,1H),0.38-0.34(m,1H). 13C NMR (126MHz, CDCl3) δ (ppm): 170.53, 165.85, 139.04, 130.34, 128.97, 122.53, 75.69, 70.93, 37.78,37.38,35.96,34.72,30.80,24.96,22.85,19.94,18.22,17.09.HRMS(ESI):m / z[M+H] + Calcd for C 18 H 25 O4:305.1753,Found:305.1745.

[0033] Example 8: Preparation of Compound 8

[0034]

[0035] Compound 2 (100 mg, 0.4 mmol) was dissolved in dichloromethane (2.5 mL), and N,N'-carbonylbis(1,2,4-triazole) (98.4 mg, 0.6 mmol) was added thereto. The mixture was reacted at 40°C for 1.5 h. The reaction of compound 2 was completed after monitoring by TLC. The reaction solution was cooled to room temperature (20°C), and methylamine (0.72 mmol) was added thereto. The mixture was reacted at room temperature (20°C) for 5 h. The reaction was completed after monitoring by TLC. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL×3). The organic layers were combined, washed with saturated sodium chloride solution (8 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The mixture was purified by 200-300 mesh silica gel column chromatography (petroleum ether:ethyl acetate=10:1-5:1) to obtain a colorless oil in a yield of 69.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.25 (d, J = 2.9Hz, 1H), 5.56 (d, J = 2.4Hz, 1H), 4.82 -4.77(m,2H),4.56(s,1H),3.20-3.13(m,1H),2.80(d,J=4.9Hz,3H),2.39-2.31 (m,1H),1.67-1.65(m,1H),1.59-1.53(m,1H),1.37-1.30(m,2H),1.22(d,J=6. 3Hz,3H),1.08(s,3H),1.00-0.90(m,2H),0.48-0.44(m,1H),0.37-0.33(m,1H). 13C NMR (126MHz, CDCl3) δ (ppm): 170.52, 157.02, 139.10, 122.48, 75.71, 71.13, 37.82, 37.4 1,36.30,34.85,30.83,27.45,25.04,22.91,20.34,18.25,17.08.HRMS(ESI):m / z[M+H] + Calcd for C 17 H 26 NO4:308.1862,Found:308.1856.

[0036] Example 9: Preparation of Compound 9

[0037]

[0038] The preparation method of compound 9 refers to compound 8. Pale yellow solid, yield is 72.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.23 (d, J = 2.8 Hz, 1H), 5.54 (d, J = 2.4, 1H), 4.81-4.75 (m, 1H), 3.18-3.12 (m, 1H), 2.38-2.29 (m, 1H), 1.74-1. 67(m,1H),1.60-1.53(m,1H),1.36-1.31(m,2H),1.22(d,J=6.3Hz),1.06(s,3H),0.98-0.88(m,2H),0.47-0.43(m,1H),0.36-0.32(m,1H). 13 CNMR (126MHz, CDCl3) δ (ppm): 170.55, 156.46, 139.07, 122.50, 75.73, 71.55, 37.82, 37.42, 36.34,36.28,35.80,34.89,30.83,25.05,22.88,20.42,18.21,17.07.HRMS(ESI):m / z[M+H] + Calcd for C 18 H 28 NO4:322.2018,Found:322.2013.

[0039] Example 10: Preparation of Compound 10

[0040]

[0041] The preparation method of compound 10 refers to compound 8. Colorless oil, yield 71.2%.1 H NMR (500MHz, CDCl3) δ (ppm): 6.23 (d, J = 2.8Hz, 1H), 5.55 (d, J = 2.5Hz, 1H), 4.81-4 .76(m,1H),4.61(s,1H),3.22-3.19(m,2H),3.17-3.14(m,1H),2.38-2.29(m,2H) ,1.66(m,1H),1.55(m,1H),1.36-1.30(m,2H),1.20(d,J=6.0Hz,3H),1.13(m,J=7 .3Hz,3H),1.07(s,1H),0.99-0.89(m,1H),0.46-0.43(m,1H),0.36-0.32(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.52, 156.28, 139.10, 122.47, 75.71, 70.92, 37.81, 37.41,36.31,35.73,34.86,30.83,25.02,22.89,20.31,18.23,17.07,15.29.[M+H] + Calcd for C 18 H 28 NO4:322.2018,Found:322.2014.

[0042] Example 11: Preparation of Compound 11

[0043]

[0044] The preparation method of compound 11 refers to compound 8. Pale yellow liquid, yield is 72.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.26 (d, J = 2.9Hz, 1H), 5.57 (d, J = 2.4Hz, 1H), 4.86-4.78(m,2H),3.28(m,4H),3.19-3.14(m,1H),2.40-3.31(m,2H),1.76 -1.68(m,1H),1.37-1.33(m,2H),1.24(d,J=6.2Hz,3H),1.13(t,J=7.1Hz, 6H),1.08(s,3H),1.00-0.91(m,2H),0.49-0.46(m,1H),0.37-0.33(m,1H). 13C NMR (126MHz, CDCl3) δ (ppm): 170.54, 155.72, 139.09, 122.47, 75.72, 71.09, 41.64, 41.15, 37.84,37.42,36.41,34.92,30.85,25.07,22.90,20.36,18.22,17.08,14.11,13.61.[M+H] + Calcd for C 20 H 32 NO4:350.2331,Found:350.2330.

[0045] Example 12: Preparation of Compound 12

[0046]

[0047] The preparation of compound 12 refers to compound 8. Pale yellow liquid, yield 55.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.25 (s, 1H), 5.56 (s, 1H), 4.84-4.77 (m, 3H), 3 .20-3.15(m,1H),2.59(s,1H),2.19-2.31(m,2H),1.68(s,1H),1.57(s,1H), 1.33-1.27(m,2H),1.23(d,J=6.1Hz,3H),1.08(s,3H),1.00-0.91(m,2H),0 .73(s,1H),0.72(s,1H),0.52(s,2H),0.48-0.44(m,1H),0.37-0.33(m,1H). 13 C NMR(126MHz, CDCl3)δ(ppm):170.60,157.13,139.06,122.55,75.75,71.15,37.79, 37.38,36.26,34.81,30.81,24.98,23.03,22.86,20.27,18.23,17.06,6.80.[M+H] + Calcd for C 19 H 28 NO4:334.2018,Found:334.2033.

