Coumarin hydrazone derivatives with antioxidant activity and use thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a coumarin hydrazone derivative with antioxidant activity and application thereof. The chemical structural formula of the coumarin derivative is shown as formula (I), wherein R is an aromatic ring containing different substituents. The in vitro antioxidant experiment results show that most of the derivatives exhibit certain scavenging capacity on DPPH, ABTS, superoxide anion and hydroxyl radicals, and have certain reducing capacity. Notably, the derivatives 5a-5d, 5i-5k, 5m and 5n have better scavenging effect on superoxide anion radicals than or equivalent to vitamin C. The method for preparing the coumarin hydrazone derivative has the advantages of simple operation, convenient post-treatment, high product separation yield and the like. The coumarin compound can be used for preparing an antioxidant.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a preparation method of coumarin analogs and use thereof in antioxidant activity. BACKGROUND

[0002] In the normal physiological metabolic process, the body will produce ions, molecules, active oxygen (ROS), carbon (RCS), nitrogen (RNS), sulfur (RSS) and other high-activity substances carrying unpaired electrons. When the body is stimulated, too many high-activity substances will be produced in the body, leading to imbalance with antioxidants, and thus causing a stress state. This oxidative stress behavior can oxidatively modify bioactive macromolecules (such as nucleic acids, lipids, proteins, etc.), resulting in the loss of their original structure and function, and thus causing adverse consequences such as cell damage, ion imbalance and organelle dysfunction. Clinical medicine shows that excessive oxidative stress is closely related to the occurrence and development of cancer, cardiovascular disease, digestive system disease, respiratory system disease and neurodegenerative disease.

[0003] Finding new antioxidants for scavenging free radicals in the body from natural products has become an inevitable trend in the development of modern medicine and health care industry. Coumarin compounds are widely present in many plants such as Leguminosae, Asteraceae and Orchidaceae, and have significant biological activity in nature. In recent years, many studies have found that coumarin derivatives exhibit great potential in free radical scavenging, membrane protection and antioxidant. In previous reports, by modifying and modifying C-3, C-4 and C-7 positions on the coumarin skeleton, many lead compounds with excellent antioxidant activity have been obtained. However, there are only a few reports on the structural derivation of C-6 position, and the antioxidant effect of such compounds has not been explored. Different positions of substituents will have different effects on the antioxidant activity of derivatives. At the same time, the applicant found that increasing the conjugated system of the coumarin structure can improve its antioxidant activity. Based on the above research background, the present application takes the coumarin skeleton as the research object, increases the conjugated system of the coumarin by introducing a "-C=N-" group, synthesizes 15 coumarin analogs, and carries out a systematic screening and evaluation of free radical scavenging ability. SUMMARY

[0004] One of the purposes of the present application is to provide a preparation method of coumarin analogs, further enriching the structural types of coumarin compounds; another purpose is to provide the application of the coumarin analogs in antioxidant, including other aspects of the use of the analogs.

[0005] To achieve the above purpose, the technical solutions adopted by the present application are as follows:

[0006] The chemical structure of the coumarin hydrazone compound is shown as formula (I)

[0007]

[0008] R is one of the following structural formulae:

[0009]

[0010] The synthesis line is as follows:

[0011]

[0012] The preparation method of the coumarin derivative is as follows:

[0013] (1) Synthesis of 6-methyl coumarin: 5-methyl salicylaldehyde, sodium acetate were placed in a round-bottom flask, Ar was degassed three times, N, N-dimethylformamide solution was added, stirred at room temperature for 30 min, acetic anhydride was added in small amounts and several times, the temperature was increased to 170°C, and the stirring was continued for 6 h, TLC thin layer detection was performed, and after the raw material was completely consumed, the reaction system was cooled to room temperature, extracted with DCM (3x10 mL), the organic layer was collected, and then washed with distilled water and saturated brine, dried over anhydrous Na2SO4, and the crude product was purified by flash chromatography (petroleum ether and EtOAc as eluent), concentrated under reduced pressure, and the target compound 2 was obtained by purification. Among them, the molar ratio of 5-methyl salicylaldehyde, acetic anhydride and sodium acetate is 1:3:2.5.

[0014] (2) Synthesis of 6-bromomethyl coumarin: 6-methyl coumarin (target compound 2) was weighed and placed in a round-bottom flask, Ar was degassed three times, carbon tetrachloride solution was added, and the temperature was increased to 85°C. At the same time, acetonitrile solution containing N-bromosuccinimide and azobisisobutyronitrile was slowly added dropwise, and the reaction was carried out overnight. TLC thin layer detection was performed, and after the raw material was completely reacted, the reaction system was cooled to room temperature. Extracted with DCM (3x10 mL), collect the DCM layer, dry over anhydrous Na2SO4, recrystallize the crude product with n-hexane and EtOAc (1:1), filter, and dry the filter cake under vacuum to obtain the target compound 3. The molar ratio of 6-methyl coumarin, N-bromosuccinimide and azobisisobutyronitrile is 1:1:0.2.

