Calabashin lactone derivatives and medical uses thereof

Abstract

The application discloses a class of calabash terpene derivatives and medical uses thereof. It relates to the field of small molecule drug discovery, and the chemical structural formula of the compound is shown as formula (I). The calabash terpene derivatives in the application can effectively improve the liver injury, liver sugar metabolism disorder, lipid metabolism disorder, liver inflammation and fibrosis degree of NASH mice, and have potential medicinal value.

Description

Technical Field

[0001] This invention relates to the field of small molecule drug discovery, and more specifically, to a class of sematopoietic sesquiterpene lactone derivatives or pharmaceutically acceptable salts thereof and their use in the preparation of drugs for non-alcoholic steatohepatitis. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide, and nonalcoholic steatohepatitis (NASH, also known as metabolic-associated steatohepatitis, MASH) is the most severe histological manifestation of NAFLD. Approximately 20% of NAFLD patients will progress to NASH, which, without intervention, will further progress to cirrhosis and malignant diseases such as liver cancer. Currently, the main treatments for NAFLD are diet control and exercise, but once it progresses to NASH, drug intervention is required. Since NASH patients often have diabetes or hyperlipidemia, hypoglycemic and lipid-lowering drugs are commonly used clinically to control the progression of NASH, such as metformin, statins, and fibrates. However, these drugs cannot fundamentally reverse NASH and inevitably have significant side effects and clinical contraindications. Although new drug development for NASH has been underway for decades, clinical trials have largely encountered setbacks, with only one new drug, Resmetirom, recently receiving FDA approval. Therefore, NASH treatment drugs are quite scarce, and there is a huge unmet clinical need in the market, making it urgent to discover some safe and effective new NASH drugs. Summary of the Invention

[0003] To address the above-mentioned problems, the present invention provides the use of a calabulane-type sesquiterpene lactone derivative as shown in formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of non-alcoholic steatohepatitis.

[0004] The technical solution of this invention is as follows: This invention discloses a class of caraburone sesquiterpene lactone derivatives or pharmaceutically acceptable salts thereof, the chemical structural formula of which is shown in formula (I):

[0005]

[0006] In the formula: Selected from = or - Forming C=O, C-OH or

[0007] X is an O or N atom;

[0008] R 1 alkyl, aryl, Where R 2 and R 3 They can be the same or different, namely hydrogen, alkyl, cycloalkyl, and aryl; R 2 R 3 It can form a 3-9 member ring structure with the N atom, and the ring structure can be substituted by one or more substituents, including hydrogen, alkyl, ester, aryl, alkylaryl, arylalkyl, arylalen, arylynyl or heterocyclic groups.

[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 calabrule-type sesquiterpene lactone derivatives of Formula I as an active ingredient, and a pharmaceutically acceptable carrier, excipient, adjuvant, excipient and / or diluent.

[0012] Another object of the present invention is to provide the use of the aforementioned calabulane-type sesquiterpene lactone derivatives or pharmaceutically acceptable salts, and pharmaceutical compositions thereof, in the preparation of medicaments for the prevention and treatment of non-alcoholic steatohepatitis.

[0013] The beneficial effects of this invention are: the carabuse-type sesquiterpene lactone derivatives in this invention can effectively improve liver damage, liver glucose metabolism disorder, lipid metabolism disorder, liver inflammation and fibrosis in NASH mice, and have potential medicinal value. Detailed Implementation

[0014] To more clearly illustrate the technical solution of the present invention, the technical solution of the present invention will be further described in detail below: Example 1: Preparation of compound 1

