Alkyl amide nuciferine derivative with uric acid reduction and kidney protection activity as well as preparation method and application of alkyl amide nuciferine derivative

Abstract

The invention belongs to the field of medicine synthesis, and particularly relates to an alkylamide nuciferine derivative with uric acid reduction and kidney protection activity as well as a preparation method and application of the alkylamide nuciferine derivative. According to the invention, nuciferine is structurally modified by introducing an active group, the biological activity of nuciferine in reduction of uric acid is improved, and cell experiments find that the alkyl amide nuciferine derivative prepared by the invention can inhibit expression of URAT1 in HK-2 cells and can promote excretion of uric acid, so that the level of uric acid is reduced, and the activity of the nuciferine in reduction of uric acid is improved. The important significance is realized on the further development of new uric acid reducing medicines; the alkylamide nuciferine derivative with the uric acid reducing and kidney protecting effects, provided by the invention, is simple in synthesis method, low in raw material price and novel in structure.

Description

Technical Field

[0001] The invention belongs to the field of drug synthesis, and specifically relates to an alkylamide nuciferine derivative having both uric acid-lowering and kidney-protecting activities, and a preparation method and application thereof. Background Art

[0002] Hyperuricemia is a disease caused by purine metabolism disorder in the body, and can cause a series of complications such as gout, hypertension, diabetes, etc. At present, clinical drugs for the treatment of hyperuricemia include allopurinol, probenecid, benzbromarone, etc. These drugs can cause chronic damage to the urinary system including the kidneys, and are prone to the formation of urate crystals and stones.

[0003] Nuciferine is a kind of Nelumbo nucifera Gaertn ) is an alkaloid extracted from the plant. At present, pharmacological studies have shown that it has multiple pharmacological activities such as antioxidant, anti-obesity, anti-inflammatory, anti-atherosclerotic, anti-viral, and anti-tumor. Due to its low toxicity and small side effects, nuciferine has become one of the hot spots in drug research and has broad development prospects.

[0004] Wang Mingxing and others discovered through experiments that nelumbo nucifera in lotus leaves can reduce serum urate and improve renal function. It can also inhibit the production of renal interleukins in mice with hyperuricemia caused by potassium oxonate. It was reported for the first time that nelumbo nucifera can play an anti-inflammatory and anti-hyperuricemia role by regulating organic ion transporters and inflammatory signals in renal hyperuricemia. The study found that daily use of nelumbo nucifera-rich supplements can effectively protect and prevent nephritic hyperuricemia. However, nelumbo nucifera has low uric acid-lowering activity and cannot be directly used in clinical uric acid-lowering drugs. The development of a more effective uric acid-lowering drug has become an urgent problem to be solved. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides an alkylamide nuciferine derivative having both uric acid lowering and kidney protecting activities.

[0006] The present invention also provides a method for preparing the alkylamide nuciferine derivative.

[0007] The present invention further provides the use of the above alkylamide nuciferine derivatives in the preparation of drugs for treating uric acid reduction or protecting kidneys.

[0008] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides an alkylamide nuciferine derivative having both uric acid lowering and kidney protection activities, and its molecular structure is as follows: ; R is , , , , , , , .

[0009] The present invention also provides a method for preparing the above-mentioned alkylamide nuciferine derivative, comprising the following steps: (1) Dissolve nephrine in methanol, then add hydrogen peroxide and stir to react; after the reaction is completed, adjust the pH value of the reaction solution, then add distilled water, extract with chloroform, and purify the chloroform layer with anhydrous Na 2 SO 4 Dry, filter to remove solids, concentrate the filtrate under reduced pressure, separate by silica gel column chromatography, and elute to obtain nuciferine- N - oxides; (2) Under ice bath conditions, N - oxide was dissolved in methanol and then FeSO 4 7H 2 O methanol solution, remove the ice bath and stir to react to obtain a reaction solution; add saturated NaHCO 3 solution, and continue the reaction; after the reaction is completed, add distilled water, extract with chloroform, and use anhydrous Na 2 SO 4 Dry, filter to remove solid, evaporate to dryness under reduced pressure, separate by silica gel column chromatography, and elute to obtain protonuciferine; (3) Dissolve proto-nuciferine and triethylamine in anhydrous DCM, slowly add RX dropwise while maintaining 0°C, and stir to react. After the reaction is completed, quench the reaction, extract the reaction solution with dichloromethane, dry it with anhydrous sodium sulfate, concentrate the filtrate, and obtain the target compound by column chromatography.