[0048] Example 13: Preparation of Compound 13

[0049]

[0050] The preparation of compound 13 refers to compound 8. Pale yellow liquid, yield is 61.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.25 (d, J = 2.8Hz, 1H), 5.56 (d, J = 2.4Hz, 1H), 4.84-4.7 7(m,2H),3.39(t,J=5.5Hz,2H),3.35-3.32(m,2H),3.18-3.14(m,1H),2.39-2.31(m, 2H),1.87(s,4H),1.76-1.70(m,1H),1.61-1.55(m,1H),1.38-1.34(m,2H),1.24(d, J=6.2Hz,3H),1.08(s,3H),1.00-0.90(m,2H),0.49-0.45(m,1H),0.37-0.33(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.55, 155.00, 139.09, 122.49, 75.74, 71.07, 46.02, 45.68, 37.84,37.44,36.41,34.94,30.85,25.72,25.06,24.95,22.90,20.50,18.23,17.08.[M+H] + Calcd for C 20 H 30 NO4:348.2175,Found:348.2169.

[0051] Example 14: Preparation of Compound 14

[0052]

[0053] The preparation of compound 14 refers to compound 8. Pale yellow liquid, yield is 72.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.26 (d, J = 2.8Hz, 1H), 5.57 (d, J = 2.4Hz, 1H), 4.87-4 .83(m,1H),4.81-4.78(m,1H),3.67(s,4H),3.47(s,4H),3.19-3.15(m,1H),2.40- 2.34(m,2H),1.75-1.71(m,1H),1.60-1.56(m,1H),1.37-1.32(m,2H),1.25(d,J= 6.2Hz,3H),1.09(s,3H),1.00-0.93(m,2H),0.48-0.45(m,1H),0.38-0.34(m,1H).13 C NMR (126MHz, CDCl3) δ (ppm): 170.50, 153.47, 140.81, 122.50, 74.92, 70.88, 68.52, 43.99, 37.77,37.37,36.27,34.76,31.42,30.79,30.17,24.98,22.87,20.27,18.22,17.06.[M+H] + Calcd for C 20 H 30 NO5,364.2124,Found:364.2113.

[0054] Example 15: Preparation of Compound 15

[0055]

[0056] The preparation of compound 15 refers to compound 8. Pale yellow liquid, yield is 65.0%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.24 (d, J = 2.8Hz, 1H), 5.56 (s, 1H), 4.84-4.76 (m, 2H),3.49(s,4H),3.19-3.13(m,1H),2.39-2.36(m,4H),2.36-2.33(m,2H),2.31 (s,1H),1.75-1.68(m,1H),1.61-1.54(m,1H),1.38-1.29(m,2H),1.23(d,J=6. 2Hz,3H),1.07(s,3H),0.99-0.89(m,2H),0.47-0.43(m,1H),0.36-0.32(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.57, 155.20, 139.04, 122.56, 75.71, 71.78, 54.74, 50.75, 46.16,43.52,37.81,37.40,36.30,34.84,30.83,25.01,22.88,20.32,18.24,17.08.[M+H] + Calcd for C 21 H 33 N2O4:377.2440,Found:377.2450.

[0057] Example 16: Preparation of Compound 16

[0058]

[0059] The preparation of compound 16 refers to compound 8. Colorless liquid, yield 54.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.25 (d, J = 2.9Hz, 1H), 5.57 (s, 1H), 5.24 (s, 1H), 4.82-4. 77(m,2H),3.29(d,J=6.0Hz,2H),3.19-3.14(m,1H),2.48(t,J=6.0Hz,2H),2.38-2.31 (m,2H),2.29(s,6H),1.73-1.68(m,1H),1.60-1.55(m,1H),1.38-1.30(m,2H),1.22(d ,J=6.2Hz,3H),1.08(s,3H),1.00-0.90(m,2H),0.46-0.44(m,1H),0.37-0.34(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.61, 156.53, 139.06, 122.55, 75.76, 71.10, 58.28, 50.74, 45.08,38.08,37.82,37.40,36.28,34.87,30.83,25.01,22.86,20.31,18.23,17.07.[M+H] + Calcd for C 20 H 33 N2O4:365.2440,Found:365.2451.

[0060] Example 17: Preparation of Compound 17

[0061]

[0062] The preparation of compound 17 refers to compound 8. Pale yellow oil, yield 61.7%. 1H NMR (500MHz, CDCl3) δ (ppm): 6.26 (d, J = 2.8Hz, 1H), 5.57 (d, J = 2.4Hz, 1H), 4.83-4.77 (m, 2H),3.40-3.35(m,2H),3.19-3.14(m,1H),2.92(d,J=12.1Hz,3H),2.48-2.43(m,2H),2.3 8-2.33(m,2H),2.28(s,6H),1.76-1.69(m,1H),1.62-1.56(m,1H),1.39-1.30(m,2H),1.2 4(d,J=6.2Hz,3H),1.08(s,3H),1.00-0.90(m,2H),0.47-0.44(m,1H),0.37-0.33(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 169.78, 156.24, 139.15, 121.36, 75.83, 71.72, 57.38, 56.86, 50. 79,46.87,45.78,37.91,37.50,36.05,34.97,30.92,25.14,22.62,20.45,18.33,17.17.[M+H] + Calcd for C 21 H 35 N2O4:379.2597,Found:379.2606.

