Coumarin hydrazone derivative with antioxidant activity and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to coumarin hydrazone derivatives with antioxidant activity and application thereof. The chemical structural formula of the coumarin derivative is as shown in formula (I), wherein R is aromatic rings containing different substituent groups. An in-vitro anti-oxidation experiment result shows that most of the derivatives have certain scavenging capacity on free radicals such as DPPH, ABTS, superoxide anions and hydroxyl and have certain reducing capacity. It is worthy of noting that the scavenging effect of the derivatives 5a-5d, 5i-5k, 5m and 5n on superoxide anion free radicals is superior to or equivalent to that of vitamin C. The method for preparing the coumarin hydrazone derivative has the advantages of simplicity in operation, convenience in post-treatment, high product separation yield and the like. The coumarin compound can be used for preparing an antioxidant. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method of a coumarin analogue and its use in antioxidant activity. Background Art

[0002] In the normal physiological metabolic process, the body will produce highly active substances such as ions, molecules, reactive oxygen species (ROS), carbon (RCS), nitrogen (RNS), and sulfur (RSS) that carry unpaired electrons. When the body is stimulated, too many highly active substances will be produced in the body, leading to an imbalance with antioxidants, which in turn causes a state of stress. This oxidative stress behavior can cause biologically active macromolecules (such as nucleic acids, lipids, proteins, etc.) to be oxidatively modified, resulting in the loss of their original structure and function, and then 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 diseases, respiratory system diseases, and neurodegenerative diseases.

[0003] Finding new antioxidants that remove free radicals in the body from natural products has become an inevitable trend in the development of modern medicine and health care industries. Coumarin compounds are widely found in many plants such as Leguminosae, Asteraceae and Orchidaceae, and have significant biological activity in nature. In recent years, a number of studies have found that coumarin derivatives have shown great potential in free radical scavenging, membrane protection and anti-oxidation. In previous reports, many lead compounds with excellent antioxidant activity were obtained by transforming and modifying the C-3, C-4 and C-7 positions on the coumarin skeleton. However, there are only a few reports on the structural derivatization research on its C-6 position, and the antioxidant effect of such compounds has not been explored, and the different positions of the substituents will also have different degrees of influence on the antioxidant activity results of the derivatives. At the same time, the applicant consulted the literature and found that the conjugated system that increases the coumarin structure can improve its antioxidant activity. Based on the above research background, the present invention takes the coumarin skeleton as the research object, introduces the "-C=N-" group to increase the conjugated system of coumarin, synthesizes 15 coumarin analogs, and conducts systematic screening and evaluation of free radical scavenging ability. Summary of the invention

[0004] One of the purposes of the present invention is to provide a method for preparing coumarin analogs, so as to further enrich the structural types of coumarin compounds; another purpose is to provide the application of the coumarin analogs in anti-oxidation, including other uses of the analogs.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The chemical structure of coumarin hydrazone compounds is shown in formula (I):

[0007]

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

[0009]

[0010] The synthesis route is as follows:

[0011]

[0012] The preparation method of coumarin derivatives comprises the following steps:

[0013] (1) Synthesis of 6-methylcoumarin: 5-methyl salicylaldehyde and sodium acetate were placed in a round-bottom flask, degassed with Ar for three times, and N,N-dimethylformamide solution was added. The mixture was stirred at room temperature for 30 min, and acetic anhydride was added dropwise in small amounts several times. The mixture was heated to 170°C and stirred for 6 h. The mixture was tested by TLC. After the raw materials were completely removed, the reaction system was cooled to room temperature and extracted with DCM (3×10 mL). The organic layer was collected and washed with distilled water and saturated brine respectively, and dried over anhydrous Na2SO4. The crude product was purified by flash chromatography (petroleum ether and EtOAc as eluents), and concentrated under reduced pressure to obtain the target compound 2. The molar ratio of 5-methyl salicylaldehyde, acetic anhydride and sodium acetate was 1:3:2.5.

[0014] (2) Synthesis of 6-bromomethylcoumarin: Weigh 6-methylcoumarin (target compound 2) and place it in a round-bottom flask. Degas with Ar for three times, add carbon tetrachloride solution, and heat to 85°C. At the same time, slowly drop acetonitrile solution containing N-bromosuccinimide and azobisisobutyronitrile, and react overnight. TLC thin layer detection, after the raw materials react completely, cool the reaction system to room temperature. Extract with DCM (3×10mL), collect the DCM layer, dry with anhydrous Na2SO4, recrystallize the crude product with n-hexane and EtOAc (1:1), filter, and vacuum dry the filter cake to obtain the target compound 3. The molar ratio of 6-methylcoumarin, N-bromosuccinimide, and azobisisobutyronitrile is 1:1:0.2.