[0010] Furthermore, in step (1), the ratio of nuciferine to hydrogen peroxide is 1 mmol:7.5 mL.

[0011] Furthermore, in step (1), the stirring reaction is carried out at room temperature for 24 hours; the pH is adjusted to 10 using a 3M NaOH aqueous solution; and the elution is carried out using an eluent consisting of chloroform: methanol: triethylamine in a volume ratio of 30:1:1‰.

[0012] Furthermore, in step (2), nuciferine- N -Oxide and FeSO 4 7H 2 O has a molar ratio of 1:2; FeSO 4 7H 2 The concentration of methanol solution of O is 1 mol / L; N -oxide and saturated NaHCO3 The ratio of the solution is 1mmol:10mL;.

[0013] Furthermore, in step (2), the stirring reaction is carried out at room temperature for 12 hours; the reaction is continued for 20 minutes; and the elution is carried out using an eluent consisting of chloroform: methanol: triethylamine in a volume ratio of 10:1:1‰.

[0014] Furthermore, in step (3), the molar ratio of proto-nuciferine, triethylamine and RX is 1:2-5:1-2.

[0015] Furthermore, in step (3), the stirring reaction is carried out at room temperature for 10-12 hours; and saturated sodium bicarbonate is used to quench the reaction.

[0016] The present invention also provides the use of the alkylamide nuciferine derivatives in the preparation of uric acid-lowering or kidney-protecting drugs.

[0017] The reaction route for preparing the alkylamide nuciferine derivatives of the present invention is: The beneficial effects of the present invention are: (1) The present invention improves the biological activity of nuciferine in lowering uric acid by introducing active groups to modify the structure of nuciferine. Cell experiments have found that the alkylamide nuciferine derivatives prepared by the present invention can inhibit the expression of URAT1 in HK-2 cells and promote uric acid excretion, thereby reducing uric acid levels, which is of great significance for the further development of new uric acid-lowering drugs; (2) The alkylamide nuciferine derivatives provided by the present invention have uric acid lowering and kidney protection effects, and have a simple synthesis method, low raw material price, and novel structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Hydrogen spectrum of compound 9 prepared in Example 1; Figure 2 Carbon spectrum of compound 9 prepared in Example 1; Figure 3 HRMS pattern of compound 9 prepared in Example 1; Figure 4 Hydrogen spectrum of compound 28 prepared in Example 2; Figure 5 Carbon spectrum of compound 28 prepared in Example 2; Figure 6 HRMS pattern of compound 28 prepared in Example 2; Figure 7 Hydrogen spectrum of compound 11 prepared in Example 3; Figure 8Carbon spectrum of compound 11 prepared in Example 3; Fig. 9 HRMS pattern of compound 11 prepared in Example 3; Fig.10 Hydrogen spectrum of compound 16 prepared in Example 4; Fig.11 Carbon spectrum of compound 16 prepared in Example 4; Fig.12 HRMS pattern of compound 16 prepared in Example 4; Fig.13 Hydrogen spectrum of compound 21 prepared in Example 5; Fig.14 Carbon spectrum of compound 21 prepared in Example 5; Fig.15 HRMS pattern of compound 21 prepared in Example 5; Fig.16 Hydrogen spectrum of compound 3 prepared in Example 6; Fig.17 Carbon spectrum of compound 3 prepared in Example 6; Fig.18 HRMS pattern of compound 3 prepared in Example 6; Fig.19 Hydrogen spectrum of compound 17 prepared in Example 7; Fig. 20 Carbon spectrum of compound 17 prepared in Example 7; Fig.21 HRMS pattern of compound 17 prepared in Example 7; Fig. 22 Hydrogen spectrum of compound 10 prepared in Example 8; Fig.23 Carbon spectrum of compound 10 prepared in Example 8; Fig.24 HRMS pattern of compound 10 prepared in Example 8; Fig.25 Hydrogen spectrum of compound 4 prepared in Example 9; Fig.26 Carbon spectrum of compound 4 prepared in Example 9; Fig. 27 HRMS chart of compound 4 prepared in Example 9. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0020] Unless otherwise specified, the raw materials used in the present invention are commercially available.