[0063] Example 18: Preparation of Compound 18

[0064]

[0065] The preparation of compound 18 refers to compound 8. Pale yellow liquid, yield 60.1%. 1H NMR (500MHz, CDCl3) δ (ppm): 7.35-7.34 (m, 2H), 7.31-7.29 (m, 3H), 6.26 (d, J = 2.8Hz, 1H), 5. 57(s,1H),4.94(s,1H),4.86(q,J=6.3Hz,1H),4.83-4.77(m,1H),4.39(d,J=4.7Hz,2H),3.1 9-3.14(m,1H),2.39-2.3(m,2H),1.73-1.67(m,1H),1.61-1.55(m,1H),1.38-1.30(m,2H),1 .25(d,J=6.2Hz,3H),1.08(s,3H),1.00-0.90(m,2H),0.48-0.44(m,1H),0.41-0.32(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.57, 156.41, 139.07, 138.67, 128.67, 127.52, 127.47, 122.54, 7 5.72,71.40,45.00,37.81,37.40,36.29,34.83,30.83,25.03,22.88,20.33,18.25,17.08.[M+H] + Calcd for C 23 H 30 NO4:384.2175,Found:384.2203.

[0066] Example 19: Preparation of Compound 19

[0067]

[0068] Compound 2 (100 mg, 0.4 mmol) was dissolved in dichloromethane (2.5 mL), p-methoxyphenyl isocyanate (90 mg, 0.6 mmol) and DIPEA (102 mg, 0.8 mmol) were added thereto, and the reaction was carried out at room temperature (20°C) for 5 h. The reaction was completed after TLC monitoring. Water (10 mL) was added to the reaction solution, and ethyl acetate was extracted (10 mL×3). The organic layers were combined, washed with saturated sodium chloride solution (8 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The target product was purified by 200-300 mesh silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 to 3:1) to obtain a white solid with a yield of 67.2%. 1H NMR (500MHz, CDCl3) δ (ppm): 7.31 (s, 2H), 6.87 (d, J = 9.0Hz, 2H), 6.45 (s, 1H), 6.25 (d, J = 2. 9Hz,1H),5.55(d,J=2.4Hz,1H),4.92(q,J=6.3Hz,1H),4.83-4.77(m,1H),3.81(s,3H),3.19 -3.13(m,1H),2.39-2.31(m,2H),1.78-1.72(m,1H),1.67-1.63(m,1H),1.44-1.34(m,2H),1 .29(d,J=6.3Hz,3H),1.09(s,3H),1.02-0.94(m,2H),0.50-0.46(m,1H),0.39-0.35(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.58, 155.88, 153.68, 139.07, 131.15, 122.56, 120.42, 114.25, 7 5.73,71.71,55.52,37.76,37.36,36.22,34.73,30.80,25.08,22.89,20.31,18.27,17.08.[MH] - Calcd for C 23 H 28 NO5:398.1967,Found:398.1959.

[0069] Example 20: Preparation of Compound 20

[0070]

[0071] The preparation of compound 20 refers to compound 19. White solid, yield 72.4%. 1H NMR (500MHz, CDCl3) δ (ppm): 7.21 (t, J = 8.1Hz, 1H), 7.15 (s, 1H), 6.87 (d, J = 10.6Hz, 1H), 6.63 (dd, J = 8.3 ,2.5Hz,1H),6.58(s,1H),6.25(d,J=2.8Hz,1H),5.55(d,J=2.4Hz,1H),4.95-4.91(m,1H),4.83-4.78(m ,1H),3.82(s,3H),3.20-3.14(m,1H),2.39-2.32(m,2H),1.80-1.73(m,1H),1.67-1.64(m,1H),1.46-1. 32(m,2H),1.30(d,J=6.3Hz,3H),1.09(s,3H),1.02-0.91(m,2H),0.50-0.46(m,1H),0.39-0.35(m,1H). 13 C NMR(126MHz, CDCl3)δ(ppm):170.52,160.33,153.18,139.30,139.08,129.75,122.52,110.72,109.09,1 04.28,75.68,71.90,55.28,37.75,37.36,36.19,34.70,30.79,25.09,22.92,20.28,18.28,17.10.[MH] - Calcd for C 23 H 28 NO5:398.1967,Found:398.1962.

[0072] Example 21: Preparation of Compound 21

[0073]

[0074] The preparation of compound 21 refers to compound 19. White solid, yield 65.3%. 1H NMR (500MHz, CDCl3) δ (ppm): 7.35 (s, 2H), 7.02 (t, J=8.6Hz, 2H), 6.53 (s, 1H), 6.25 (d, J= 2.9Hz,1H),5.55(d,J=2.5Hz,1H),4.92(q,J=6.3Hz,1H),4.83-4.78(m,1H),3.20-3.15( m,1H),2.39-2.32(m,2H),1.80-1.73(m,1H),1.67-1.63(m,1H),1.43-1.35(m,2H),1.30 (d,J=6.2Hz,3H),1.09(s,3H),1.03-0.93(m,2H),0.50-0.46(m,1H),0.39-0.35(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.57, 159.86, 157.93, 153.46, 139.08, 134.05, 122.57, 120.25, 115.7 5,115.57,75.70,71.99,37.72,37.33,36.18,34.63,30.77,25.07,22.90,20.26,18.29,17.08.[MH] - Calcd for C 22 H 25 FNO4:386.1768,Found:368.1753.

[0075] Example 22: Preparation of Compound 22

[0076]

[0077] The preparation of compound 22 refers to compound 19. White solid, yield 62.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 7.35 (d, J = 8.1Hz, 2H), 7.27 (d, J = 8.7Hz, 2H), 6.68 (s ,1H),6.24(d,J=2.8Hz,1H),5.55(d,J=2.2Hz,1H),4.92(q,J=6.3Hz,1H),3.19-3 .14(m,1H),2.38-2.31(m,2H),1.79-1.72(m,1H),1.67-1.62(m,1H),1.29(d,J=6 .3Hz,4H),1.08(s,3H),1.02-0.92(m,2H),0.49-0.45(m,1H),0.37-0.34(m,1H). 13C NMR (126MHz, CDCl3) δ (ppm): 170.58, 153.18, 139.07, 136.70, 129.03, 128.25, 122.59, 119.7 6,75.71,72.13,37.70,37.31,36.15,34.59,30.75,25.06,22.89,20.25,18.29,17.07.[MH] - Calcd for C 22 H 25 ClNO4:402.1472,Found:402.1474.