[0015] The medicinal material of *Hemiberlesia lataniae* (600g) was immersed in 80% ethanol (6L) and extracted three times by reflux at 95℃ for 1h each time. The filtrate was collected and concentrated under reduced pressure to obtain a dark brown extract. The extract was dissolved in 20% ethanol (200ml) and water (200mL) was added. The extract was extracted with ethyl acetate (300mL×3), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a yellow oily substance. The extract was then purified by rapid silica gel column chromatography with petroleum ether-ethyl acetate gradient elution. Fractions rich in compound 1 were collected, combined, and concentrated to obtain a 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 procedure was the same as that for compound 1. The extract was purified by rapid silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient, and fractions rich in compound 2 were collected, combined, and concentrated to obtain a 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. The mixture was reacted at room temperature (20 °C) for 3 h. The reaction of compound 2 was monitored by TLC until it was complete. 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 under vacuum and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 5:1) to give the product: 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 for compound 4 is the same as that for compound 3. It is a white solid with a yield of 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] For the preparation method of compound 5, please refer to compound 3. It is a colorless liquid with a yield of 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 for compound 6 is the same as that for compound 3. It is a colorless liquid with a yield of 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] For the preparation method of compound 7, please refer to compound 3. It is a pale yellow liquid with a yield of 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. The mixture was reacted at 40 °C for 1.5 h. The reaction was monitored by TLC until the reaction of compound 2 was complete. The reaction solution was cooled to room temperature (20 °C), and methylamine (0.72 mmol) was added. The mixture was reacted at room temperature (20 °C) for 5 h. The reaction was monitored by TLC until the reaction was complete. 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 under vacuum and purified by silica gel column chromatography (200-300 mesh) (petroleum ether:ethyl acetate = 10:1 to 5:1) to give a colorless oil with 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] For the preparation method of compound 9, please refer to compound 8. It is a pale yellow solid with a yield of 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] For the preparation method of compound 10, please refer to compound 8. It is a colorless oil with a yield of 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] For the preparation method of compound 11, please refer to compound 8. It is a pale yellow liquid with a yield of 72.3%. 1 H NMR (500MHz, CDCl3) δ (ppm): 6.26 (d, J = 2.9 Hz, 1H), 5.57 (d, J = 2.4 Hz, 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] For the preparation of compound 12, refer to compound 8. It is a pale yellow liquid with a yield of 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] For the preparation of compound 13, refer to compound 8. It is a pale yellow liquid with a yield of 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] For the preparation of compound 14, refer to compound 8. It is a pale yellow liquid with a yield of 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] For the preparation of compound 15, refer to compound 8. It is a pale yellow liquid with a yield of 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] For the preparation of compound 16, refer to compound 8. It is a colorless liquid with a yield of 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 forC 20 H 33 N2O4:365.2440, Found:365.2451.

[0060] Example 17: Preparation of Compound 17

[0061]

[0062] For the preparation of compound 17, refer to compound 8. It is a pale yellow oil with a yield of 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] For the preparation of compound 18, refer to compound 8. It is a pale yellow liquid with a yield of 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), and p-methoxyphenyl isocyanate (90 mg, 0.6 mmol) and DIPEA (102 mg, 0.8 mmol) were added. The mixture was reacted at room temperature (20 °C) for 5 h. The reaction was monitored by TLC until completion. Water (10 mL) was added to the reaction mixture, 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 under vacuum and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 3:1) to obtain the target product. The product was a white solid with a yield of 67.2%. 1H NMR (500MHz, CDCl3) δ (ppm): 7.31 (s, 2H), 6.87 (d, J = 9.0 Hz, 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] For the preparation of compound 20, refer to compound 19. It is a white solid with a yield of 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 forC 23 H 28 NO5:398.1967, Found:398.1962.

[0072] Example 21: Preparation of compound 21

[0073]

[0074] For the preparation of compound 21, refer to compound 19. It is a white solid with a yield of 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] For the preparation of compound 22, refer to compound 19. It is a white solid with a yield of 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] For the preparation of compound 23, refer to compound 19. It is a white solid with a yield of 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. The mixture was stirred at room temperature (20 °C) for 5 min, and then 4-amino-N,N-dimethylaniline (82 mg, 0.6 mmol) was added. The mixture was reacted at room temperature for 4 h, and the reaction was monitored by TLC until completion. Water (10 mL) was added to the reaction mixture, 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 under vacuum and purified by silica gel column chromatography (200-300 mesh) (dichloromethane:methanol = 100:1 to 50:1) to give a pale yellow solid with 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] For the preparation of compound 25, refer to compound 24. It is a pale yellow solid with a yield of 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 forC 27 H 36 N3O4:466.2706, Found:466.2703.

[0087] Example 26: Preparation of Compound 26

[0088]

[0089] For the preparation of compound 26, refer to compound 24. It is a white solid with a yield of 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. 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. After the reaction of compound 2 was completed by TLC, 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 under vacuum and purified by silica gel column chromatography (200-300 mesh) (petroleum ether:ethyl acetate = 10:1 to 5:1) to give 360 ​​mg of 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: Intermediate I-1 (200 mg, 0.45 mmol) was dissolved in dichloromethane (2 ml), and trifluoroacetic acid (0.5 ml) was added. The reaction was carried out at room temperature (20 °C) for 2 h. The reaction was monitored by TLC until the reaction was complete. The reaction solution was concentrated under reduced pressure to remove the solvent, and 140 mg of yellow oil was obtained with a yield of 89.7%. It was used directly in the next step of the reaction without purification.