[0021] Example 1 (1) Dissolve nephrine (0.295 g, 1 mmol) in 15 mL of methanol, and then add 7.5 mL of 30% H 2 O 2 After the reaction, 3M NaOH aqueous solution was added to the reaction solution to adjust the pH to 10, and then 150 mL of distilled water was added. The solution was extracted with chloroform three times (600 mL × 3). The chloroform layer was washed with anhydrous NaOH. 2 SO 4 Dry, filter to remove solids, and concentrate the filtrate under reduced pressure. Separate by silica gel column chromatography, elute with chloroform: methanol: triethylamine (30:1:1‰ v / v / v), and finally obtain nuciferine- N -Oxide.

[0022] (2) Under ice bath conditions, N -oxide (0.310 g, 1 mmol) was dissolved in 2 mL of methanol, and then FeSO 4 7H 2 O (0.304 g, 2 mmol) in methanol (2 mL). Remove the ice bath and stir the reaction at room temperature for 12 h. Finally, add 10 mL of saturated NaHCO 3 After the reaction, add 90 mL of distilled water to the three-necked flask and extract with chloroform three times (100 mL × 3). 2 SO 4 After drying, the solid was removed by filtration and evaporated to dryness under reduced pressure. The product was separated by silica gel column chromatography and eluted with chloroform: methanol: triethylamine (10:1:1‰) to obtain protonuciferine.

[0023] (3) Dissolve proto-nuciferine (0.10 g, 0.31 mmol) and triethylamine (0.12 mL, 0.86 mmol) in anhydrous DCM (20 mL), slowly add 4-bromobutyryl chloride (0.52 mmol) at 0 °C, and stir the reaction solution overnight at room temperature. After the reaction is completed, quench the reaction with saturated sodium bicarbonate (25 mL), extract the reaction solution with dichloromethane (2×25 mL), and dry it over anhydrous sodium sulfate. Concentrate the filtrate and column chromatography to obtain the target compound 9 as a white powder, mp 242.4 – 243.4 °C.

[0024] The structural formula of compound 9 is: like Figure 1-Figure 3 As shown: 1 H NMR (600 MHz, CDCl3 ) δ 8.48 (s, 1H), 7.45 – 7.13 (m, 3H), 6.71 (s,1H), 5.21 – 4.35 (m, 2H), 3.91 (s, 3H), 3.67 (s, 3H), 3.45 – 3.08 (m, 1H), 3.05 – 2.60 (m, 4H), 1.95 – 1.65 (m, 1H), 1.20 – 0.95 (m, 2H), 0.90 – 0.65(m, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 172.96, 152.27, 145.69, 136.28, 131.82,128.75, 128.54, 128.04, 127.79, 127.43, 126.93, 125.73, 111.63, 60.06, 55.99,52.92, 41.01, 37.03, 29.98, 11.57, 8.17, 7.11. HRMS calculation for C 22 H 24 BrNO 3 : [M-HBr+Na] + 372.1576, found 372.1581. Example 2 Steps (1) and (2) are the same as in Example 1; (3) Dissolve proto-nuciferine (0.10 g, 0.31 mmol) and triethylamine (0.12 mL, 0.86 mmol) in anhydrous DCM (20 mL), slowly add bromoacetyl chloride (0.06 g, 0.38 mmol) at 0 °C, and stir the reaction solution overnight at room temperature. After the reaction is completed, quench the reaction with saturated sodium bicarbonate (25 mL), extract the reaction solution with dichloromethane (2×25 mL), and dry it with anhydrous sodium sulfate. Concentrate the filtrate and column chromatography to obtain the target compound 28 as a white powder, mp 180.9-181.9 °C.