[0078] Example 23: Preparation of Compound 23

[0079]

[0080] The preparation of compound 23 refers to compound 19. White solid, yield 74.6%. 1 H NMR (500MHz, CDCl3) δ (ppm): 7.27 (s, 2H), 7.13 (d, J = 8.2Hz, 2H), 6.49 (s, 1H), 6.25 (d, J = 2. 9Hz,1H),5.55(d,J=2.5Hz,1H),4.92(q,J=6.3Hz,1H),4.83-4.78(m,1H),3.20-3.15(m,1H) ,2.39-2.34(m,2H),2.32(s,3H),1.80-1.74(m,1H),1.67-1.63(m,1H),1.43-1.36(m,2H),1 .30(d,J=6.3Hz,3H),1.09(s,3H),1.02-0.91(m,2H),0.50-0.46(m,1H),0.39-0.36(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.56, 153.43, 139.07, 135.43, 132.90, 129.54, 122.54, 118.69, 7 5.71,71.73,37.77,37.37,36.22,34.74,30.80,25.09,22.91,20.73,20.30,18.27,17.09.[MH] - Calcd for C 22 H 28 NO4:382.2018,Found:382.2007.

[0081] Example 24: Preparation of Compound 24

[0082]

[0083] Triphosgene (71 mg, 0.24 mmol) was dissolved in dichloromethane (3 mL), and compound 2 (100 mg, 0.4 mmol) and triethylamine (122 mg, 1.2 mmol) were added thereto. After stirring at room temperature (20°C) for 5 min, 4-amino-N,N-dimethylaniline (82 mg, 0.6 mmol) was added, and the reaction was carried out at room temperature for 4 h. The reaction was completed after TLC monitoring. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL×3). The organic layers were combined, washed with saturated sodium chloride solution (8 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The filtrate was purified by 200-300 mesh silica gel column chromatography (dichloromethane: methanol = 100:1 to 50:1) to obtain a light yellow solid in a yield of 23.2%. 1 H NMR (500MHz, CDCl3) δ (ppm): 7.25 (s, 2H), 6.73 (d, J = 8.4Hz, 2H), 6.39 (s, 1H), 6.25 (d, J = 2. 8Hz,1H),5.55(d,J=2.5Hz,1H),4.95-4.87(m,1H),4.83-4.77(m,1H),3.20-3.13(m,1H),2 .93(s,6H),2.39-2.32(m,2H),1.79-1.72(m,1H),1.66-1.62(m,1H),1.42-1.37(m,2H),1. 29(d,J=6.2Hz,3H),1.09(s,3H),1.01-0.93(m,2H),0.50-0.46(m,1H),0.39-0.35(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.58, 153.81, 142.28, 139.07, 136.79, 122.55, 120.66, 113.54, 7 5.74,71.53,41.18,37.79,37.39,36.26,34.80,30.82,25.09,22.90,20.33,18.27,17.09.[MH] - Calcd for C 24 H 31 N2O4:411.2284,Found:411.2266.

[0084] Example 25: Preparation of Compound 25

[0085]

[0086] The preparation of compound 25 refers to compound 24. Pale yellow solid, yield 26.5%. 1 H NMR (500MHz, CDCl3) δ (ppm): 7.30 (s, 2H), 6.91 (d, J = 9.0Hz, 2H), 6.42 (s, 1H), 6.25 (d, J = 2.9Hz, 1H), 5.55(d,J=2.4Hz,1H),4.92(q,J=6.4Hz,1H),4.83-4.77(m,1H),3.24(t,J=5.0Hz,4H),3.20-3.13(m ,1H),2.71(s,4H),2.45(s,3H),2.39-2.31(m,2H),1.78-1.72(m,1H),1.67-1.61(m,1H),1.38-1.34 (m,2H),1.28(d,J=4.0Hz,3H),1.09(s,3H),1.02-0.94(m,2H),0.50-0.67(m,1H),0.39-0.35(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.60, 153.66, 147.27, 139.06, 131.34, 122.57, 120.12, 117.28, 75.74, 7 1.66,55.64,49.24,45.55,37.75,37.36,36.21,34.72,30.79,25.07,22.89,20.30,18.27,17.07.[MH] - Calcd for C 27 H 36 N3O4:466.2706,Found:466.2703.

[0087] Example 26: Preparation of Compound 26

[0088]

[0089] The preparation of compound 26 refers to compound 24. White solid, yield 53.2%. 1H NMR (500MHz, CDCl3) δ (ppm): 8.07 (s, 1H), 7.82 (s, 1H), 6.74 (d, J = 8.8Hz, 1H), 6.45 (s, 1H), 6.2 5(d,J=2.9Hz,1H),5.56(d,J=2.5Hz,1H),4.92(q,J=6.3Hz,1H),4.83-4.78(m,1H),3.93(s,3H) ,3.20-3.15(m,1H),2.42-2.32(m,2H),1.80-1.73(m,1H),1.67-1.64(m,1H),1.43-1.35(m,2H ),1.30(d,J=6.2Hz,3H),1.09(s,3H),1.03-0.93(m,2H),0.50-0.46(m,1H),0.39-0.35(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.56, 160.70, 153.77, 139.07, 137.47, 131.29, 128.73, 122.58, 110. 70,75.70,72.19,53.56,37.72,37.33,36.17,34.63,30.77,25.04,22.89,20.24,18.28,17.08.[MH] - Calcd for C 22 H 29 N2O5:399.1920,Found:399.1918.