[0015] (3) Synthesis of 6-formyl coumarin: Dissolve 6-bromomethyl coumarin (target compound 3) in acetic acid and distilled water, stir evenly, slowly add urotropine, and raise the temperature to 100°C for 5 hours. Detect by TLC thin layer until the reaction is complete. The generated compound can make DNP (2,4-dinitrophenylhydrazine) appear yellow. Cool to room temperature, extract with EtOAc (3×10mL), collect the organic layer, wash with distilled water and saturated brine respectively, dry with anhydrous Na2SO4, recrystallize the crude product with a mixed solvent of n-hexane and EtOAc, filter, and vacuum dry the filter cake to obtain the target compound 4. The molar ratio of 6-bromomethyl coumarin to urotropine is 1:2.

[0016] (4) Synthesis of compounds 5a-5o: Dissolve the hydrazine compound in anhydrous ethanol and acetic acid, stir at room temperature for 30 minutes, slowly drop 6-formyl coumarin dissolved in ethanol, raise the temperature to 85°C, and continue the reaction for 8 hours. TLC thin layer detection until the reaction is complete. Filter by suction to obtain a solid crude product, which is recrystallized from a mixed solvent of n-hexane and ethanol, and the pure product obtained by recrystallization is further filtered by suction. The filter cake is vacuum dried to obtain the target compound 5a-5o. The molar ratio of the 6-formyl coumarin, hydrazine compound, and acetic acid is 1:1.5-1.8:0.5.

[0017] The application of coumarin derivatives in the preparation of antioxidant activities, including their scavenging effects on free radicals such as DPPH, ABTS, superoxide anion and hydroxyl, and their effects on reducing ability.

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

[0019]

[0020] Preferably, the use of coumarin hydrazone compounds in the preparation of drugs for scavenging DPPH free radicals, the structural formula of the coumarin hydrazone compounds is:

[0021]

[0022] Preferably, the use of coumarin hydrazone compounds in the preparation of drugs for scavenging hydroxyl radicals has a structural formula of:

[0023] Preferably, the use of coumarin hydrazone compounds in the preparation of drugs for scavenging ABTS free radicals has a structural formula of:

[0024] Among them, the structure of compound 5i is novel and is reported for the first time in the present invention. Although 5c and 5d are known compounds, their good antioxidant activity is discovered for the first time in the present invention.

[0025] The beneficial effect obtained by the present invention is that the chemical structure of coumarin analogs and the corresponding synthesis route are provided. More importantly, their scavenging ability to free radicals such as DPPH, ABTS, superoxide anion and hydroxyl is detected by an ELISA instrument. At the same time, a lead compound with a significant free radical scavenging effect is screened out, indicating that coumarin skeleton analogs have the potential to be developed into antioxidants. DETAILED DESCRIPTION

[0026] The present invention is described in detail below by specific examples. It should be understood that the following examples are only for explanation and illustration and do not limit the scope of the present invention in any form. In the following embodiments, biochemical reagents not specifically described are all conventional reagents in the art and 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-formyl coumarin are as follows:

[0030] (1) Weigh 1.00 g (7.34 mmol) of 5-methyl salicylaldehyde and 1.51 g (18.36 mmol) of sodium acetate into a round-bottom flask, degas with Ar for three times, add 15 mL of DMF solution, stir at room temperature for 30 min, heat the system to 170°C, slowly dropwise add 1.39 mL of acetic anhydride solution (22.02 mmol), and continue stirring for 6 h. After TLC thin layer detection, after the reaction of the raw materials is complete, cool the reaction system to room temperature, extract with DCM (3×10 mL), wash with distilled water and saturated NaCl solution, dry with anhydrous Na2SO4, and purify the crude product by flash chromatography (PE and EtOAc as eluents), and concentrate under reduced pressure to obtain the target compound 2.

[0031] (2) Weigh 1.00 g (6.24 mmol) of 6-methylcoumarin into a 50 mL round-bottom flask, degas with Ar for three times, add 15 mL of CCl4 solution, raise the reaction temperature to 85°C, slowly drop 1.11 g (6.24 mmol) of N-bromosuccinimide and 0.21 g (1.25 mmol) of azobisisobutyronitrile dissolved in acetonitrile (15 mL), and continue stirring for 6 h. Extract with DCM (3×10 mL), wash with distilled water and saturated brine, combine the organic layers, and dry over anhydrous Na2SO4. The crude product is recrystallized with n-hexane and EtOAc (1:1), filtered, and the filter cake is dried under vacuum to obtain the target compound 3, i.e., 6-bromomethylcoumarin.

