Gallic acid derivatives containing urea-like structures, synthetic methods and uses thereof

Abstract

The application is a gallic acid derivative containing a urea structure. The application also relates to a synthesis method of the derivative, which specifically comprises the following steps: first, preparing 3,4,5-trihydroxybenzhydrazide from gallic acid, and then reacting with a substituted isocyanate to prepare N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide compounds. The raw material of the application is easy to obtain, the reaction condition is mild, the synthesis method is simple, safe, small in environmental pollution and convenient in post-treatment. The synthesized compounds have inhibitory effects on Vibrio harveyi and Vibrio parahaemolyticus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical preparation, in particular to gallic acid derivatives containing urea structure and the preparation method and the use for inhibiting marine vibrio. BACKGROUND

[0002] With the continuous development of aquaculture, the disease problem is getting worse. Among them, vibrio disease as one of the earliest discovered and most serious water-borne infectious diseases has gradually become the focus of attention. Marine vibrio is a kind of short heterotrophic gram-negative bacteria, facultative anaerobic, widely distributed in seawater and marine animals, many of which are halophilic, motile and oxidase positive. Among marine vibrio, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus and Vibrio anguillarum have pathogenicity, which not only causes infection and death of cultured fish and shrimp, but also causes cholera, gastroenteritis and inflammation and other diseases in humans. Therefore, the demand for new antibacterial drugs is increasing in seawater aquaculture production, and finding new lead compounds or compound skeletons to modify, simplify and develop new aquatic antibacterial drugs has become a hot spot in current aquatic disease prevention and control research.

[0003] Gallnut is one of traditional Chinese medicinal materials in China, which has the effects of astringing lung and reducing fire, astringing sweat, stopping bleeding, etc. It is also widely used in aquaculture. Many studies have shown that Chinese herbal medicines such as gallnut have good bacteriostatic and bactericidal effects on water-borne pathogens such as Vibrio harveyi, Vibrio anguillarum and Vibrio parahaemolyticus. Gallic acid is the main active ingredient of gallnut, which has rich biological activities, including antioxidant, anticancer, antibacterial, antiviral, anti-inflammatory, antidiabetic, etc. It has a wide inhibitory effect on gram-negative and positive bacteria, and has good medical value.

[0004] Urea compounds are important chemicals, which are widely used in herbicides, insecticides, plant growth regulators, gasoline antioxidants and preservatives, etc. At the same time, they are also important intermediates for synthesizing carbamates. Many studies have shown that compounds containing urea structure have obvious effect on antibacterial. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a new gallic acid derivative containing urea structure with higher biological activity and better inhibitory effect on marine vibrio, aiming at the deficiencies of the prior art.

[0006] Another technical problem to be solved by the present application is to provide a method for rapidly and efficiently synthesizing gallic acid derivatives containing urea structure.

[0007] Another object of the present application is to provide the use of the gallic acid derivative with urea structure in inhibiting Vibrio hollisae and Vibrio parahaemolyticus.

[0008] To solve the above technical problems, the present application provides a gallic acid derivative with urea structure, whose structural formula is shown in the following formula:

[0009]

[0010] R is 4-CH3-, 3-CH3-, 2-Cl-, 4-F-, 4-OCH3-, 3-Cl- or 4-Cl-.

[0011] The technical problems to be solved by the present application can also be further realized by the following technical solutions. The present application further discloses a synthesis method of the gallic acid derivative with urea structure as described in the above technical solutions, whose steps are as follows:

[0012] (1) Gallic acid, H2SO4 and anhydrous ethanol are first reacted to generate ethyl 3,4,5-trihydroxybenzoate;

[0013] (2) Ethyl 3,4,5-trihydroxybenzoate is reacted with hydrazine hydrate to obtain 3,4,5-trihydroxybenzohydrazide;

[0014] 3,4,5-trihydroxybenzohydrazide is reacted with substituted isocyanate to obtain N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide; the substituent of the substituted isocyanate is selected from 4-CH3-, 3-CH3-, 2-Cl-, 4-F-, 4-OCH3-, 3-Cl-, 4-Cl-.

[0015] The synthesis method described above has the following further preferred technical solutions:

[0016] 1. In step (1), the reaction is carried out in anhydrous ethanol as a solvent, H2SO4 is used as a catalyst, the pH is 1-5, the reaction temperature is 40-60°C, and the reaction time is 20-22h.

[0017] 2. In step (2), the reaction solvent is anhydrous ethanol, hydrazine hydrate is used in excess, the molar ratio of ethyl 3,4,5-trihydroxybenzoate to hydrazine hydrate is (1-1.5):(7-10), the reaction temperature is 20-30°C, and the reaction time is 34-36h.