[0090] Example 27: Preparation of Compound 27

[0091]

[0092] Synthesis of intermediate I-1: Compound 2 (500 mg, 2 mmol) was dissolved in anhydrous THF (8 mL), and bis(tert-butyloxycarbonyl)amine (651.8 mg, 3 mmol) and triphenylphosphine (786.9 mg, 3 mmol) were added thereto under N2 protection. DIAD (606.6 mg, 3 mmol) was slowly added dropwise at 0°C, and the reaction was carried out at room temperature (20°C) for 1.5 h. The reaction of compound 2 was completed after TLC monitoring. Water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL×3). The organic layers were combined, washed with saturated sodium chloride solution (18 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The mixture was purified by 200-300 mesh silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 to 5:1) to obtain 360 mg of a white solid with a yield of 40.1%. 1H NMR (500MHz, CDCl3) δ (ppm): 6.25 (d, J = 2.5Hz, 1H), 5.57 (d, J = 1.8Hz, 1H), 4.82 -4.77(m,1H),4.28-4.21(m,1H),3.19-3.14(m,1H),2.40-2.31(m,2H),2.00-1 .92(m,1H),1.51(s,18H),1.39-1.34(m,1H),1.30(d,J=6.8Hz,3H),1.27-1.21 (m,1H),1.08(s,3H),0.99-0.89(m,2H),0.46-0.43(m,1H),0.37-0.33(m,1H).

[0093] Synthesis of intermediate I-2: The intermediate I-1 (200 mg, 0.45 mmol) was dissolved in dichloromethane (2 ml), trifluoroacetic acid (0.5 ml) was added thereto, and the reaction was carried out at room temperature (20°C) for 2 h. The reaction of the raw material was completed after TLC monitoring. The reaction solution was concentrated under reduced pressure and the solvent was removed to obtain 140 mg of a yellow oil with a yield of 89.7%. It was directly used in the next step without purification.

[0094]

[0095] Synthesis of compound 27: The intermediate I-2 (100 mg, 0.29 mmol) was dissolved in dichloromethane (2 ml), the corresponding acid chloride (0.44 mmol) was added thereto, DIPEA (148 mg, 1.16 mmol) was added dropwise, and the reaction was carried out at room temperature (20°C) for 2 h. The reaction of the intermediate was completed after TLC monitoring. Water (5 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL×3). The organic layers were combined, washed with saturated sodium chloride solution (10 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The residue was purified by 200-300 mesh silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 to 3:1) to give a white solid in a yield of 63.6%. 1H NMR (500MHz, CDCl3) δ (ppm): 7.73 (d, J = 8.5Hz, 2H), 6.94 (d, J = 8.4Hz, 2H), 6.25 (d, J = 2.8Hz, 1H), 5.77 ( d,J=10.0Hz,1H),5.57(d,J=2.4Hz,1H),4.82-4.76(m,1H),4.28-4.22(m,1H),3.87(m,3H),3.20-3.14( m,1H),2.42-2.37(m,1H),2.32(dd,J=13.7,6.1Hz,1H),1.68-1.65(m,2H),1.52-1.44(m,1H),1.37-1. 32(m,1H),1.26(d,J=6.6Hz,3H),1.08(s,3H),1.01-0.93(m,2H),0.48-0.45(m,1H),0.40-0.37(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.60, 166.39, 162.06, 139.08, 128.58, 127.22, 122.57, 113.72, 7 5.75,55.42,45.31,37.75,37.38,37.27,34.74,30.83,25.68,23.00,21.44,18.35,16.88.[M+H] + Calcd for C 23 H 30 NO4:384.2175,Found:384.2194.

[0096] Example 28: Preparation of Compound 28

[0097]

[0098] The preparation method of compound 28 refers to compound 27. White solid, yield 59.7%. 1H NMR (500MHz, CDCl3) δ (ppm): 7.64 (d, J = 8.3Hz, 2H), 7.23 (d, J = 7.9Hz, 2H), 6.23 (d, J = 2.9Hz, 1H), 5.80 ( d,J=8.5Hz,1H),5.55(d,J=2.5Hz,1H),4.79-4.74(m,1H),4.26-4.21(m,1H),3.18-3.11(m,1H),2.39( s,3H),2.37-2.34(m,1H),2.30(dd,J=13.7,6.1Hz,1H),1.66-1.62(m,2H),1.50-1.43(m,1H),1.34-1. 30(m,1H),1.24(d,J=6.6Hz,3H),1.05(s,3H),0.98-0.89(m,2H),0.46-0.42(m,1H),0.38-0.34(m,1H). 13 C NMR(126MHz, CDCl3)δ(ppm):170.59,166.81,141.74,139.08,132.09,129.21,126.77,122.5 6,75.74,45.32,37.76,37.38,37.27,34.72,30.82,25.66,23.03,21.42,18.35,16.89.[M+H] + Calcd for C 23 H 30 NO3:368.2226,Found:368.2245.

[0099] Example 29: Preparation of Compound 29

[0100]

[0101] The preparation method of compound 29 refers to compound 27. White solid, yield is 63.1%. 1H NMR (500MHz, CDCl3) δ (ppm): 7.78 (dd, J = 8.7, 5.3Hz, 2H), 7.08 (t, J = 8.6Hz, 2H), 6.21 (d, J = 2.8Hz, 1H ),6.17(d,J=8.4Hz,1H),5.55(d,J=2.4Hz,1H),4.79-4.74(m,1H),4.23-4.18(m,1H),3.17-3.11(m,1 H),2.39-2.34(m,1H),2.28(dd,J=13.7,6.1Hz,1H),1.65-1.61(m,2H),1.48-1.41(m,1H),1.33-1.29 (m,1H),1.23(d,J=6.6Hz,3H),1.05(s,3H),0.96-0.88(m,2H),0.44-0.41(m,1H),0.37-0.33(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.63, 165.91, 165.58, 163.58, 139.07, 131.12, 131.09, 129.23, 129.15, 122.6 0,115.56,115.39,75.78,45.57,37.71,37.36,37.10,34.69,30.80,25.69,23.00,21.26,18.33,16.87.[M+H] + Calcdfor C 22 H 27 FNO3:372.1975Found:372.2002.