[0032] (3) Weigh 1.00 g (4.18 mmol) of 6-bromomethylcoumarin into a 50 mL round-bottom flask, add 15 mL of acetic acid and water respectively, stir evenly, add 1.17 g (8.31 mmol) of urotropine in small amounts several times, raise the temperature to 100 °C and react for 5 h. Detect by TLC thin layer until the reaction is complete. The generated compound can make DNP (2,4-dinitrophenylhydrazine) appear yellow. Cool to room temperature, extract with EtOAc (3×10 mL), wash with distilled water and saturated brine, combine the organic layers, dry the organic layers with anhydrous Na2SO4, filter, evaporate to dryness under reduced pressure, recrystallize the crude product with a mixed solvent of n-hexane and EtOAc, filter, and vacuum dry the filter cake to obtain the target compound 4, i.e., 6-formylcoumarin.

[0033] Example 1

[0034] 5a:

[0035] Weigh p-toluoyl hydrazide (0.69 mmol) and place it in a 20 mL Shrek tube, add 2 mL of anhydrous ethanol and acetic acid (0.23 mmol) respectively. After stirring at room temperature for 30 min, slowly drop 6-formyl coumarin (0.46 mmol) dissolved in ethanol (0.5 mL), raise the temperature to 85 ° C, and continue to react for 8 h. TLC thin layer detection, until the reaction is complete, filter and obtain a solid crude product. The crude product is recrystallized from a mixed solvent of n-hexane and ethanol, and the pure product obtained by recrystallization is further filtered. The filter cake is vacuum dried 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-benzopyran-6-yl)methylene)benzohydrazide (5a): 1H NMR (300MHz, DMSO-d6): δ (ppm) 11.89 (s, 1H), 8.49 (s, 1H), 8.18 (d, J = 9.6Hz, 1H), 8.08 (d, J = 2.0Hz, 1H), 7.97 (dd, J = 8. 6,2.0Hz,1H),7.84(d,J=7.9Hz,2H),7.49(d,J=8.4Hz,1H),7.34(d,J=7.9Hz,2H),6.56(d,J=9.6Hz,1H),2.38(s,3H). 13 C NMR (75MHz, 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] The synthesis method is the same as that of Example 1, except that p-toluoylhydrazide is replaced by p-methoxybenzoylhydrazide to obtain the target product 5b, which is a yellow solid with a yield of 86.9%.

[0040] (E)-4-Methoxy-N'-((2-oxo-2H-benzopyran-6-yl)methylene)benzohydrazide (5b): 1 H NMR (300MHz, DMSO-d6): δ (ppm) 11.85 (s, 1H), 8.49 (s, 1H), 8.19 (d, J = 9.6Hz, 1H), 8.09 (s, 1H), 7.97 (d, J = 9.0H z,1H),7.93(d,J=8.7Hz,2H),7.49(d,J=8.7Hz,1H),7.08(d,J=8.7Hz,2H),6.57(d,J=9.6Hz,1H),3.84(s,3H). 13 C NMR (75MHz, 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 synthesis method is the same as that of Example 1, except that p-toluoylhydrazide is replaced by p-nitrobenzoylhydrazide to obtain the target product 5c, which is a yellow solid with a yield of 93.5%.

[0044] (E)-4-Nitro-N'-((2-oxo-2H-benzopyran-6-yl)methylene)benzohydrazide (5c): 1 H NMR (300MHz, DMSO-d6): δ (ppm) 12.26 (s, 1H), 8.52 (s, 1H), 8.40 (d, J = 8.9Hz, 2H), 8.20 (d, J = 7.5Hz, 2H), 8.16 (d ,J=6.9Hz,1H),8.12(d,J=2.1Hz,1H),8.00(dd,J=8.7,2.1Hz,1H),7.51(d,J=8.7Hz,1H),6.58(d,J=9.6Hz,1H). 13 C NMR (75MHz, DMSO-d6): δ (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 synthesis method is the same as that of Example 1, except that p-toluohydrazide is replaced by p-bromobenzohydrazide to obtain the target product 5d, which is a white solid with a yield of 97.9%.