[0018] 3. In step (3), the reaction is carried out in acetone as a solvent, the molar ratio of 3,4,5-trihydroxybenzohydrazide to substituted benzoyl isothiocyanate is 1:(1-2), the reaction temperature is 50-70°C, and the reaction time is 6-8h.

[0019] 4. In step (1): After the reaction is completed, water is added to the reaction solution, the pH is adjusted with NaHCO3, ethyl acetate is extracted, the organic phase is dried and concentrated to obtain ethyl 3,4,5-trihydroxybenzoate.

[0020] 5. In step (2): After the reaction is complete, a large amount of solid is precipitated. Anhydrous ethanol is added, the mixture is filtered, and washed three times with water to obtain a white solid 3,4,5-trihydroxybenzoylhydrazine.

[0021] 6. In step (3): After the reaction is complete, acetone is removed by rotary evaporation, and a solid is precipitated. The solid is extracted with ethyl acetate, and the aqueous phase is extracted with ethyl acetate. The organic phase is combined, dried and concentrated to obtain N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide compounds.

[0022] The present invention also discloses the use of the gallic acid derivative containing urea structure or the gallic acid derivative containing urea structure synthesized by any of the above synthesis methods, wherein the use is that the gallic acid derivative containing urea structure is used in the preparation of antibacterial drugs, and the bacteria inhibited are Vibrio harveyi and Vibrio parahaemolyticus of the marine Vibrio species.

[0023] The following is the route of the synthesis method of the present invention:

[0024]

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. This invention provides a gallic acid derivative containing a urea structure that exhibits antibacterial activity against Vibrio marineis. It possesses higher biological activity and demonstrates better inhibitory effects against Vibrio marineis.

[0027] 2. The synthesis method described in this invention is simple to operate, safe, and has mild reaction conditions; the raw materials are readily available, the reaction adopts traditional temperature control, the experimental steps are simple, and the post-processing is convenient, thus broadening the scope of application. Detailed Implementation

[0028] The specific technical solutions of the present invention are further described below to provide a better understanding of the invention, without constituting a limitation on its rights.

[0029] Example 1: A method for synthesizing gallic acid derivatives containing urea structures, the steps of which are as follows:

[0030] (1) Preparation of 3,4,5-trihydroxybenzoylhydrazine: Gallic acid, H2SO4 and anhydrous ethanol were reacted to generate ethyl 3,4,5-trihydroxybenzoate. Anhydrous ethanol was used as solvent, H2SO4 was used as catalyst, pH was 2, reaction temperature was 50℃ and reaction time was 20h.

[0031] 3,4,5-trihydroxybenzoic acid ethyl ester was reacted with hydrazine hydrate to obtain 3,4,5-trihydroxybenzohydrazide; the reaction solvent was anhydrous ethanol, hydrazine hydrate was in excess, the molar ratio of 3,4,5-trihydroxybenzoic acid ethyl ester to hydrazine hydrate was 1:9, the reaction temperature was 25°C, and the reaction time was 35h.

[0032] (2) Preparation of N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide compounds: 3,4,5-trihydroxybenzohydrazide was reacted with substituted isocyanate to obtain N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide compounds; the reaction solvent was acetone, the molar ratio of 3,4,5-trihydroxybenzohydrazide to substituted isocyanate was 1:1.7, the reaction temperature was 55°C, and the reaction time was 6h. The substituent of the substituted isocyanate was selected from 4-CH3-, 3-CH3-, 2-Cl-, 4-F-, 4-OCH3-, 3-Cl-, and 4-Cl-.

[0033] Example 2, a synthesis method of gallic acid derivatives containing a urea structure, the steps of which are as follows:

[0034] (1) Preparation of 3,4,5-trihydroxybenzohydrazide: gallic acid, H2SO4, and anhydrous ethanol were first reacted to obtain 3,4,5-trihydroxybenzoic acid ethyl ester; the reaction solvent was anhydrous ethanol, H2SO4 was used as a catalyst, the pH was 3, the reaction temperature was 50°C, and the reaction time was 20h.

[0035] 3,4,5-trihydroxybenzoic acid ethyl ester was reacted with hydrazine hydrate to obtain 3,4,5-trihydroxybenzohydrazide; the reaction solvent was anhydrous ethanol, hydrazine hydrate was in excess, the molar ratio of 3,4,5-trihydroxybenzoic acid ethyl ester to hydrazine hydrate was 1:9, the reaction temperature was 25°C, and the reaction time was 35h.

[0036] (2) Preparation of N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide compounds: 3,4,5-trihydroxybenzohydrazide was reacted with substituted isocyanate to obtain N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide compounds; the reaction solvent was acetone, the molar ratio of 3,4,5-trihydroxybenzohydrazide to substituted isocyanate was 1:1.7, the reaction temperature was 55°C, and the reaction time was 6h. The substituent of the substituted isocyanate was selected from 4-CH3-, 3-CH3-, 2-Cl-, 4-F-, 4-OCH3-, 3-Cl-, and 4-Cl-.