[0025] The structural formula of compound 28 is: like Figure 4-Figure 6 As shown: 1 H NMR (600 MHz, CDCl 3 ) δ 8.44 (t, J= 9.8 Hz, 1H), 7.37 – 7.23 (m, 3H), 6.68 (s, 1H), 5.00 (t, J = 37.1 Hz, 1H), 4.70 – 4.20 (m, 1H), 4.20 – 4.00 (m,2H), 3.91 (s, 3H), 3.67 (s, 3H), 3.40 – 4.10 (m, 1H), 3.10 – 2.67 (m, 4H). 13 C NMR (151 MHz, DMSO) δ 165.51, 152.28, 145.54, 137.00, 131.67,129.87, 128.95, 128.39, 128.19, 127.41, 126.93, 125.58, 112.76, 60.02, 56.29,50.90, 43.08, 41.42, 33.82, 30.29. HRMS calculation for C 20 H 20 NO 3 Br: [M+Na] + 424.0524, found 424.0522. Example 3 Steps (1) and (2) are the same as in Example 1; (3) Dissolve proto-nuciferine (0.10 g, 0.31 mmol) and triethylamine (0.12 mL, 0.86 mmol) in anhydrous DCM (20 mL), slowly add 2-chloropropionyl chloride (0.05 g, 0.39 mmol) at 0 °C, and stir the reaction solution overnight at room temperature. After the reaction is completed, quench the reaction with saturated sodium bicarbonate (25 mL), extract the reaction solution with dichloromethane (2×25 mL), and dry it with anhydrous sodium sulfate. Concentrate the filtrate and column chromatography to obtain the target compound 11 as a white powder, mp 216.8 – 217.8 °C.

[0026] The structural formula of compound 11 is: like Figure 7-Figure 9 As shown: 1 H NMR (600 MHz, DMSO) δ 8.30 (d, J = 7.7 Hz, 1H), 7.33 (dd, J= 16.6,9.1 Hz, 2H), 7.27 (t, J = 7.2 Hz, 1H), 6.92 (s, 1H), 5.21 (dq, J = 18.8, 6.2 Hz,1H), 4.97 – 4.67 (m, 1H), 4.22 (d, J = 9.9 Hz, 1H), 3.84 (s, 3H), 3.60 (s, 3H), 3.23 (t, J = 12.1 Hz, 1H), 3.01 – 2.75 (m, 3H), 1.57 (dd, J = 12.2, 6.4 Hz, 3H). 13 C NMR (151 MHz, CDCl 3 ) δ 166.27, 151.15, 144.81, 135.38, 130.46,127.99, 127.51, 127.38, 126.82, 126.74, 126.03, 124.95, 110.20, 59.02, 54.96,50.42, 49.15, 40.44, 33.05, 29.31, 19.87. HRMS calculation for C 21 H 22 NO 3 Br: [M+Na] + 438.0681, found 438.0684. Example 4 Steps (1) and (2) are the same as in Example 1; (3) Dissolve proto-nuciferine (0.10 g, 0.31 mmol) and triethylamine (0.12 mL, 0.86 mmol) in anhydrous DCM (20 mL), slowly add cyclopentanecarbonyl chloride (0.05 g, 0.38 mmol) at 0 °C, and stir the reaction solution overnight at room temperature. After the reaction is completed, quench the reaction with saturated sodium bicarbonate (25 mL), extract the reaction solution with dichloromethane (2×25 mL), and dry it with anhydrous sodium sulfate. Concentrate the filtrate and column chromatography to obtain the target compound 16 as a white powder, mp 180.3 – 181.3 °C.

[0027] The structural formula of compound 16 is: like Figure 10-12 As shown: 1 H NMR (600 MHz, CDCl 3 ) δ 8.46 (dd, J = 41.1, 7.6 Hz, 1H), 7.44 – 7.18(m, 3H), 6.69 (d, J = 26.7 Hz, 1H), 5.03 (dd, J = 57.7, 10.7 Hz, 1H), 4.44 (dd, J =306.4, 12.9 Hz, 1H), 3.90 (s, 3H), 3.67 (s, 3H), 3.30 – 3.09 (m, 1H), 3.07 –2.96 (m, 1H), 2.94 – 2.61 (m, 4H), 2.10 – 1.45 (m, 8H). 13 C NMR (151 MHz, CDCl 3 ) δ 174.46, 151.99, 145.81, 137.01, 131.56,128.58, 128.34, 127.85, 127.69, 127.42, 126.88, 125.83, 111.20, 60.02, 55.99,50.76, 42.10, 40.93, 33.99, 31.01, 30.14, 29.73, 26.50, 26.03. HRMS calculation for C 24 H 27 NO 3 : [M+Na] + 400.1889, found 400.1891. Example 5 Steps (1) and (2) are the same as in Example 1; (3) Dissolve proto-nuciferine (0.10 g, 0.31 mmol) and triethylamine (0.12 mL, 0.86 mmol) in anhydrous DCM (20 mL), slowly add cyclohexanecarbonyl chloride (0.06 g, 0.41 mmol) at 0°C, and stir the reaction solution overnight at room temperature. After the reaction is completed, quench the reaction with saturated sodium bicarbonate (25 mL), extract the reaction solution with dichloromethane (2×25 mL), and dry it with anhydrous sodium sulfate. Concentrate the filtrate and obtain the target compound 21 by column chromatography as a light yellow powder with mp 157.8 – 158.8°C.