[0015] (3) Synthesis of 6-Formyl Coumarin: 6-Bromomethyl coumarin (target compound 3) was dissolved in acetic acid and distilled water, stirred uniformly, urotropine was added slowly, and the temperature was raised to 100℃ for 5h. TLC thin layer detection until the reaction was complete. The generated compound can make DNP (2,4-dinitrophenylhydrazine) yellow. After cooling to room temperature, it was extracted with EtOAc (3x10mL) in turn, the organic layer was collected, washed with distilled water and saturated brine respectively, dried over anhydrous Na2SO4, and the crude product was recrystallized with a mixed solvent of n-hexane and EtOAc, filtered and dried in vacuum to obtain the target compound 4. The molar ratio of 6-bromomethyl coumarin and urotropine was 1:2.

[0016] (4) Synthesis of compounds 5a-5o: hydrazine compounds were dissolved in anhydrous ethanol and acetic acid, stirred at room temperature for 30min, and then 6-formyl coumarin dissolved in ethanol was added dropwise slowly. The temperature was raised to 85℃ and the reaction was continued for 8h. TLC thin layer detection until the reaction was complete. Filtration gave a solid crude product, which was recrystallized with a mixed solvent of n-hexane and ethanol. The pure product obtained by recrystallization was further filtered and dried in vacuum to obtain the target compounds 5a-5o. The molar ratio of 6-formyl coumarin, hydrazine compounds and acetic acid was 1:1.5-1.8:0.5.

[0017] The application of coumarin derivatives in the preparation of antioxidant activity, including their scavenging efficiency on DPPH, ABTS, superoxide anion and hydroxyl radicals, and the influence on reducing power.

[0018] Preferably, derivatives 5c, 5d and 5i have good ability to scavenge superoxide anion radicals, and the structural formula is:

[0019]

[0020] Preferably, the application of coumarin hydrazone compounds in the preparation of DPPH radical scavenging drugs, and the structural formula of coumarin hydrazone compounds is:

[0021]

[0022] Preferably, the application of coumarin hydrazone compounds in the preparation of hydroxyl radical scavenging drugs, and the structural formula of coumarin hydrazone compounds is:

[0023] Preferably, the application of coumarin hydrazone compounds in the preparation of ABTS radical scavenging drugs, and the structural formula of coumarin hydrazone compounds is:

[0024] Among them, the structure of compound 5i is novel, and is reported for the first time in the application. 5c and 5d are known compounds, but the application first found that they have good antioxidant activity.

[0025] The application has the advantages that the chemical structure of coumarin analogs and the corresponding synthetic route are provided. More importantly, the radical scavenging abilities of the coumarin analogs on DPPH, ABTS, superoxide anion and hydroxyl radicals are detected by an enzyme label instrument. Meanwhile, the lead compounds with significant radical scavenging effects are screened, which indicates that the coumarin skeleton analogs have the potential to be developed into antioxidants. DETAILED DESCRIPTION

[0026] The application will be described in detail below through specific examples, and it should be understood that the following examples are only used for explanation and illustration, and do not limit the scope of the application in any form. In the following examples, the biochemical reagents not specifically described are conventional reagents in the art, which can be prepared according to conventional methods in the art or commercially available, and the specifications are laboratory pure grade.

[0027] Preparation of compounds 5a-5o

[0028]

[0029] The preparation steps of 6-aldehyde coumarin are as follows:

[0030] (1) 5-methyl salicylaldehyde 1.00 g (7.34 mmol), sodium acetate 1.51 g (18.36 mmol) were weighed into a round-bottom flask, degassed three times under Ar, 15 mL of DMF solution was added, stirred at room temperature for 30 min, the system was heated to 170℃, 1.39 mL of acetic anhydride solution (22.02 mmol) was slowly added, and the stirring was continued for 6 h. TLC thin layer detection was performed, and after the raw material was completely reacted, the reaction system was cooled to room temperature, extracted with DCM (3x10 mL), washed with distilled water and saturated NaCl solution, dried over anhydrous Na2SO4, and the crude product was purified by flash chromatography (PE and EtOAc as eluent), concentrated under reduced pressure to obtain the target compound 2.

[0031] (2) Take 6-methylcoumarin 1.00 g (6.24 mmol) into a 50 mL round-bottom flask, Ar degassing three times, add CCl4 solution 15 mL, the reaction temperature rises to 85 °C, slowly drop N-bromosuccinimide 1.11 g (6.24 mmol) and azobisisobutyronitrile 0.21 g (1.25 mmol) dissolved in acetonitrile (15 mL), continue to stir for 6 h. First, extract with DCM (3x10 mL), wash with distilled water and saturated brine, combine the organic layer, dry with anhydrous Na2SO4. The crude product is recrystallized with n-hexane and EtOAc (1:1), suction filtration, and the filter cake is dried in vacuum to obtain the target compound 3, 6-bromomethylcoumarin.

[0032] (3) Take 6-bromomethylcoumarin 1.00 g (4.18 mmol) into a 50 mL round-bottom flask, add acetic acid and water 15 mL respectively, stir well, add a small amount of urotropine 1.17 g (8.31 mmol) in several times, and the temperature is raised to 100 °C for 5 h. TLC thin layer detection until the reaction is complete. The generated compound can make DNP (2,4-dinitrophenylhydrazine) yellow. Cool to room temperature, extract with EtOAc (3x10 mL) in turn, wash with distilled water and saturated brine, combine the organic layer, dry the organic layer with anhydrous Na2SO4, filter, and evaporate under reduced pressure. The crude product is recrystallized with n-hexane and EtOAc mixed solvent, suction filtration, and the filter cake is dried in vacuum to obtain the target compound 4, 6-aldehyde coumarin.