[0048] (E)-4-Bromo-N'-((2-oxo-2H-benzopyran-6-yl)methylene)benzohydrazide (5d): 1 H NMR (300MHz, DMSO-d6): δ (ppm) 11.96 (s, 1H), 8.43 (s, 1H), 8.12 (d, J = 9.3Hz, 1H), 8.04 (d, J = 2.0Hz, 1H), 7.91 (d d,J=8.7,2.1Hz,1H),7.82(d,J=9.3Hz,2H),7.70(d,J=8.4Hz,2H),7.43(d,J=8.5Hz,1H),6.50(d,J=9.6Hz,1H). 13C NMR (75MHz, DMSO-d6): δ (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 synthesis method is the same as that of Example 1, except that p-toluohydrazide 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-benzopyran-6-yl)methylene)benzohydrazide (5e): 1 H NMR (300MHz, DMSO-d6): δ (ppm) 11.98 (s, 1H), 8.49 (s, 1H), 8.18 (d, J = 9.6Hz, 1H), 8.09 (d, J = 2.1Hz, 1H), 8.02 (d ,J=5.4Hz,1H),7.97(dd,J=6.9,1.8Hz,2H),7.49(d,J=8.7Hz,1H),7.38(t,J=8.8Hz,2H),6.57(d,J=9.6Hz,1H). 13 C NMR (75MHz, DMSO-d6): δ (ppm) 164,7 (d, J = 248.0Hz), 162.6, 160.1, 154.9, 146.8, 144.6, 131.2, 130. 9,130.8(d,J=7.1Hz,2C),130.3(d,J=2.5Hz),127.6,119.6,117.5,117.3,116.0(d,J=21.8Hz,2C). 19 F NMR (282MHz, DMSO-d6): δ (ppm)-108.2 (brs).

[0053] Example 6

[0054] 5f:

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

[0056] (E)-N'-((2-oxo-2H-benzopyran-6-yl)methylene)-4-(trifluoromethyl)benzohydrazide (5f): 1 HNMR (300MHz, DMSO-d6): δ (ppm) 12.13 (s, 1H), 8.51 (s, 1H), 8.18 (d, J = 9.6Hz, 1H), 8.12 (d, J = 8.1Hz, 2H), 8. 10(s,1H),7.99(dd,J=8.6,2.1Hz,1H),7.92(d,J=8.2Hz,2H),7.49(d,J=8.6Hz,1H),6.56(d,J=9.6Hz,1H). 13 C NMR (75MHz, DMSO-d6): δ (ppm) 162.1, 159.7, 154.5, 147.1, 144.1, 137.2, 131.2 (q, J = 31.4Hz), 130 .6,130.3,128.6(2C),127.2,125.5(q,J=3.5Hz,2C),123.9(q,J=267.3Hz),119.2,117.1,116.9. 19 F NMR (282MHz, DMSO-d6): δ (ppm)-61.4.

[0057] Example 7

[0058] 5g:

[0059] The synthesis method is the same as that of Example 1, except that p-toluohydrazide is replaced by p-trifluoromethoxybenzohydrazide to obtain 5 g of the target product in the form of a white solid with a yield of 96.9%.

[0060] (E)-N'-((2-oxo-2H-benzopyran-6-yl)methylene)-4-(trifluoromethoxy)benzohydrazide (5 g): 1 H NMR (400MHz, DMSO-d6): δ (ppm) 12.05 (s, 1H), 8.49 (s, 1H), 8.18 (d, J = 9.6Hz, 1H), 8.11 (d, J = 2.0Hz, 1H), 8.05 (d ,J=8.4Hz,2H),7.98(dd,J=8.8,2.4Hz,1H),7.55(d,J=8.4Hz,2H),7.50(d,J=8.8Hz,1H),6.57(d,J=9.6Hz,1H). 13C NMR (101MHz, DMSO-d6): δ (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=258.2Hz),119.2,117.1,116.9. 19 F NMR (376MHz, DMSO-d6): δ (ppm)-57.1.

[0061] Example 8

[0062] 5h:

[0063] The synthesis method is the same as that of Example 1, except that p-toluoyl 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-benzopyran-6-yl)methylene)isonicotinylhydrazide (5h): 1 H NMR (300MHz, DMSO-d6): δ (ppm) 12.19 (s, 1H), 8.81 (dd, J = 4.5, 1.8Hz, 2H), 8.51 (s, 1H), 8.19 (d, J = 9.6Hz, 1H), 8.13 (d ,J=2.0Hz,1H),8.00(dd,J=8.7,2.0Hz,1H),7.84(dd,J=4.5,1.5Hz,2H),7.51(d,J=8.4Hz,1H),6.58(d,J=9.6Hz,1H). 13 C NMR (75MHz, DMSO-d6): δ (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 that of Example 1, except that p-toluoylhydrazide is replaced by benzenesulfonylhydrazide, and the molar ratio of the 6-formylcoumarin, sulfonylhydrazide compound, and acetic acid is 1:1.8:0.5. The target product 5i is obtained as a white solid with a yield of 89.4%.