[0094]

[0095] Synthesis of compound 27: Intermediate I-2 (100 mg, 0.29 mmol) was dissolved in dichloromethane (2 mL), and the corresponding acyl chloride (0.44 mmol) was added. DIPEA (148 mg, 1.16 mmol) was added dropwise. The reaction was carried out at room temperature (20 °C) for 2 h. The reaction of the intermediate was monitored by TLC until it was complete. 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 under vacuum and purified by silica gel column chromatography (200-300 mesh) (petroleum ether:ethyl acetate = 10:1 to 3:1) to give a white solid in 63.6% yield. 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] For the preparation method of compound 28, please refer to compound 27. It is a white solid with a yield of 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] For the preparation method of compound 29, please refer to compound 27. It is a white solid with a yield of 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 FNO3:372.1975 Found:372.2002.

[0102] Example 30: Preparation of compound 30

[0103]

[0104] 4-Morpholine methylbenzoic acid (97 mg, 0.44 mmol) was dissolved in dichloromethane (2 mL), and HATU (165.3 mg, 0.44 mmol) was added. 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, and the reaction was continued at room temperature (20 °C) for 2 h. The reaction of the intermediate was monitored by TLC until it was completely reacted. 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 under vacuum and purified by silica gel column chromatography (200-300 mesh) (dichloromethane:methanol = 100:1 to 30:1) to obtain a white solid with 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 forC 27 H 35 N2O4:451.2597, Found:451.2599.

[0105] Example 31: Preparation of compound 31

[0106]

[0107] For the preparation of compound 31, refer to compound 30. It is a white solid with a yield of 48.9%. 1 H NMR (500MHz, CDCl3) δ (ppm): 7.68 (d, J = 8.9 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 6.25 (d, J = 2.9 Hz, 1H), 5.75 (d, J = 8.5 Hz ,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] For the preparation of compound 32, refer to compound 31. It is a pale yellow liquid with a yield of 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] For the preparation of compound 33, refer to compound 31. It is a colorless liquid with a yield of 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 forC 19 H 19 N2O3: 333.2178, Found: 333.2192.

[0114] Example 34: Preparation of compound 34

[0115]

[0116] Intermediate I-2 (100 mg, 0.29 mmol) was dissolved in dichloromethane (2 mL), and dimethylcarbamoyl chloride (47 mg, 0.44 mmol) was added. DIPEA (148 mg, 1.16 mmol) was then added dropwise. The reaction was carried out at room temperature (20 °C) for 3 h. TLC was used to monitor the reaction of intermediate I-2 until it was complete. 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 under vacuum and purified by silica gel column chromatography (200-300 mesh) (petroleum ether:ethyl acetate = 10:1 to 2:1) to give 59 mg of 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 the in vitro anti-NASH activity of compounds 1-34

[0118] Primary hepatocytes are the main structural component of the liver and the basic unit for liver metabolic disorders. To test the in vitro anti-NASH activity of compounds, a C57 mouse primary hepatocyte NASH model was constructed by inducing NASH in the primary hepatocytes with palmitic acid (PA, 400 μM) and oleic acid (OA, 400 μM). The effect of the test compounds on the expression level of inflammation-related mRNAs in the mouse primary hepatocyte NASH model was measured.

[0119] Experimental methods

[0120] Preparation of oleic acid or palmitic acid solutions: Solution A (Vehicle): Dissolve BSA in PBS solution to prepare a 40% BSA solution; Solution B: Use solution A as the solvent to prepare a 20 mmol / L palmitic acid stock solution (solution B1) and a 20 mmol / L oleic acid stock solution (solution B2); Solution C: Mix 1 mL of solution B1 and 1 mL of solution B2, and dilute the mixture to 50 mL using DMEM 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 using DMEM medium as a control.

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

[0122] Hepatocyte modeling and drug administration: Primary hepatocytes were seeded in 6-well plates and cultured for 24 h to allow them to adhere fully. The culture medium was aspirated, and the cells were washed once with PBS. Different solutions were added to the hepatocytes according to the groups: control group: 2 mL solution D per well; model group: 2 mL solution C per well; drug administration group: 2 mL solution E per well. After incubation for 24 h, the supernatant was discarded, and the cells were collected for further experiments.

[0123] Experimental results

[0124] As shown in Table 1, most of the compounds showed good inhibition rates of Il1b and Il6 mRNA in the primary hepatocyte NASH model. Among them, compounds 3, 5, 14, and 33 showed similar inhibition rates of PO-induced Il1b and Il6 gene expression in hepatocytes to compound 1, while compounds 19 and 20 were superior to compound 1.