[0033] Example 1

[0034] 5a:

[0035] Take p-toluoylhydrazine (0.69 mmol) into a 20 mL Schlenk tube, add anhydrous ethanol 2 mL, acetic acid (0.23 mmol) respectively. After stirring at room temperature for 30 min, slowly drop 6-aldehyde coumarin (0.46 mmol) dissolved in ethanol (0.5 mL), and the temperature is raised to 85 °C for 8 h. TLC thin layer detection until the reaction is complete, suction filtration to obtain the solid crude product, and the pure product obtained by recrystallization is further suction filtered. The filter cake is dried in vacuum to obtain the target product 5a, which is a white solid with a yield of 80.8%.

[0036] (E)-4-methyl-N'-((2-oxo-2H-chromen-6-yl)methylene)benzohydrazide (5a): 1H NMR (300 MHz, DMSO-d6): δ (ppm) 11.89 (s, 1H), 8.49 (s, 1H), 8.18 (d, J = 9.6 Hz, 1H), 8.08 (d, J = 2.0 Hz, 1H), 7.97 (dd, J = 8.6, 2.0 Hz, 1H), 7.84 (d, J = 7.9 Hz, 2H), 7.49 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 7.9 Hz, 2H), 6.56 (d, J = 9.6 Hz, 1H), 2.38 (s, 3H). 13 C NMR (75 MHz, DMSO-d6): δ (ppm) 163.0, 159.8, 154.4, 146.0, 144.2, 142.0, 130.9, 130.5, 130.3, 129.1 (2C), 127.7 (2C), 127.1, 119.2, 117.1, 116.9, 21.1.

[0037] Example 2

[0038] 5b:

[0039] Synthesis method is same as Example 1, except that p-toluoyl hydrazine is replaced by p-methoxybenzoyl hydrazine, to obtain the target product 5b, the property is yellow solid, the yield is 86.9%.

[0040] (E)-4-methoxy-N'-((2-oxo-2H-chromen-6-yl)methylidene)benzohydrazide (5b): 1 H NMR (300 MHz, DMSO-d6): δ (ppm) 11.85 (s, 1H), 8.49 (s, 1H), 8.19 (d, J = 9.6 Hz, 1H), 8.09 (s, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 8.7 Hz, 2H), 7.49 (d, J = 8.7 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 6.57 (d, J = 9.6 Hz, 1H), 3.84 (s, 3H). 13 C NMR (75 MHz, DMSO-d6): δ (ppm) 164.1, 162.1, 159.8, 154.4, 145.6, 144.2 (2C), 131.0, 130.2, 129.6, 127.0, 125.4, 119.2, 117.1, 116.9, 113.8 (2C), 55.5.

[0041] Example 3

[0042] 5c:

[0043] The synthetic procedure was the same as in Example 1 except that p-toluylhydrazide was replaced by p-nitrobenzohydrazide to give the target product 5c as a yellow solid in 93.5% yield.

[0044] (E)-4-nitro-N'-((2-oxo-2H-chromen-6-yl)methylene)benzohydrazide (5c): 1 H NMR (300 MHz, DMSO-d6): d (ppm) 12.26 (s, 1H), 8.52 (s, 1H), 8.40 (d, J = 8.9 Hz, 2H), 8.20 (d, J = 7.5 Hz, 2H), 8.16 (d, J = 6.9 Hz, 1H), 8.12 (d, J = 2.1 Hz, 1H), 8.00 (dd, J = 8.7, 2.1 Hz, 1H), 7.51 (d, J = 8.7 Hz, 1H), 6.58 (d, J = 9.6 Hz, 1H). 13 C NMR (75 MHz, DMSO-d6): d (ppm) 162.0, 160.1, 155.0, 149.8, 147.8, 144.6, 139.4, 131.0, 130.8, 129.7 (2C), 127.8, 124.2 (2C), 119.6, 117.6, 117.4.

[0045] Example 4

[0046] 5d:

[0047] The synthetic procedure was the same as in Example 1 except that p-toluylhydrazide was replaced by p-bromobenzohydrazide to give the target product 5d as a white solid in 97.9% yield.

[0048] (E)-4-bromo-N'-((2-oxo-2H-chromen-6-yl)methylene)benzohydrazide (5d): 1 H NMR (300 MHz, DMSO-d6): d (ppm) 11.96 (s, 1H), 8.43 (s, 1H), 8.12 (d, J = 9.3 Hz, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.91 (dd, J = 8.7, 2.1 Hz, 1H), 7.82 (d, J = 9.3 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.5 Hz, 1H), 6.50 (d, J = 9.6 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): d (ppm) 162.7, 160.1, 154.9, 147.0, 144.6, 132.8, 132.0 (2C), 131.1, 130.7, 130.2 (2C), 127.6, 126.1, 119.6, 117.6, 117.3.

[0049] Example 5

[0050] 5e:

[0051] The synthetic method is the same as Example 1, except that p-toluic hydrazide is replaced by p-fluorobenzohydrazide to obtain the target product 5e, which is a yellow solid with a yield of 94.6%.