[0068] (E)-N'-((2-oxo-2H-benzopyran-6-yl)methylene)benzenesulfonylhydrazide (5i): 1 H NMR (300MHz, DMSO-d6): δ (ppm) 11.68 (s, 1H), 8.12 (d, J = 9.6Hz, 1H), 7.98 (s, 1H), 7.92 (t, J = 1.2Hz, 2H), 7.90 (d, J = 1.9H) z,1H),7.80(dd,J=8.7,2.1Hz,1H),7.68-7.65(m,1H),7.65-7.59(m,2H),7.42(d,J=8.7Hz,1H),6.54(d,J=9.6Hz,1H). 13 C NMR (75MHz, 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] The synthesis method is the same as that of Example 9, except that benzenesulfonylhydrazide is replaced by p-toluenesulfonylhydrazide to obtain the target product 5j, which is a yellow solid with a yield of 85.0%.

[0072] (E)-4-Methyl-N'-((2-oxo-2H-benzopyran-6-yl)methylene)benzenesulfonylhydrazide (5j): 1 H NMR (400MHz, DMSO-d6): δ (ppm) 11.55 (s, 1H), 8.11 (d, J = 9.6Hz, 1H), 7.96 (s, 1H), 7.91 (d, J = 2.0Hz, 1H), 7.79 (m,1H),7.78(d,J=8.0Hz,2H),7.42(s,1H),7.41(dd,J=8.4,2.8Hz,2H),6.53(d,J=9.6Hz,1H),2.36(s,3H). 13 C NMR (75MHz, 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] Embodiment 11

[0074] 5k:

[0075] The synthesis method is the same as that of Example 9, except that benzenesulfonyl hydrazide is replaced by p-methoxybenzenesulfonyl 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-benzopyran-6-yl)methylene)benzenesulfonylhydrazide (5k): 1 H NMR (400MHz, DMSO-d6): δ (ppm) 11.48 (s, 1H), 8.11 (d, J = 9.6Hz, 1H), 7.96 (s, 1H), 7.91 (d, J = 2.0Hz, 1H), 7.82 (dt, J = 9.2, 2. 6Hz,2H),7.79(dd,J=8.8,2Hz,1H),7.42(d,J=8.4Hz,1H),7.12(dt,J=8.8,2.4Hz,2H),6.53(d,J=9.6Hz,1H),3.81(s,3H). 13 C NMR (101MHz, 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 synthesis method is the same as that of Example 9, except that benzenesulfonylhydrazide is replaced by p-bromobenzenesulfonylhydrazide to obtain the target product 51 as a white solid with a yield of 94.7%.

[0080] (E)-4-Bromo-N'-((2-oxo-2H-benzopyran-6-yl)methylene)benzenesulfonylhydrazide (51): 1 H NMR (400MHz, DMSO-d6): δ (ppm) 11.70 (s, 1H), 8.08 (d, J = 9.7Hz, 1H), 7.96 (s, 1H), 7.89 (d, J = 2.0Hz, 1H), 7.82 (dd, J = 8.7, 2.1Hz,1H),7.80(d,J=3.2Hz,2H),7.79-7.78(m,1H),7.77(d,J=2.0Hz,1H),7.39(d,J=8.6Hz,1H),6.51(d,J=9.6Hz,1H). 13C NMR (101MHz, DMSO-d6): δ (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] Embodiment 13

[0082] 5m:

[0083] The synthesis method is the same as that of Example 9, except that benzenesulfonylhydrazide is replaced by p-nitrobenzenesulfonylhydrazide. The product is a yellow solid with a yield of 90.4%.

[0084] (E)-4-Nitro-N'-((2-oxo-2H-benzopyran-6-yl)methylene)benzenesulfonylhydrazide (5p): 1 H NMR (400MHz, DMSO-d6): δ (ppm) 11.96 (s, 1H), 8.40 (dt, J = 9.2, 2.2Hz, 1H), 8.22 (dt, J = 9.2, 2.6Hz, 1H), 8.13 (dt, J = 9.2, 2.4H z,2H),8.08(d,J=9.6Hz,1H),7.99(s,1H),7.91(d,J=2.1Hz,1H),7.79(m,1H),7.40(d,J=8.6Hz,1H),6.51(d,J=9.6Hz,1H). 13 C NMR (101MHz, DMSO-d6): δ (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] Embodiment 14

[0086] 5n:

[0087] The synthesis method is the same as that of Example 9, except that benzenesulfonylhydrazide is replaced by p-fluorobenzenesulfonylhydrazide. The product is a yellow solid with a yield of 92.5%.