[0028] The structural formula of compound 21 is: like Figure 13-Figure 15 As shown: 1 H NMR (600 MHz, CDCl 3 ) δ 8.34 (d, J = 6.0 Hz, 1H), 7.39 – 7.21 (m, 2H), 7.17 – 7.00 (m, 1H), 6.61 (s, 1H), 5.00 (d, J = 12.5 Hz, 1H), 4.06 (d, J = 10.8Hz, 1H), 3.83 (s, 3H), 3.60 (s, 3H), 3.21 – 3.17 (m, 1H), 2.99 – 2.58 (m,4H), 2.50 – 2.38 (m, 1H), 1.87 – 1.43 (m, 8H), 1.26 – 1.19 (m, 2H). 13 C NMR (151 MHz, DMSO) δ 174.18, 152.12, 145.53, 137.42, 131.75, 130.00, 128.88, 128.35, 128.07, 127.25, 126.98, 126.39, 112.66, 59.99, 56.28,50.18, 34.05, 30.93, 29.76, 29.31, 26.11, 25.72, 25.63. HRMS calculation for C 25 H 29 NO 3 : [M+Na] + 414.2045, found 414.2049. Example 6 Steps (1) and (2) are the same as in Example 1; (3) Dissolve proto-nuciferine (0.10 g, 0.31 mmol) and triethylamine (0.12 mL, 0.86 mmol) in anhydrous DCM (20 mL), slowly add acetoxyacetyl chloride (0.05 g, 0.37 mmol) at 0 °C, and stir the reaction solution overnight at room temperature. After the reaction is completed, quench the reaction with saturated sodium bicarbonate (25 mL), extract the reaction solution with dichloromethane (2×25 mL), and dry it over anhydrous sodium sulfate. Concentrate the filtrate and column chromatography to obtain the target compound 3 as a white powder, mp 163.8 – 164.8 °C.

[0029] The structural formula of compound 3 is: like Figure 16-Figure 18 As shown: 1 H NMR (600 MHz, CDCl 3 ) δ 8.36 (s, 1H), 7.36 – 7.19 (m, 3H), 6.60 (s,1H), 5.12 – 4.82 (m, 1H), 4.80 – 4.70 (m, 2H), 4.63 – 4.37 (m, 1H), 3.83 (s,3H), 3.60 (s, 3H), 3.33 – 2.94 (m, 2H), 2.84 – 2.66 (m, 49.3 Hz, 3H), 2.13 (s, 3H). 13 C NMR (151 MHz, CDCl 3 ) δ 170.70, 164.98, 152.24, 145.95, 136.40,131.43, 128.58, 128.42, 127.98, 127.85, 127.59, 127.06, 125.75, 111.26,62.01, 60.04, 55.98, 50.95, 40.18, 33.95, 30.51, 20.69. HRMS calculation for C 22 H 23 NO 5 : [M+Na] + 404.1474, found 404.1478. Example 7 Steps (1) and (2) are the same as in Example 1; (3) Dissolve proto-nuciferine (0.10 g, 0.31 mmol) and triethylamine (0.12 mL, 0.86 mmol) in anhydrous DCM (20 mL), slowly add ethyl chloroformate (0.06 g, 0.40 mmol) dropwise at 0 °C, and stir the reaction solution overnight at room temperature. After the reaction is completed, quench the reaction with saturated sodium bicarbonate (25 mL), extract the reaction solution with dichloromethane (2×25 mL), and dry it over anhydrous sodium sulfate. Concentrate the filtrate and column chromatography to obtain the target compound 17 as a white powder, mp 126.3 – 127.3 °C.