[0052] (E)-4-fluoro-N'-((2-oxo-2H-chromen-6-yl)methylene)benzohydrazide (5e): 1 H NMR (300 MHz, DMSO-d6): d (ppm) 11.98 (s, 1H), 8.49 (s, 1H), 8.18 (d, J = 9.6 Hz, 1H), 8.09 (d, J = 2.1 Hz, 1H), 8.02 (d, J = 5.4 Hz, 1H), 7.97 (dd, J = 6.9, 1.8 Hz, 2H), 7.49 (d, J = 8.7 Hz, 1H), 7.38 (t, J = 8.8 Hz, 2H), 6.57 (d, J = 9.6 Hz, 1H). 13 C NMR (75 MHz, DMSO-d6): d (ppm) 164,7 (d, J = 248.0 Hz), 162.6, 160.1, 154.9, 146.8, 144.6, 131.2, 130.9, 130.8 (d, J = 7.1 Hz, 2C), 130.3 (d, J = 2.5 Hz), 127.6, 119.6, 117.5, 117.3, 116.0 (d, J = 21.8 Hz, 2C). 19 F NMR (282 MHz, DMSO-d6): d (ppm) -108.2 (brs).

[0053] Example 6

[0054] 5f:

[0055] The synthetic method is the same as Example 1, except that p-toluic hydrazide is replaced by p-trifluoromethylbenzohydrazide to obtain the target product 5f, which is a white solid with a yield of 96.9%.

[0056] (E)-N'-((2-oxo-2H-chromen-6-yl)methylene)-4-(trifluoromethyl)benzohydrazide (5f): 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 12.05 (s, 1H), 8.49 (s, 1H), 8.18 (d, J = 9.6 Hz, 1H), 8.11 (d, J = 2.0 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.98 (dd, J = 8.8, 2.4 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.8 Hz, 1H), 6.57 (d, J = 9.6 Hz, 1H). 13 C NMR (75 MHz, DMSO-d6): δ (ppm) 162.1, 159.7, 154.5, 147.1, 144.1, 137.2, 131.2 (q, J = 31.4 Hz), 130.6, 130.3, 128.6 (2C), 127.2, 125.5 (q, J = 3.5 Hz, 2C), 123.9 (q, J = 267.3 Hz), 119.2, 117.1, 116.9. 19 F NMR (282 MHz, DMSO-d6): δ (ppm) -61.4.

[0057] Example 7

[0058] 5g:

[0059] Synthesis method is same as Example 1, except that p-toluoyl hydrazide is replaced by p-trifluoromethoxybenzoyl hydrazide, to obtain the target product 5g, white solid, yield of 96.9%.

[0060] (E)-N'-((2-oxo-2H-chromen-6-yl)methylene)-4-(trifluoromethyl)benzohydrazide (5f): 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 12.05 (s, 1H), 8.49 (s, 1H), 8.18 (d, J = 9.6 Hz, 1H), 8.11 (d, J = 2.0 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.98 (dd, J = 8.8, 2.4 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.8 Hz, 1H), 6.57 (d, J = 9.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6): d (ppm) 162.0, 159.7, 154.5, 150.6, 146.7, 144.2, 132.5, 130.7, 130.3, 130.1 (2C), 127.2, 120.9 (2C), 120.0 (q, J = 258.2 Hz), 119.2, 117.1, 116.9. 19 F NMR (376 MHz, DMSO-d6): d (ppm) -57.1.

[0061] Example 8

[0062] 5h:

[0063] The synthesis method is the same as Example 1, except that p-toluyl hydrazide is replaced by isoniazid, to obtain the target product 5h, which is a white solid with a yield of 85.0%.

[0064] (E)-N'-((2-oxo-2H-chromen-6-yl)methylene)isonicotinohydrazide (5h): 1 H NMR (300 MHz, DMSO-d6): d (ppm) 12.19 (s, 1H), 8.81 (dd, J = 4.5, 1.8 Hz, 2H), 8.51 (s, 1H), 8.19 (d, J = 9.6 Hz, 1H), 8.13 (d, J = 2.0 Hz, 1H), 8.00 (dd, J = 8.7, 2.0 Hz, 1H), 7.84 (dd, J = 4.5, 1.5 Hz, 2H), 7.51 (d, J = 8.4 Hz, 1H), 6.58 (d, J = 9.6 Hz, 1H). 13 C NMR (75 MHz, DMSO-d6): d (ppm) 161.7, 159.7, 154.6, 150.4 (2C), 147.5, 144.2, 140.4, 130.5, 130.5, 127.4, 121.6 (2C), 119.2, 117.2, 117.0.

[0065] Example 9

[0066] 5i:

[0067] The synthesis method is the same as Example 1, except that p-toluyl hydrazide is replaced by phenylsulfonyl hydrazide, and the molar ratio of 6-aldehyde coumarin, sulfonyl hydrazide, acetic acid is 1:1.8:0.5. The target product 5i is obtained, which is a white solid with a yield of 89.4%.