[0102] Example 30: Preparation of Compound 30

[0103]

[0104] 4-Morpholinemethylbenzoic acid (97 mg, 0.44 mmol) was dissolved in dichloromethane (2 ml), HATU (165.3 mg, 0.44 mmol) was added thereto, DIPEA (148 mg, 1.16 mmol) was added dropwise, and the mixture was stirred at room temperature (20°C) for 10 min. Intermediate 9 (100 mg, 0.29 mmol) was added thereto, and the reaction was continued at room temperature (20°C) for 2 h. The reaction of the intermediate was completed after TLC monitoring. Water (5 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL×3). The organic layers were combined, washed with saturated sodium chloride solution (10 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The product was purified by 200-300 mesh silica gel column chromatography (dichloromethane: methanol = 100: 1 to 30: 1) to give a white solid in a yield of 58.2%. 1 H NMR (500MHz, CDCl3) δ (ppm): 7.72 (d, J = 8.3Hz, 2H), 7.41 (d, J = 7.9Hz, 2H), 6.24 (d, J = 2.9Hz, 1H), 5.90 (d, J = 8.5 Hz,1H),5.57(d,J=2.5Hz,1H),4.81-4.76(m,1H),4.28-4.22(m,1H),3.72(t,J=4.6Hz,4H),3.54(s,2H),3.20-3 .13(m,1H),2.45(s,4H),2.42-2.36(m,1H),2.31(dd,J=13.7,6.1Hz,1H),1.67-1.62(m,2H),1.52-1.46(m,1H), 1.34-1.30(m,1H),1.26(d,J=6.6Hz,3H),1.07(s,3H),0.98-0.89(m,2H),0.47-0.44(m,1H),0.40-0.36(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.55, 166.68, 141.60, 139.09, 133.86, 129.21, 126.80, 122.55, 75.71, 6 6.97,62.94,53.62,45.39,37.74,37.37,37.27,34.69,30.81,25.68,23.05,21.40,18.37,16.89.[MH] - Calcd for C 27 H 35 N2O4:451.2597,Found:451.2599.

[0105] Example 31: Preparation of Compound 31

[0106]

[0107] The preparation of compound 31 refers to compound 30. White solid, yield 48.9%. 1 H NMR (500MHz, CDCl3) δ (ppm): 7.68 (d, J = 8.9Hz, 2H), 6.90 (d, J = 8.9Hz, 2H), 6.25 (d, J = 2.9Hz, 1H), 5.75 (d, J = 8.5Hz ,1H),5.56(d,J=2.5Hz,1H),4.81-4.75(m,1H),4.28-4.21(m,1H),3.32(t,J=5.1Hz,4H),3.19-3.12(m,1H),2.59( t,J=5.0Hz,4H),2.42-2.39(m,1H),2.33-2.29,2.31(dd,J=13.7,6.1Hz,1H),1.66-1.61(m,2H),1.51-1.44(m,1H ),1.36-1.28(m,1H),1.24(d,J=6.6Hz,3H),1.07(s,3H),1.00-0.90(m,2H),0.47-0.44(m,1H),0.39-0.35(m,1H). 13 CNMR(126MHz, CDCl3)δ(ppm):170.56,166.45,153.24,139.11,128.15,124.70,122.51,114.35,75.73,5 4.82,47.90,46.11,45.14,37.77,37.39,37.39,34.77,30.84,25.66,23.03,21.53,18.35,16.89.[M+Cl] - Calcd for C 27 H 37 ClN3O3:486.2523,Found:286.2527.

[0108] Example 32: Preparation of Compound 32

[0109]

[0110] The preparation of compound 32 refers to compound 31. Pale yellow liquid, yield is 64.7%. 1H NMR (500MHz, CDCl3) δ (ppm): 6.87 (d, J = 8.8Hz, 1H), 6.25 (q, J = 2.6Hz, 1H), 5.57 (t, J = 2.1Hz, 1H) ,4.82-4.76(m,1H),4.06-4.01(m,1H),3.73(s,4H),3.17(q,J=9.6Hz,1H),3.00(s,2H),2.54(s, 4H),2.41-2.36(m,1H),2.34-2.30(m,1H),1.59-1.53(m,2H),1.41-1.35(m,1H),1.28-1.23(m,1 H),1.16(d,J=6.6Hz,3H),1.07(s,3H),1.01-0.93(m,2H),0.46-0.42(m,1H),0.38-0.34(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.51, 168.93, 139.06, 122.56, 75.65, 66.94, 62.10, 53.86, 50.84,44.40,37.74,37.35,37.12,34.60,30.78,25.71,23.08,21.32,18.33,16.88.[M+H] + Calcd for C 21 H 33 N2O4:377.2440,Found:377.2468.

[0111] Example 33: Preparation of Compound 33

[0112]

[0113] The preparation of compound 33 refers to compound 31. Colorless liquid, yield 57.3%. 1H NMR (500MHz, CDCl3) δ (ppm): 6.91 (d, J = 9.1Hz, 1H), 6.23 (d, J = 2.8Hz, 1H), 5.55 (d, J = 2.4Hz, 1H),4.80-4.74(m,1H),4.06-4.00(m,1H),3.17-3.11(m,1H),2.92(s,2H),2.40-2.34(m,1H ),2.32-2.29(m,1H),2.27(s,6H),1.57-1.51(m,2H),1.43-1.38(m,1H),1.26-1.20(m,1H), 1.15(d,J=6.5Hz,3H),1.05(s,3H),0.98-0.89(m,2H),0.44-0.40(m,1H),0.36-0.32(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.62, 169.39, 139.05, 122.62, 75.76, 63.07, 45.84, 44.35,37.78,37.38,37.04,34.71,30.83,25.68,23.04,21.31,18.33,16.88.[MH] - Calcd for C 19 H 19 N2O3:333.2178,Found:333.2192.