[0030] The structural formula of compound 17 is: like Figure 19-21 As shown: 1 H NMR (600 MHz, CDCl 3 ) δ 8.45 (dd, J = 26.4, 7.9 Hz, 1H), 7.40 – 7.20(m, 3H), 6.68 (d, J = 24.2 Hz, 1H), 5.13 – 4.92 (m, 1H), 4.30 – 4.13 (m, 2H), 3.96 – 3.86 (m, 4H), 3.67 (s, 3H), 3.63 – 3.44 (m, 2H), 3.33 (t, J = 12.4 Hz,1H), 3.18 – 3.05 (m, 1H), 2.97 – 2.65 (m, 3H), 1.26 (dt, J = 49.0, 7.1 Hz, 3H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.68, 164.64, 152.17, 145.89, 136.59,131.47, 128.63, 128.39, 127.85, 127.79, 127.55, 127.01, 126.09, 111.22,61.56, 60.03, 55.99, 50.96, 42.42, 42.16, 33.72, 30.57, 14.19. HRMS calculation for C 23 H 25 NO 5 : [M+Na]+ 418.1630, found 418.1633. Example 8 Steps (1) and (2) are the same as in Example 1; (3) Dissolve proto-nuciferine (0.10 g, 0.31 mmol) and triethylamine (0.12 mL, 0.86 mmol) in anhydrous DCM (20 mL), slowly add ethyl oxalyl chloride (0.05 g, 0.37 mmol) at 0 °C, and stir the reaction solution overnight at room temperature. After the reaction is completed, quench the reaction with saturated sodium bicarbonate (25 mL), extract the reaction solution with dichloromethane (2×25 mL), and dry it with anhydrous sodium sulfate. Concentrate the filtrate and column chromatography to obtain the target compound 10 as a white powder, mp 167.7 – 168.7 °C.

[0031] The structural formula of compound 10 is: like Figure 22-Figure 24 As shown: 1 H NMR (600 MHz, CDCl 3 ) δ 8.44 (d, J = 8.0 Hz, 1H), 7.37 – 7.31 (m, 1H), 7.30 – 7.22 (m, 2H), 6.68 (d, J = 17.7 Hz, 1H), 5.07 – 4.75 (m, 1H), 4.43 –4.37 (m, 1H), 4.33 – 4.18 (m, 1H), 3.93 – 3.84 (m, 4H), 3.67 (d, J = 4.9 Hz,3H), 3.38 (td, J = 12.8, 2.2 Hz, 1H), 3.17 – 3.06 (m, 1H), 3.06 – 2.95 (m, 1H), 2.95 – 2.81 (m, 1H), 2.78 – 2.66 (m, 1H), 1.34 (dt, J = 87.6, 7.2 Hz, 3H). 13 C NMR (151 MHz, CDCl 3) δ 162.91, 161.11, 152.42, 146.04, 135.99,131.39, 128.67, 128.49, 127.93, 127.83, 127.43, 127.21, 124.96, 111.39,62.22, 60.06, 56.00, 50.48, 42.06, 33.82, 30.45, 14.07. HRMS calculation for C 22 H 23 NO 5 : [M+Na] + 404.1474, found 404.1476. Example 9 Steps (1) and (2) are the same as in Example 1; (3) Dissolve proto-nuciferine (0.10 g, 0.31 mmol) and triethylamine (0.12 mL, 0.86 mmol) in anhydrous DCM (20 mL), slowly add 2-bromopropionyl chloride (0.06 g, 0.35 mmol) at 0 °C, and stir the reaction solution overnight at room temperature. After the reaction is completed, quench the reaction with saturated sodium bicarbonate (25 mL), extract the reaction solution with dichloromethane (2×25 mL), and dry it with anhydrous sodium sulfate. Concentrate the filtrate and column chromatography to obtain the target compound 4 as a white powder, mp 148.7-149.7 °C.