[0068] (E)-N'-((2-oxo-2H-chromen-6-yl)methylene)benzenesulfonohydrazide (5i): 1 H NMR (300 MHz, DMSO-d6): δ (ppm) 11.68 (s, 1H), 8.12 (d, J = 9.6 Hz, 1H), 7.98 (s, 1H), 7.92 (t, J = 1.2 Hz, 2H), 7.90 (d, J = 1.9 Hz, 1H), 7.80 (dd, J = 8.7, 2.1 Hz, 1H), 7.68-7.65 (m, 1H), 7.65-7.59 (m, 2H), 7.42 (d, J = 8.7 Hz, 1H), 6.54 (d, J = 9.6 Hz, 1H). 13 C NMR (75 MHz, DMSO-d6): δ (ppm) 160.1, 154.8, 146.2, 144.5, 139.4, 133.6, 130.5, 130.3, 129.8 (2C), 127.6 (2C), 127.4, 119.5, 117.5, 117.3.

[0069] Example 10

[0070] 5j:

[0071] Synthesis method is the same as Example 9, except that the benzene sulfonohydrazide is replaced by p-toluenesulfonohydrazide, to obtain the target product 5j, the property is yellow solid, the yield is 85.0%.

[0072] (E)-4-methyl-N'-((2-oxo-2H-chromen-6-yl)methylene)benzenesulfonohydrazide (5j): 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 11.55 (s, 1H), 8.11 (d, J = 9.6 Hz, 1H), 7.96 (s, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.79 (m, 1H), 7.78 (d, J = 8.0 Hz, 2H), 7.42 (s, 1H), 7.41 (dd, J = 8.4, 2.8 Hz, 2H), 6.53 (d, J = 9.6 Hz, 1H), 2.36 (s, 3H). 13 C NMR (75 MHz, DMSO-d6): δ (ppm) 159.6, 154.4, 145.5, 144.0, 143.5, 136.1, 130.1, 129.7, 129.7 (2C), 127.2 (2C), 126.9, 119.0, 117.0, 116.8, 21.0.

[0073] Example 11

[0074] 5k:

[0075] The synthetic method is the same as Example 9, except that benzene sulfonyl hydrazide is replaced by p-methoxybenzene sulfonyl hydrazide to obtain the target product 5k, which is a white solid with a yield of 89.9%.

[0076] (E)-4-methoxy-N'-((2-oxo-2H-chromen-6-yl)methylidene)benzenesulfonohydrazide (5k): 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 11.48 (s, 1H), 8.11 (d, J = 9.6 Hz, 1H), 7.96 (s, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.82 (dt, J = 9.2, 2.6 Hz, 2H), 7.79 (dd, J = 8.8, 2 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.12 (dt, J = 8.8, 2.4 Hz, 2H), 6.53 (d, J = 9.6 Hz, 1H), 3.81 (s, 3H). 13 C NMR (101 MHz, DMSO-d6): δ (ppm) 162.7, 159.7, 154.4, 145.4, 144.1, 130.6, 130.2, 129.8, 129.4 (2C), 126.9, 119.0, 117.1, 116.9, 114.5 (2C), 55.7.

[0077] Example 12

[0078] 5l:

[0079] The synthetic method is the same as Example 9, except that benzene sulfonyl hydrazide is replaced by p-bromobenzene sulfonyl hydrazide to obtain the target product 5l, which is a white solid with a yield of 94.7%.

[0080] (E)-4-bromo-N'-((2-oxo-2H-chromen-6-yl)methylidene)benzenesulfonohydrazide (5l): 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 11.70 (s, 1H), 8.08 (d, J = 9.7 Hz, 1H), 7.96 (s, 1H), 7.89 (d, J = 2.0 Hz, 1H), 7.82 (dd, J = 8.7, 2.1 Hz, 1H), 7.80 (d, J = 3.2 Hz, 2H), 7.79-7.78 (m, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 6.51 (d, J = 9.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6): d (ppm) 159.6, 154.5, 146.3, 144.1, 138.2, 132.5 (2C), 130.0, 129.9, 129.2 (2C), 127.1, 127.0, 119.0, 117.1, 116.9.

[0081] Example 13

[0082] 5m:

[0083] Synthesis as in Example 9, except that benzene sulfonyl hydrazide was replaced by p-nitrobenzene sulfonyl hydrazide. It was obtained as a yellow solid in 90.4% yield.

[0084] (E)-4-nitro-N'-((2-oxo-2H-chromen-6-yl)methylidene)benzenesulfonohydrazide (5p): 1 H NMR (400 MHz, DMSO-d6): d (ppm) 11.96 (s, 1 H), 8.40 (dt, J = 9.2, 2.2 Hz, 1 H), 8.22 (dt, J = 9.2, 2.6 Hz, 1 H), 8.13 (dt, J = 9.2, 2.4 Hz, 2H), 8.08 (d, J = 9.6 Hz, 1 H), 7.99 (s, 1 H), 7.91 (d, J = 2.1 Hz, 1 H), 7.79 (m, 1 H), 7.40 (d, J = 8.6 Hz, 1 H), 6.51 (d, J = 9.6 Hz, 1 H). 13 C NMR (101 MHz, DMSO-d6): d (ppm) 159.6, 154.6, 150.0, 144.1, 130.0, 129.8, 128.9 (2C), 127.1, 126.8, 124.7 (2C), 124.6, 119.0, 117.1, 116.9.

[0085] Example 14

[0086] 5n:

[0087] Synthesis as in Example 9, except that benzene sulfonyl hydrazide was replaced by p-fluorobenzene sulfonyl hydrazide. It was obtained as a yellow solid in 92.5% yield.