[0114] Example 34: Preparation of Compound 34

[0115]

[0116] The intermediate I-2 (100 mg, 0.29 mmol) was dissolved in dichloromethane (2 ml), dimethylaminoformyl chloride (47 mg, 0.44 mmol) was added thereto, and DIPEA (148 mg, 1.16 mmol) was added dropwise. The mixture was reacted at room temperature (20°C) for 3 h. The reaction of the intermediate 9 was completed by TLC monitoring. Water (5 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL×3). The organic layers were combined, washed with saturated sodium chloride solution (10 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The mixture was purified by 200-300 mesh silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 to 2:1) to obtain 59 mg of a white solid with a yield of 63.2%. 1H NMR (500MHz, CDCl3) δ (ppm): 6.24 (d, J = 2.9Hz, 1H), 5.56 (d, J = 2.5Hz, 1H), 4.81-4.76 (m, 1 H),4.08(d,J=8.1Hz,1H),3.91-3.85(m,1H),3.19-3.13(m,1H),2.89(s,6H),2.40-2.34( m,1H),2.33-2.29(m,1H),1.52(t,J=7.1Hz,2H),1.43-1.37(m,1H),1.32-1.25(m,1H),1. 14(d,J=6.5Hz,3H),1.07(s,3H),0.99-0.90(m,2H),0.46-0.42(m,1H),0.39-0.35(m,1H). 13 C NMR (126MHz, CDCl3) δ (ppm): 170.57, 157.97, 139.12, 122.50, 75.76, 46.25, 37. 80,37.77,37.42,36.18,34.91,30.86,25.70,22.92,22.05,18.32,16.92.[MH] - Calcd for C 18 H 27 N2O3:319.2022,Found:319.2040.

[0117] Example 35: Evaluation of in vitro anti-NASH activity of compounds 1-34

[0118] Primary hepatocytes are the main component of the liver structure and the basic unit of liver metabolic disorders. In order to test the in vitro anti-NASH activity of the compounds, a C57 mouse primary hepatocyte NASH model was constructed by inducing palmitic acid (PA, 400μM) and oleic acid (OA, 400μM), and the effects of the test compounds on the expression levels of inflammation-related mRNA in the mouse primary hepatocyte NASH model were measured.

[0119] Experimental methods

[0120] Preparation of oleic acid or palmitic acid solution: Solution A (Vehicle): Dissolve BSA in PBS solution to prepare a 40% BSA solution; Solution B: Use solution A as solvent to prepare 20 mmol / L palmitic acid mother solution (solution B1) and 20 mmol / L oleic acid mother solution (solution B2); Solution C: Mix 1 mL of solution B1 and 1 mL of solution B2, and dilute the mixture to 50 mL with DMEM culture medium to obtain a mixed solution of 400 μmol / L palmitic acid and 400 μmol / L oleic acid; Solution D: Take 2 mL of solution A and dilute it to 50 mL with DMEM culture medium as a control.

[0121] Preparation of test compound solution: Solution E: Prepare the test compound into a 10 mmol / L stock solution with DMSO, and then use Solution C to dilute the compound stock solution to the test concentration.

[0122] Hepatocyte modeling and drug administration: primary hepatocytes were seeded in 6-well plates and cultured for 24 hours to allow them to fully adhere to the wall, the culture medium was aspirated, and the cells were washed once with PBS; different solutions were added to the hepatocytes according to the grouping: control group: 2 mL of solution D was added to each well; model group: 2 mL of solution C was added to each well; drug administration group: 2 mL of solution E was added to each well; after incubation for 24 hours, the supernatant was discarded and the cells were collected for further experiments.

[0123] Experimental Results

[0124] As shown in Table 1, most compounds have good inhibition rates on Il1b and Il6 mRNA in the primary hepatocyte NASH model, among which compounds 3, 5, 14, and 33 have inhibition rates on PO-induced hepatocyte Il1b and Il6 gene expression similar to that of compound 1, while compounds 19 and 20 are better than compound 1.

[0125] Table 1. Inhibition rate of compounds 1 to 34 on Il1b / Il6 mRNA in primary hepatocyte NASH model

[0126]

[0127]

[0128] *Inhibition rate of inflammation-related mRNA in PA- and OA-induced NASH models in primary mouse hepatocytes at 100 nM

[0129] Example 36: In vivo anti-NASH pharmacodynamics study of compound 1

[0130] A high fat, high cholesterol diet (HFHC) can simulate the formation mechanism of human NAFLD / NASH. Long-term HFHC diet can lead to liver fatty degeneration, liver inflammation, and fibrosis in mice.

[0131] Experimental methods

[0132] Establishment and grouping of NASH mouse model induced by HFHC diet:

[0133] Male C57BL / 6J mice (6-8 weeks old) were fed with a high-fat diet (HFD) for 16 weeks. A normal diet (NCD) control group of C57 mice was also set up and fed with ordinary diet. The C57 mice fed with a high-fat diet were divided into 3 groups, with 6 mice in each group, namely the HFD model group, the compound 1 (1 mg / kg / d) group and the compound 1 (10 mg / kg / d) group. The compound 1 (1 mg / kg / d) group and the compound 1 (10 mg / kg / d) group were orally administered with compound 1 (1 mg / kg) or compound 1 (10 mg / kg) daily, and the C57 mice in the NCD group and the HFD model control group were orally administered with solvent control (0.25% CMC-Na) daily for 16 weeks. After the 16-week experiment, blood samples were collected, serum was separated, and frozen at -80°C for future use. The liver was separated, the main lobe was fixed and embedded, and the rest was frozen at -80°C for future use.

[0134] Glucose tolerance and insulin tolerance tests:

[0135] On the first day of the 15th week, after each group of mice had been deprived of food but not water for 12 hours, their fasting plasma glucose was measured as the 0-min starting point, and each group of mice was orally administered glucose (1 g / kg), and their blood sugar was measured at 15 minutes, 30 minutes, 1 hour, and 2 hours, and the data were recorded; on the third day of the 15th week, after each group of mice had been deprived of food but not water for 6 hours, their fasting plasma glucose was measured as the 0-min starting point, and each group of mice was intraperitoneally injected with insulin (0.75 U / kg), and their blood sugar was measured at 15 minutes, 30 minutes, 1 hour, and 2 hours, and the data were recorded.