[0032] The structural formula of compound 4 is: like Figure 25-27 As shown: 1 H NMR (600 MHz, DMSO) δ 8.30 (d, J = 7.7 Hz, 1H), 7.33 (dd, J = 16.6,9.1 Hz, 2H), 7.27 (t, J = 7.2 Hz, 1H), 6.92 (s, 1H), 5.21 (dq, J = 18.8, 6.2 Hz,1H), 4.97 – 4.67 (m, 1H), 4.22 (d, J = 9.9 Hz, 1H), 3.84 (s, 3H), 3.60 (s, 3H), 3.23 (t, J= 12.1 Hz, 1H), 3.01 – 2.75 (m, 3H), 1.57 (dd, J = 12.2, 6.4 Hz, 3H). 13 C NMR (151 MHz, CDCl 3 ) δ 166.27, 151.15, 144.81, 135.38, 130.46,127.99, 127.51, 127.38, 126.82, 126.74, 126.03, 124.95, 110.20, 59.02, 54.96,50.42, 49.15, 40.44, 33.05, 29.31, 19.87. HRMS calculation for C 21 H 22 NO 3 Br: [M+Na] + 438.0681, found 438.0684. Effect verification 1. Cell culture and drug administration Cell passaging: When HK-2 cells grow to a density of 85%-90%, the cells are processed for cell passaging. Place the cells in a clean bench, discard the old culture medium with a pipette, add 4.0 mL of PBS to wash, discard the PBS, and repeat 2-3 times. Use a pipette to add 1.0 mL of 0.25% trypsin to digest for 40 seconds, remove the trypsin with a pipette, add culture medium and repeatedly blow until it becomes a single cell suspension. Take 1.0 mL of cell suspension and transfer it to a new culture dish, add 7 mL of culture medium, shake well, and transfer the culture dish to a cell culture incubator after microscopic observation.

[0033] Uric acid induction and administration. When the cells were successfully revived and stably cultured for more than three generations, and the cell density of the culture dish reached 85%-90%, the HK-2 cells were taken out of the incubator and placed on an ultra-clean bench. The culture medium was discarded, and the cells were washed three times with PBS to remove dead cells. The culture dishes of the model group and the drug-treated group were added with a culture medium containing 100 mg / L uric acid and a certain amount of the compound solution prepared in the example (the final concentration of the compound was 50 μM), shaken well, marked, and transferred to the incubator for 48 hours for protein extraction and quantitative analysis.

[0034] (II) Cell protein quantification experiment Take the protein standard preparation solution of the BCA kit, add it to the protein standard, prepare a 1 mg / mL protein solution, and dilute the concentration to 0.5, 0.4, 0.2, 0.1, and 0.05 mg / mL for subsequent sample preparation. Take a 50.0 mL centrifuge tube and prepare the BCA colorimetric agent according to the instructions of the kit. Add the diluted protein solution to a 96-well plate, 25 µL per well, and set three replicates for each concentration. Thaw the extracted cell protein in advance and dilute it 40 to 80 times, add it to a 96-well plate, 25 µL per well, and set three replicates for each group. After the protein is loaded, add 125 µL of BCA colorimetric agent, incubate in a constant temperature incubator for half an hour, detect 562 nm with an enzyme marker, make a protein standard curve based on the absorbance value, and substitute the absorbance value of the protein to be tested to obtain the protein concentration.

[0035] (III) Western-Blot 1. Make gel. Take a professional gel glass plate, wash it clean and dry it, clamp it on the mold, inject the lower layer gel prepared according to the instructions for making polyacrylamide gel (PAGE), seal and flatten the gel surface with Wahaha water, let it stand at room temperature for 20-30 min, pour out the Wahaha water after obvious traces of gel and water layer appear, inject the 5% upper layer gel prepared according to the instructions, insert a 10-hole comb, and let it stand at room temperature again for 20-30 min. Insert the prepared gel into the electrophoresis tank, fill it with the prepared 1× electrophoresis solution, and pull out the comb vertically. Add 5 µL of protein marker to one well, and add 60 µg of the above quantified protein to the experimental well. Connect the power supply, constant voltage 120 V, set the time as required, and perform electrophoresis. After the electrophoresis is completed, transfer to the membrane. Cut a 10-hole strip-sized polyvinylidene fluoride (PVDF) membrane, activate it in methanol, and then transfer it to the membrane. After electrophoresis, the constant current was 300 mA and electrotransfer was performed for 60-75 min.