[0088] (E)-4-fluoro-N'-((2-oxo-2H-chromen-6-yl)methylidene)benzenesulfonohydrazide (5n): 1H NMR (400 MHz, DMSO-d6): d (ppm) 11.67 (s, 1H), 8.11 (d, J = 9.6 Hz, 1H), 7.99 (s, 1H), 7.96 (tt, J = 8.7, 2.7 Hz, 2H), 7.93 (d, J = 2.1 Hz, 1H), 7.81 (dd, J = 8.4, 2.1 Hz, 1H), 7.46 (tt, J = 8.8, 2.6 Hz, 2H), 7.42 (d, J = 8.4 Hz, 1H), 6.54 (d, J = 9.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6): d (ppm) 161.5 (d, J = 363.2 Hz), 156.5, 154.8, 146.1, 144.1, 135.3 (d, J = 3.1 Hz), 130.3 (d, J = 9.7 Hz, 2C), 130.0, 129.9, 127.0, 119.0, 117.1, 116.9, 116.6 (d, J = 23.2 Hz, 2C). 19 F NMR (376 MHz, DMSO-d6): d (ppm) -105.7.

[0089] Example 15

[0090] 5o:

[0091] Synthesis as in Example 9, except that benzene sulfonylhydrazide is replaced by p-trifluoromethylbenzene sulfonylhydrazide. White solid, 97.2% yield.

[0092] (E)-N'-((2-oxo-2H-chromen-6-yl)methylene)-4-(trifluoromethyl)benzenesulfonylhydrazide (5o): 1 H NMR (400 MHz, DMSO-d6): d (ppm) 11.91 (s, 1H), 8.12 (d, J = 5.4 Hz, 2H), 8.10 (d, J = 6.7 Hz, 2H), 8.03 (s, 1H), 8.01 (s, 1H), 8.01 (s, 1H), 7.93 (d, J = 2.0 Hz, 1H), 7.81 (dd, J = 8.4, 2.1 Hz, 1H), 6.53 (d, J = 9.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6): d (ppm) 159.7, 154.5, 146.6, 144.1, 142.8, 132.8, 129.9, 129.9, 128.3 (2C), 127.1, 126.6 (2C), 123.4, 119.4, 117.1, 116.9. 19F NMR (376 MHz, DMSO-d6): δ (ppm) -61.7.

[0093] Example 16 Evaluation of antioxidant activity in vitro

[0094] According to the hydrazine coumarin compounds described in Examples 1-15, the present application takes vitamin C (Vc) as a positive control, and evaluates the antioxidant activity of the hydrazine coumarin compounds by taking DPPH free radical, hydroxyl radical, superoxide anion radical, ABTS radical scavenging capacity and total reducing power as indexes.

[0095] Test Example 1: DPPH free radical scavenging capacity determination

[0096] 1. Experimental principle

[0097] 1,1-diphenyl-2-trinitrobenzene hydrazine (DPPH) is a stable free radical with a nitrogen atom at the center and three benzene rings around it. The alcohol solution of DPPH· is dark purple and has strong absorption at 517 nm, and the concentration and absorbance value are linearly related. When there is a free radical scavenger in the solution, DPPH· can combine or replace with the free radical scavenger, the number of DPPH· decreases, resulting in a decrease in absorbance and a gradual change in color, i.e. the degree of discoloration is quantitatively related to the number of electrons accepted. Therefore, the DPPH· scavenging rate of the compound can be calculated according to the absorbance value at this wavelength, so as to evaluate the DPPH free radical scavenging effect of the drug.

[0098] 2. Experimental method

[0099] The experiment is divided into three groups: sample group, control group and blank group.

[0100] Sample group: 100 μL of DPPH solution (0.2 mM) was added to a 96-well plate, and then 50 μL of 600 μM of the sample to be tested was added, with a total volume of 150 μL per well, so that the maximum final concentration of the sample was 200 μM.

[0101] Control group: 100 μL of anhydrous ethanol was added to a 96-well plate, and then 50 μL of 600 μM of the sample to be tested was added, with a total volume of 150 μL per well.

[0102] Blank group: 100 μL of DPPH solution (0.2 mM) and 50 μL of DMSO were added in turn.

[0103] After the above grouping is completed, the 96-well plate is placed at room temperature in the dark for 30 min, and after the reaction is completed, it is mixed well by shaking. The OD value of each well at 517 nm wavelength is detected by an enzyme marker, and the DPPH free radical scavenging rate of the sample to be tested is calculated according to formula (1).

[0104] DPPH radical scavenging rate (%) = [1-(OD 样品 -OD 对照 ) / OD 空白 ] x 100% Formula (1)

[0105] 3. Experimental results and analysis

[0106] Table 1 Derivative scavenging rate of DPPH radical

[0107]

[0108]

[0109] The DPPH method was used to detect the DPPH radical scavenging ability of the derivatives. The results are shown in Table 1. At the maximum concentration of 200 μM, the derivatives obtained in the present application showed different degrees of scavenging effect on DPPH radical (20.96-59.49%), but there was still a large gap compared with the scavenging ability of Vc (93.01%). Among the derivatives 5a-5o, derivative 5c showed moderate scavenging ability on DPPH radical (59.49%). In the acylhydrazone coumarin derivatives, the introduction of electron-withdrawing groups on the aromatic ring can significantly improve the scavenging ability (5c-5f vs. 5a, 5b, 5g). Conversely, for the sulfonylhydrazone coumarin derivatives, the introduction of electron-donating groups on the aromatic ring can improve their scavenging effect (5j vs. 5i; 5j vs. 5l-5o).