[0088] (E)-4-Fluoro-N'-((2-oxo-2H-benzopyran-6-yl)methylene)benzenesulfonylhydrazide (5n): 1H NMR (400MHz, DMSO-d6): δ (ppm) 11.67 (s, 1H), 8.11 (d, J = 9.6Hz, 1H), 7.99 (s, 1H), 7.96 (tt, J = 8.7, 2.7Hz, 2H), 7.93 (d ,J=2.1Hz,1H),7.81(dd,J=8.4,2.1Hz,1H),7.46(tt,J=8.8,2.6Hz,2H),7.42(d,J=8.4Hz,1H),6.54(d,J=9.6Hz,1H). 13 C NMR (101MHz, DMSO-d6): δ (ppm) 161.5 (d, J = 363.2Hz), 156.5, 154.8, 146.1, 144.1, 135.3 (d, J = 3. 1Hz),130.3(d,J=9.7Hz,2C),130.0,129.9,127.0,119.0,117.1,116.9,116.6(d,J=23.2Hz,2C). 19 F NMR (376MHz, DMSO-d6): δ (ppm)-105.7.

[0089] Embodiment 15

[0090] 5o:

[0091] The synthesis method is the same as that of Example 9, except that benzenesulfonyl hydrazide is replaced by p-trifluoromethylbenzenesulfonyl hydrazide. The product is a white solid with a yield of 97.2%.

[0092] (E)-N'-((2-oxo-2H-benzopyran-6-yl)methylene)-4-(trifluoromethyl)benzenesulfonylhydrazide (5o): 1 HNMR (400MHz, DMSO-d6): δ (ppm) 11.91 (s, 1H), 8.12 (d, J = 5.4Hz, 2H), 8.10 (d, J = 6.7Hz, 2H), 8.03 (s, 1H ),8.01(s,1H),8.01(s,1H),7.93(d,J=2.0Hz,1H),7.81(dd,J=8.4,2.1Hz,1H),6.53(d,J=9.6Hz,1H). 13 C NMR (101MHz, DMSO-d6): δ (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 (376MHz, DMSO-d6): δ (ppm)-61.7.

[0093] Example 16 Evaluation of in vitro antioxidant activity

[0094] According to the hydrazine coumarin compounds described in Examples 1-15, vitamin C (Vc) is used as a positive control, and the antioxidant activity is evaluated using DPPH free radical, hydroxyl free radical, superoxide anion free radical, ABTS free radical scavenging ability and total reducing power as indicators.

[0095] Test Example 1: Determination of DPPH free radical scavenging ability

[0096] 1. Experimental Principle

[0097] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable free radical with a nitrogen atom as the center and three benzene rings around it. The alcohol solution of DPPH· is dark purple, has strong absorption at 517nm, and its concentration is linearly related to the absorbance value. When there is a free radical scavenger in the solution, DPPH· can combine with or replace the free radical scavenger, and the number of DPPH· decreases, resulting in a decrease in absorbance, and the color gradually becomes lighter, that is, the degree of fading is quantitatively related to the number of electrons accepted. Therefore, the scavenging rate of the compound on DPPH· can be calculated based on the absorbance value at this wavelength, so as to evaluate the scavenging of the drug on the DPPH free radical.

[0098] 2. Experimental Methods

[0099] The experimental grouping was set into 3 groups: sample group, control group and blank group.

[0100] Sample group: Add 100 μL of DPPH solution (0.2 mM) to a 96-well plate, and then add 50 μL of 600 μM test sample, respectively. The total volume of each well is 150 μL, so that the maximum final concentration of the sample is 200 μM.

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

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

[0103] After adding the above groupings, the 96-well plate was placed at room temperature and away from light for 30 minutes. After the reaction was completed, the plate was shaken and mixed. The OD value of each well at a wavelength of 517 nm was measured by an ELISA instrument, and the DPPH free radical scavenging rate of the sample to be tested was calculated according to formula (1).