[0036] The transferred PVDF membrane was cut out according to the protein marker ruler to obtain the target band URAT1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band, placed in 3.0 mL of pre-cooled 5% milk blocking solution, and blocked on a shaker for 2 h. The blocked target band was placed in an antibody incubation box, and the URAT1 (GLUT9) antibody and GAPDH antibody diluted according to the instructions were added, and incubated in a refrigerator at 4°C overnight. The next day, the URAT1 (GLUT9) and GAPDH solutions were discarded, TBST was added and placed on a shaker for washing 3 times, 5 min each time, and the corresponding secondary antibody was added, and continued to be placed on a shaker for incubation for 2 h. After the binding of the secondary antibody was completed, it was washed, and the enhanced chemiluminescence agent was added and placed in a chemiluminescence imaging system to scan the membrane and take pictures. ImageJ was used to analyze the grayscale value of the protein band, and GraphPad Prism 8.0 software was used to draw and analyze the expression of URAT1 (GLUT9) in each group of cells. The protein expression level percentage was obtained based on the URAT1 protein expression level in the blank group. The specific results are shown in Table 1 Table 1 Experimental results of compounds (50 μM) inhibiting URAT1 expression in HK-2 cells

Claims

1. An alkylamide nuciferine derivative having both uric acid lowering and kidney protection activities, characterized in that: Its molecular structure is: ； R is , , , , , , , .

2. A method for preparing the alkylamide nuciferine derivative according to claim 1, characterized in that: The following steps are involved: (1) Dissolve nuciferine in methanol, then add hydrogen peroxide and stir to react; after the reaction is completed, adjust the pH value of the reaction solution, then add distilled water, extract with chloroform, dry the chloroform layer with anhydrous Na2SO4, filter to remove the solid, concentrate the filtrate under reduced pressure, separate by silica gel column chromatography, and elute to obtain nuciferine- N - oxides; (2) Under ice bath conditions, N - oxide is dissolved in methanol, and then a methanol solution of FeSO4·7H2O is added, and the reaction is stirred after removing the ice bath to obtain a reaction solution; a saturated NaHCO3 solution is added to the reaction solution to continue the reaction; after the reaction is completed, distilled water is added, and chloroform is extracted, the chloroform layer is dried over anhydrous Na2SO4, the solid is removed by filtration, and the mixture is evaporated to dryness under reduced pressure, and separated by silica gel column chromatography and eluted to obtain proto-nuciferine; (3) Dissolve proto-nuciferine and triethylamine in anhydrous DCM, slowly add RX dropwise while maintaining 0°C, and stir to react. After the reaction is completed, quench the reaction, extract the reaction solution with dichloromethane, dry it with anhydrous sodium sulfate, concentrate the filtrate, and obtain the target compound by column chromatography.

3. The preparation method according to claim 2, characterized in that: In step (1), the ratio of nuciferine to hydrogen peroxide is 1 mmol:7.5 mL.

4. The preparation method according to claim 2 or 3, characterized in that: In step (1), the stirring reaction is carried out at room temperature for 24 hours; the pH is adjusted to 10 using a 3M NaOH aqueous solution; and the elution is carried out using an eluent consisting of chloroform: methanol: triethylamine in a volume ratio of 30:1:1‰.

5. The preparation method according to claim 2, characterized in that: In step (2), nuciferine- N -oxide and FeSO4·7H2O molar ratio is 1:2; the concentration of FeSO4·7H2O methanol solution is 1 mol / L; N -The ratio of oxide and saturated NaHCO3 solution is 1mmol:10mL;.

6. The preparation method according to claim 2 or 5, characterized in that: In step (2), the stirring reaction is carried out at room temperature for 12 hours; the reaction is continued for 20 minutes; and the eluent composed of chloroform: methanol: triethylamine in a volume ratio of 10:1:1‰ is used for elution.

7. The preparation method according to claim 2, characterized in that: In step (3), the molar ratio of proto-nuciferine, triethylamine and RX is 1:2-5:1-2.

8. The preparation method according to claim 2 or 7, characterized in that: In step (3), the stirring reaction is carried out at room temperature for 10-12 hours; and saturated sodium bicarbonate is used to quench the reaction.

9. Use of the alkylamide nuciferine derivative as claimed in claim 1 in the preparation of uric acid-lowering or kidney-protecting drugs.

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