[0110] Test Example 2: Determination of hydroxyl radical scavenging ability

[0111] 1. Experimental principle

[0112] H2O2 and ferrous ions generate ·OH radicals through Fenton reaction, which have high reactivity. When the reaction system contains salicylic acid, it can quickly capture hydroxyl radicals to produce purple 2,3-dihydroxybenzoic acid, which has a large absorption peak at a wavelength of 510 nm. The absorbance value is positively correlated with the concentration of ·OH radicals in the system. When a radical scavenger is added to the reaction system, it will prevent the combination of salicylic acid and ·OH radicals, and the oxidized salicylic acid will decrease, the color of the reaction system will become lighter or disappear, resulting in a decrease in absorbance. Based on this, the ·OH radical scavenging rate of the compound can be calculated according to the absorbance value at this wavelength.

[0113] 2. Experimental method

[0114] The present application uses Fe 2+ -H2O2-salicylic acid system to detect the ·OH radical scavenging ability of the sample. The experiment is divided into three groups: sample group, control group and blank group.

[0115] Sample group: 50 μL of 600 μM sample to be tested was added to a 96-well plate, and then 50 μL of 9 mM FeSO4 solution, 50 μL of salicylic acid-ethanol solution and 50 μL of 8.8 mM H2O2 solution were added in sequence, and the total reaction volume was 200 μL.

[0116] Control group: 50 μL of 600 μM sample to be tested was added first, and then 50 μL of FeSO4 solution, 50 μL of salicylic acid-ethanol solution and 50 μL of distilled water were added in sequence.

[0117] Blank group: 50 μL of DMSO solution, 50 μL of FeSO4 solution, 50 μL of salicylic acid-ethanol solution and 50 μL of H2O2 solution were added in sequence.

[0118] After all the reaction systems were added, they were incubated at 37°C on a shaker for 30 min in the dark. The OD value of each well at 510 nm was detected by an enzyme marker, and the hydroxyl radical scavenging rate of the sample to be tested was calculated according to formula (2).

[0119] ·OH radical scavenging rate (%) = [1-(OD 样品 -OD 对照 ) / OD 空白 ] × 100% formula (2)

[0120] 3. Experimental results and analysis

[0121] Table 2 Derivatives of hydroxyl radical scavenging rate

[0122]

[0123] The present application detects the scavenging effect of coumarin derivatives on ·OH radicals by means of Fe 2+ -H2O2-salicylic acid system method. As shown in Table 2, the scavenging rate of derivatives 5a-5o on ·OH radicals is between 0.39-17.50%, but its scavenging effect is obviously weaker than that of Vc (25.07%). Whether it is acylhydrazone or sulfonylhydrazone derivative, the introduction of electron-donating groups on the aromatic ring can significantly improve its scavenging ability on hydroxyl radicals (5b vs. 5c-5f; 5j vs. 5i; 5j vs. 5l-5o).

[0124] Test example 3: Superoxide anion radical scavenging capacity determination

[0125] 1. Experimental principle

[0126] Pyrogallol can undergo autoxidation under alkaline conditions to release O2 - ·, which in turn generates an orange-red intermediate. The intermediate has a strong absorption at 320 nm. When there is O2 -• The scavenger can rapidly react with O2 - • The reaction, thereby preventing the accumulation of intermediate products, results in a decrease in the absorbance of the solution at a wavelength of 320 nm. That is, the amount of intermediate product is positively correlated with the strength of the absorbance. Thus, the present application can determine the effect of a compound on O2 - • The scavenging effect of free radicals.

[0127] 2. Experimental method

[0128] The present application uses the autoxidation method of pyrogallol to detect the scavenging ability of a sample on O2 - • Free radicals. The experiment is divided into three groups: a sample group, a control group, and a blank group.

[0129] Sample group: 30 μL of the sample (200 μM) and 150 μL of Tris-HCl solution (50 mM) are sequentially added to a 96-well plate. The plate is placed in a shaker at 25°C and shaken for 20 min. Then, 30 μL of pyrogallol solution (3 mM) is added to make the total reaction volume 210 μL, and the mixture is shaken for another 5 min.

[0130] Control group: The experimental operation is the same as described above, except that 30 μL of distilled water is used instead of the pyrogallol solution.

[0131] Blank group: The experimental operation is the same as described in the sample group, except that 30 μL of DMSO solution is used instead of the sample solution.

[0132] After the reaction is completed, the absorbance value of each well at 320 nm is measured using an enzyme marker. The scavenging rate of the sample on superoxide anion free radicals is calculated according to formula (3).