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

[0126]

[0127]

[0128] * Inhibition rate of inflammation-related mRNAs in PA and OA-induced mouse primary hepatocyte NASH model at 100 nM

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

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

[0131] Experimental methods

[0132] Establishment and grouping of HFHC diet-induced NASH mouse model for drug administration:

[0133] Male C57BL / 6J mice (6-8 weeks old) were fed a high-fat diet (HFD) for 16 weeks. A control group of C57 mice fed a normal diet (NCD) was also included. The C57 mice fed the high-fat diet were divided into three groups of six mice each: 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) and compound 1 (10 mg / kg / d) groups were orally administered compound 1 (1 mg / kg) or compound 1 (10 mg / kg) daily. The NCD group and the HFD model control group were orally administered the solvent control (0.25% CMC-Na) daily for 16 weeks. After 16 weeks of experiment, blood samples were collected, serum was separated, and stored at -80℃ for later use. The liver was separated, fixed, and embedded in the primary lobe. The remaining liver was stored at -80℃ for later use.

[0134] Glucose tolerance and insulin tolerance test:

[0135] On the first day of week 15, after 12 hours of fasting without water, fasting plasma glucose was measured as the starting point of 0 min for each group of mice. Each group of mice was orally administered glucose (1 g / kg), and their blood glucose was measured at 15 min, 30 min, 1 h, and 2 h, and the data were recorded. On the third day of week 15, after 6 hours of fasting without water, fasting plasma glucose was measured as the starting point of 0 min for each group of mice. Each group of mice was intraperitoneally injected with insulin (0.75 U / kg), and their blood glucose was measured at 15 min, 30 min, 1 h, and 2 h, and the data were recorded.

[0136] Serum biochemical parameter determination:

[0137] Serum triglyceride (TG) and total cholesterol (TC) tests: Serum samples were added to 96-well plates, the corresponding working solution was added and the plates were shaken to mix; the plates were incubated at 37°C for 15 min, and the absorbance was measured at 500 nm. The triglyceride or total cholesterol content was calculated based on the weight of the liver tissue.

[0138] Serum AST and ALT test: Serum sample was added to the test well and control well respectively, along with the corresponding reagent 1 and reagent 2, and finally NaOH solution was added. The absorbance value was detected at a wavelength of 510 nM, and the serum AST and ALT content was calculated according to the standard curve.

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

[0140] Experimental results

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

[0142]

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

[0144]

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

[0146]

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

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

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

[0150] Experimental methods

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

[0152] Male C57BL / 6J mice (6-8 weeks old) were fed a methionine- and choline-deficient diet (MCD) for 8 consecutive weeks. A control group of C57 mice on a normal diet (NCD) was also included. The C57 mice on the MCD diet were divided into three groups of six mice each: an MCD model group, a compound 1 (10 mg / kg / d) group, and a compound 20 (10 mg / kg / d) group. All C57 mice in these groups continued to be fed the MCD diet. The normal control group continued to be fed a normal diet. The compound 1 (10 mg / kg / d) and compound 20 (10 mg / kg / d) groups received oral administration of either compound 1 (10 mg / kg) or compound 20 (10 mg / kg) daily. The normal control and MCD model control groups received oral 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℃ for later use; liver was separated, fixed and embedded in the primary lobe, and the remainder was stored at -80℃ for later use.

[0153] For the remaining methods, see Example 36.

[0154] Experimental results

[0155] After administration of compounds 1 and 20, serum transaminases AST and ALT (Table 5) and other liver injury indicators were significantly reduced in MCD diet mice; compounds 1 and 20 also effectively reduced the levels of mRNAs associated with inflammation and fibrosis markers in MCD diet mice (Table 6).

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

[0157]

[0158] Table 6. Effects of compounds 1 and 20 on liver inflammation and fibrosis mRNA in MCD-fed mice

[0159]

[0160]

Claims

1. A class of caraburone sesquiterpene lactone derivatives or pharmaceutically acceptable salts thereof, characterized in that, It is selected from the following compounds: .

2. A pharmaceutical composition comprising a carabuse sesquiterpene lactone derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1, and at least one pharmaceutically acceptable carrier.

3. Use of a class of caraburolide sesquiterpene lactone derivatives as described in claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the prevention / treatment of hepatitis.

4. Use of the pharmaceutical composition of claim 2 in the preparation of a medicament for the prevention / treatment of hepatitis.

5. The use as described in claim 3 or 4, characterized in that: The hepatitis mentioned is non-alcoholic steatohepatitis.

Images