[0136] Serum biochemical parameters determination:

[0137] Serum triglyceride (TG) and total cholesterol (TC) test: serum samples were added to a 96-well plate, and the corresponding working solution was added and mixed by shaking; incubated at 37°C for 15 min, the absorbance value was detected at 500 nm, and the triglyceride or total cholesterol content was calculated based on the liver tissue weight.

[0138] Serum AST, ALT test: add serum samples to the test wells and control wells respectively, add the corresponding reagents 1 and 2, and finally add NaOH solution, detect the absorbance value at a wavelength of 510nM, and calculate the serum AST, ALT content according to the standard curve.

[0139] Determination of TG and TC content in the liver: Take 50-100 mg of liver tissue sample, add anhydrous ethanol as the homogenization medium at a weight: volume ratio of 1:9, homogenize at 60 Hz for 1 min, centrifuge the obtained homogenate at 2500 rpm for 10 min, get the supernatant, add the supernatant to a 96-well plate, add the corresponding working solution, and shake to mix; incubate at 37°C for 15 min, detect the absorbance at 500 nm, and calculate the triglyceride content based on the weight of the liver tissue.

[0140] Experimental Results

[0141] Table 2. Effects of compound 1 on lipid metabolism in mice fed HFHC diet

[0142]

[0143] Table 3. Effects of compound 1 on mRNAs of liver inflammation, fibrosis and lipid metabolism in mice fed HFHC diet

[0144]

[0145] Table 4. Effects of compound 1 on glucose metabolism in mice fed HFHC diet

[0146]

[0147] As shown in Tables 2-4, the administration of compound 1 effectively reduced the liver damage indicators such as TG and TC in the serum and liver of mice fed a HFHC diet (Table 2); compound 1 also effectively reduced the mRNA levels related to lipid synthesis in the liver of mice fed a HFHC diet, increased the mRNA levels related to fatty acid oxidation, and reduced the mRNA levels related to inflammation and fibrosis markers (Table 3); in addition, the HFHC diet mice that received compound 1 had lower blood glucose levels in OGTT and ITT tests, indicating that compound 1 can improve glucose tolerance and insulin sensitivity in mice fed a HFHC diet (Table 4).

[0148] Example 37: In vivo anti-NASH pharmacodynamics study of compound 20

[0149] The methionine-and choline deficient diet (MCD) is a commonly used feed for establishing NASH models in mice and rats. Due to the lack of choline and methionine that can be converted into choline in the feed, fat in the liver cannot be normally transported into the blood, and will accumulate in liver cells and intercellular spaces to form lipid droplets, leading to fatty liver. In order to verify the therapeutic effect of compound 20 on NASH in vivo, a C57 mouse NASH model induced by a choline and methionine deficient (MCD) diet was constructed.

[0150] Experimental methods

[0151] Establishment of MCD diet-induced NASH mouse model and grouping and drug administration

[0152] Male C57BL / 6J mice (6-8 weeks old) were fed with methionine and choline deficient diet (MCD) for 8 consecutive weeks. A normal diet (NCD) control group of C57 mice was set up and fed with ordinary diet. The C57 mice fed with MCD diet were divided into 3 groups, 6 mice in each group, namely MCD model group, compound 1 (10 mg / kg / d) group, and compound 20 (10 mg / kg / d) group. The C57 mice in each group continued to be fed with MCD diet. The C57 mice in the normal control group continued to be fed with ordinary diet. The compound 1 (10 mg / kg / d) group and the compound 20 (10 mg / kg / d) group were orally administered with compound 1 (10 mg / kg) or compound 20 (10 mg / kg) daily. The C57 mice in the normal control group and the MCD model control group were orally administered with solvent control (0.25% CMC-Na) for 8 consecutive weeks; after the 8-week experiment, blood samples were collected, serum was separated, and stored at -80°C for future use; the liver was separated, the main lobe was fixed and embedded, and the rest was stored at -80°C for future use.

[0153] For other methods, see Example 36.

[0154] Experimental Results

[0155] After administration of compounds 1 and 20, liver injury indicators such as serum transaminase AST and ALT (Table 5) were significantly reduced in mice fed a MCD diet; compounds 1 and 20 also effectively reduced the mRNA levels related to inflammatory and fibrosis markers in mice fed a MCD diet (Table 6).

[0156] Table 5. Effects of Compounds 1 and 20 on AST and ALT in MCD-fed mice

[0157]

[0158] Table 6. Effects of Compounds 1 and 20 on mRNAs of inflammation and fibrosis in the liver of mice fed a MCD diet

[0159]

[0160]

Claims

1. A calaburane sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof, characterized in that: The chemical structural formula of the compound is shown in formula (I): Where: Selected from Formation of C=O, C-OH or X is an O or N atom; R 1 Alkyl, aryl, Where R 2 and R 3 may be the same or different, and are hydrogen, alkyl, cycloalkyl, or aryl; R 2 , R 3 The N atom and the alkyl group can form a 3-9 membered ring structure, which can be substituted by one or more substituents, including hydrogen, alkyl, ester, aryl, alkylaryl, arylalkyl, arylalkenyl, arylalkynyl or heterocyclic group.

2. The calaburane sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound is selected from the following compounds:

3. A pharmaceutical composition comprising a calaburane sesquiterpene lactone derivative according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical composition comprising one or more of the calaburane sesquiterpene lactone derivatives or pharmaceutically acceptable salts thereof as claimed in claim 3 as active ingredients, and a pharmaceutically acceptable carrier, excipient, adjuvant, auxiliary material and / or diluent.

5. Use of a calaburane sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 2 in the preparation of a drug for preventing / treating hepatitis.

6. Use of the pharmaceutical composition according to claim 3 in the preparation of a drug for preventing / treating hepatitis.

7. The use according to claim 5 or 6, characterized in that: The hepatitis is non-alcoholic fatty hepatitis.

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