[0104] DPPH free radical scavenging rate (%) = [1-(OD 样品 -OD 对照 ) / OD 空白 ]×100% formula (1)

[0105] 3. Experimental results and analysis

[0106] Table 1 Scavenging rate of derivatives on DPPH free radicals

[0107]

[0108]

[0109] The DPPH method was used to detect the scavenging ability of the derivatives on DPPH free radicals. The results are shown in Table 1. At the maximum concentration of 200 μM, the derivatives obtained by the present invention showed different degrees of scavenging effect on DPPH free radicals (20.96-59.49%), but there was still a large gap compared with the scavenging ability of Vc (93.01%). Among derivatives 5a-5o, derivative 5c showed a moderate scavenging ability (59.49%) on DPPH free radicals. In 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). On the contrary, for 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: Hydroxyl radical scavenging ability determination

[0111] 1. Experimental Principle

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

[0113] 2. Experimental Methods

[0114] The present invention adopts Fe 2+ -H2O2-salicylic acid system was used to detect the scavenging ability of samples on ·OH free radicals. The experiment was divided into 3 groups: sample group, control group and blank group.

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

[0116] Control group: first add 50 μL of 600 μM test sample, then add 50 μL of FeSO4 solution, 50 μL of salicylic acid-ethanol solution and 50 μL of distilled water in sequence.

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

[0118] After adding all the reaction systems, place in a 37°C shaker in the dark and incubate for 30 minutes. Use an enzyme marker to measure the OD value of each well at 510 nm, and calculate the hydroxyl radical scavenging rate of the sample to be tested according to formula (2).

[0119] OH free 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 Scavenging rate of hydroxyl radicals by derivatives

[0122]

[0123] The present invention relies on Fe 2+ -H2O2-salicylic acid system method was used to detect the scavenging effect of coumarin derivatives on ·OH free radicals. As shown in Table 2, the scavenging rates of derivatives 5a-5o on ·OH free radicals ranged from 0.39% to 17.50%, but their scavenging effects were significantly weaker than those of Vc (25.07%). Whether it is an acylhydrazone or sulfonylhydrazone derivative, the introduction of electron-donating groups on the aromatic ring can significantly improve their scavenging ability on hydroxyl free radicals (5b vs. 5c-5f; 5j vs. 5i; 5j vs. 5l-5o).

[0124] Test Example 3: Determination of superoxide anion free radical scavenging ability

[0125] 1. Experimental Principle

[0126] Pyrogallol can undergo self-oxidation under alkaline conditions, releasing O2 - ·, and then an orange-red intermediate product is generated. This intermediate has a strong absorption at 320nm. -· In the presence of scavengers, it can quickly react with O2 - · reaction, thereby preventing the accumulation of intermediate products, resulting in a weakened absorption of the solution at a wavelength of 320nm. That is, the amount of intermediate products is positively correlated with the strength of absorbance. Therefore, the present invention can determine the O2 - · Free radical scavenging effect.

[0127] 2. Experimental Methods

[0128] The present invention utilizes the pyrogallol self-oxidation method to detect the O2 - · Free radical scavenging ability. The experiment was divided into 3 groups: sample group, control group and blank group.

[0129] Sample group: Add 30 μL of the sample to be tested (200 μM) and 150 μL of Tris-HCl solution (50 mM) to the 96-well plate. Place in a shaker at 25°C and shake for 20 min. Add 30 μL of pyrogallol solution (3 mM) to make the total reaction volume 210 μL, and continue shaking for 5 min.

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

[0131] Blank group: The operation method of the sample group was followed, except that 30 μL DMSO solution was used instead of the sample solution to be tested.

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

[0133] O2 - · Free radical scavenging rate (%) = [1-(OD 样品 -OD 对照 ) / OD 空白 ]×100% Formula (3)

[0134] 3. Experimental results and analysis

[0135] The results of the present invention are shown in Table 3. The derivatives 5a-5o have a - · Free radicals showed different degrees of scavenging effects (65.63-97.19%). At the maximum initial concentration of 200 μM, derivatives 5c, 5d and 5i had no significant effects on O2 - The free radical scavenging percentage is between 94.71-97.19%, which is better than or equivalent to Vc (95.67%). For sulfonylhydrazone coumarin compounds, the introduction of electron-withdrawing groups on the aromatic ring will not be conducive to improving the scavenging rate (5l-5o vs. 5k; 5l-5o vs. 5i).

[0136] Table 3 Derivatives for O2 - Free radical scavenging rate

[0137]

[0138] Test Example 4: Determination of ABTS free radical cation scavenging ability

[0139] 1. Experimental Principle

[0140] Under the action of oxidants such as K2S2O8 and H2O2, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) can be oxidized to green or blue-green ABTS + ·, which has the maximum characteristic absorption peak at 734nm or 405nm. When the antioxidant in the system is + When the reaction occurs, the system will turn into colorless ABTS, that is, ABTS in the system + The content of · is linearly related to the absorbance value. Therefore, the present invention determines the activity of the compound to ABTS based on the measured absorbance value. + Free radical scavenging effect.