[0133] O2 - • Free radicals (%) = [1-(OD 样品 - OD 对照 ) / OD 空白 ] x 100% formula (3)

[0134] 3. Experimental results and analysis

[0135] The results of the present application are shown in Table 3, and the derivatives 5a-5o exhibit different degrees of scavenging effect on O2 - • Free radicals (65.63-97.19%). At a maximum initial concentration of 200 μM, the derivatives 5c, 5d, and 5i exhibit a scavenging percentage on O2 - • Free radicals between 94.71 and 97.19%, which is better than or equivalent to Vc (95.67%). For the sulfonyl hydrazone coumarin compounds, the introduction of electron-withdrawing groups on the aromatic ring will not improve the scavenging rate (5l-5o vs. 5k; 5l-5o vs. 5i).

[0136] Table 3 Derivative on O2 - · radical scavenging rate

[0137]

[0138] Test Example 4: ABTS radical cation scavenging capacity determination

[0139] 1. Experimental principle

[0140] Under the action of oxidants such as K2S2O8, H2O2, 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) can be oxidized to green or blue-green ABTS + ·, which has a maximum characteristic absorption peak at 734 nm or 405 nm. When the antioxidant in the system reacts with ABTS + ·, the system will become colorless ABTS, that is, the content of ABTS + · in the system is linearly related to the absorbance value. Therefore, according to the determined absorbance value, the compound is determined according to the ABTS + · radical scavenging effect.

[0141] 2. Experimental method

[0142] The ABTS method is used to detect the ABTS + · radical scavenging capacity of the compound, and the experiment is set up into three groups: sample group, control group and blank group.

[0143] Sample group: In a 96-well plate, 20 μL of the sample solution (200 μM) to be tested and 200 μL of ABTS working solution (2.6 mM K2S2O8 solution and 7.4 mM ABTS solution mixed in equal volumes) are sequentially added, and the reaction is carried out at room temperature for 10 min in the dark. The total volume of the reaction is 220 μL.

[0144] The control group is 200 μL of pure water added with 20 μL of each sample to be tested.

[0145] The blank group is 200 μL of ABTS + · solution added with 20 μL of DMSO.

[0146] Subsequently, the absorbance value is measured at a wavelength of 734 nm, and the ABTS + · radical scavenging rate of the sample is calculated according to formula (4).

[0147] ABTS + · radical scavenging rate = [1-(OD 样品 - OD 对照 ) / OD空白 ] x 100% Formula (4)

[0148] 3. Experimental results and analysis

[0149] ABTS + The results of the radical scavenging experiment are shown in Table 4. The derivatives 5a-5o showed ABTS + The radical scavenging rate was between 4.45-42.65%, which was significantly weaker than Vc (70.36%). Compared with other derivatives, derivative 5i showed stronger scavenging effect at a maximum initial concentration of 200 μM. Among the hydrazine hydrazone derivatives, the introduction of electron-withdrawing groups on the aromatic ring would be beneficial to improve the scavenging capacity (5c-5f vs. 5a, 5g).

[0150] Table 4 Derivatives on ABTS + Radical scavenging rate

[0151]

[0152] Test Example 5: Total reducing capacity determination

[0153] 1. Experimental principle

[0154] When there is a substance with reducing capacity in the system, Fe 3+ can be reduced to Fe 2+ , and then Fe 2+ can generate a purple red substance with phenanthroline, and has a maximum absorption peak at a wavelength of 510 nm. Conversely, Fe 3+ and phenanthroline produce colorless substances, and there is no maximum characteristic peak at this wavelength. Therefore, the absorbance value is closely related to the reducing capacity of the sample, and the larger the OD value, the stronger the reducing capacity of the sample.

[0155] 2. Experimental method

[0156] The total reducing capacity of the compound was detected by the phenanthroline-Fe 2+ reduction method, and the experiment was set up into 3 groups: sample group, control group and blank group.

[0157] Sample group: 180 μL of determination application liquid (2 mM phenanthroline solution mixed with 2 mM FeCl3 solution in equal volume) and 20 μL of sample to be tested (200 μM) were sequentially added to the 96-well plate, and the total reaction volume was 200 μL.

[0158] Control group: The difference is that 180 μL of control application liquid (2 mM phenanthroline solution mixed with 2 mM FeSO4 solution in equal volume) is used instead of the determination application liquid.

[0159] Blank group: the difference is that 200 μL DMSO solution is added.

[0160] After the addition, the sample is placed at room temperature in the dark for 30 min, then shaken and mixed, and the absorbance value at 510 nm is measured by an enzyme marker.

[0161] 3. Experimental results and analysis

[0162] The total reducing capacity of the sample detected by the present application is shown in Table 5. At the maximum initial concentration of 200 μM, the derivative 5c has stronger total reducing capacity (OD = 0.2673) compared with the derivatives 5a, 5b and 5d-5o, but there is still a significant gap in the total reducing capacity with Vc (OD = 0.3916). Overall, the acylhydrazones (OD = 0.1920-0.2673) have better total reducing capacity than the sulfonhydrazones derivatives (OD = 0.1408-0.1860) (5a-5h vs. 5i-5o).

[0163] Table 5 Total reducing capacity of the derivatives

[0164]

[0165] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A coumarilic hydrazone compound, characterized by, The chemical structure of the compound is shown as follows: ； wherein R is .

2. Use of the coumarilic hydrazone compound according to claim 1 in the preparation of a medicine for scavenging superoxide anion.

3. Use according to claim 2, characterized in that: The dosage form of the medicine is powder, granule, tablet, capsule, pill or injection.

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