[0141] 2. Experimental Methods

[0142] The present invention adopts ABTS method to detect the effect of compound on ABTS + The free radical scavenging ability was determined by setting the experiment into three groups: sample group, control group and blank group.

[0143] Sample group: In a 96-well plate, add 20 μL of the sample solution (200 μM) and 200 μL of ABTS working solution (equal volumes of 2.6 mM K2S2O8 solution and 7.4 mM ABTS solution) in sequence, and let stand at room temperature in the dark for 10 min to make the total reaction volume 220 μL.

[0144] The control group was composed of 200 μL of pure water with 20 μL of various test samples added.

[0145] The blank group was 200 μL ABTS + Add 20 μL DMSO to the solution.

[0146] Then, the absorbance value was measured at a wavelength of 734 nm, and the sample's ABTS was calculated according to formula (4): + · Free radical scavenging rate.

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

[0148] 3. Experimental results and analysis

[0149] ABTS + The results of the free radical scavenging experiment are shown in Table 4. The derivatives 5a-5o have no + The free radical scavenging rate was between 4.45% and 42.65%, and its scavenging effect was significantly weaker than that of Vc (70.36%). Compared with other derivatives, derivative 5i showed a stronger scavenging effect at the maximum initial concentration of 200 μM. In hydrazide hydrazone derivatives, the introduction of electron-withdrawing groups on the aromatic ring will help improve the scavenging ability (5c-5f vs.5a,5g).

[0150] Table 4 Derivatives to ABTS + Free radical scavenging rate

[0151]

[0152] Test Example 5: Determination of total reducing capacity

[0153] 1. Experimental Principle

[0154] When there are reducing substances in the system, Fe 3+ Can be reduced to Fe 2+ , followed by Fe 2+ It can react with o-phenanthroline to form a purple-red substance with a maximum absorption peak at a wavelength of 510nm. 3+ It reacts with o-phenanthroline to produce a colorless substance with no maximum characteristic peak at this wavelength. Therefore, the absorbance value is closely related to the reducing ability of the sample. The larger the OD value, the stronger the reducing ability of the sample.

[0155] 2. Experimental Methods

[0156] The present invention adopts o-phenanthroline-Fe 2+ The reduction method detected the total reducing ability of the compound, and the experiment was set up into three groups: sample group, control group and blank group.

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

[0158] Control group: The difference is that 180 μL of control application solution (equal volumes of 2 mM o-phenanthroline solution and 2 mM FeSO4 solution mixed) was used instead of the test application solution.

[0159] Blank group: The difference is that a total of 200 μL DMSO solution was added.

[0160] After the addition, the mixture was allowed to stand at room temperature in the dark for 30 min, then vortexed to mix, and the absorbance at a wavelength of 510 nm was measured using an enzyme-labeled instrument.

[0161] 3. Experimental results and analysis

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

[0163] Table 5 Total reducing capacity of derivatives

[0164]

[0165] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of coumarin hydrazone compounds, characterized in that: The chemical structure of the coumarin hydrazone compound is shown in formula (I): In the formula, R is one of the following structural formulas:

2. Use of the coumarin hydrazone compound according to claim 1 in the preparation of antioxidant drugs.

3. The use of the coumarin hydrazone compound in the preparation of an antioxidant drug according to claim 2, characterized in that: The general chemical structure of coumarin hydrazone compounds is shown in formula (I): R is 4. The use of the coumarin hydrazone compound according to claim 2 in the preparation of an antioxidant drug, characterized in that: Application of coumarin hydrazone compounds in the preparation of drugs for scavenging DPPH free radicals, wherein the structural formula of the coumarin hydrazone compounds is:

5. The use of the coumarin hydrazone compound in the preparation of an antioxidant drug according to claim 2, characterized in that: Application of coumarin hydrazone compounds in the preparation of drugs for scavenging hydroxyl free radicals, the structural formula of coumarin hydrazone compounds is:

6. The use of the coumarin hydrazone compound according to claim 2 in the preparation of an antioxidant drug, characterized in that: Application of coumarin hydrazone compounds in the preparation of drugs for scavenging ABTS free radicals. The structural formula of coumarin hydrazone compounds is:

7. The use of the coumarin hydrazone compound according to claim 2 in the preparation of an antioxidant drug, characterized in that: Application of coumarin hydrazone compounds in the preparation of drugs for scavenging superoxide anions, the structural formula of coumarin hydrazone compounds is:

8. Use of the coumarin hydrazone compound according to any one of claims 2 to 7 in the preparation of an antioxidant drug, characterized in that: The dosage form of the drug is powder, granule, tablet, capsule, pill, solution, suspension or injection.

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