[0037] Example 3, a synthesis method of gallic acid derivatives containing a urea structure, the steps of which are as follows:

[0038] (1) Preparation of 3,4,5-trihydroxybenzohydrazide: gallic acid, H2SO4 and anhydrous ethanol were reacted to form 3,4,5-trihydroxybenzoic acid ethyl ester; the reaction was carried out in anhydrous ethanol as solvent, H2SO4 as catalyst, pH 4, reaction temperature 50°C, reaction time 22h.

[0039] 3,4,5-trihydroxybenzoic acid ethyl ester was reacted with hydrazine hydrate to obtain 3,4,5-trihydroxybenzohydrazide; the reaction solvent was anhydrous ethanol, hydrazine hydrate was in excess, the molar ratio of 3,4,5-trihydroxybenzoic acid ethyl ester to hydrazine hydrate was 1:9.5, reaction temperature was 27°C, reaction time was 36h.

[0040] (2) Preparation of N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide: 3,4,5-trihydroxybenzohydrazide was reacted with substituted isocyanate to obtain N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide; the reaction was carried out in acetone as solvent, the molar ratio of 3,4,5-trihydroxybenzohydrazide to substituted isocyanate was 1:1.3, reaction temperature was 65°C, reaction time was 7h. The substituent of substituted isocyanate was selected from 4-CH3-, 3-CH3-, 2-Cl-, 4-F-, 4-OCH3-, 3-Cl-, 4-Cl-.

[0041] Example 4, synthesis test of gallic acid derivatives containing urea structure:

[0042] In a 50mL round bottom flask, 0.19g of compound III (the code of the compound is consistent with the above reaction scheme, the same below), 0.35g of compound IV, i.e. 1-isocyanide-4-toluene and 15mL of acetone were sequentially added, and the reaction was carried out at 50°C for 6h. After the reaction, the solution was cooled to room temperature, the solvent was spin-dried, ethyl acetate (3x10mL) was added for extraction, the water phase was combined and extracted with ethyl acetate (3x30mL), the organic phase was combined and spin-dried to obtain compound V, i.e. N-(p-tolyl)-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide, white solid, yield 75%, m.p. 238-239°C; IR (KBr), v / cm -1 : 3429, 3294, 2985, 2025, 1666, 1613, 1551, 1514, 1442, 1396, 1352, 1206, 872; 1H NMR (500 MHz, DMSO) δ 9.85 (s, 1H, NH), 9.00 (s, 3H, OH), 8.65 (s, 1H, NH), 7.92 (s, 1H, NH), 7.33 (d, J = 8.0 Hz, 2H, Ar-R), 7.04 (d, J = 7.9 Hz, 2H, Ar-R), 6.90 (s, 2H, Ar-R), 2.21 (s, 3H, CH3); HRMS (ESI): m / z [M+H]- calcd for: C15H14N3O5-: 316.0939; Found: 316.0937. 15 H 14 N3O5 - :316.0939;Found:316.0937.

[0043] Example 5, Synthesis of Gallic Acid Derivatives Containing Urea Structure, Synthesis Test Two:

[0044] Into a 50 mL round bottom flask was added compound III (the code of the compound is consistent with the previous reaction scheme, the same below) 0.20 g, compound IV, i.e. 1-isocyanyl-3-methylbenzene 0.30 g and 15 mL acetone, and reacted at 60 °C for 6 h. After the reaction, it was cooled to room temperature, the solvent was spin-dried, extracted with ethyl acetate (3 x 10 mL), the aqueous phase was extracted with ethyl acetate (3 x 30 mL), the organic phase was combined and spin-dried to obtain compound V, i.e. N-(m-tolyl)-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide, white solid, yield 77%, m.p. 195-197 °C; IR (KBr), v / cm -1 :3727, 3516, 3440, 3258, 2974, 1700, 1654, 1596, 1560, 1493, 1359, 1224, 874; 1 H NMR (500 MHz, DMSO) δ 9.84 (s, 1H, NH), 9.08 (s, 2H, OH), 8.74 (s, 1H, OH), 8.66 (s, 1H, NH), 7.94 (s, 1H, NH), 7.28-7.23 (m, 2H, Ar-R), 7.11 (t, J = 7.8 Hz, 1H, Ar-R), 6.89 (s, 2H, Ar-R), 6.75 (d, J = 7.4 Hz, 1H, Ar-R), 2.24 (s, 3H, CH3); HRMS (ESI): m / z [M+H]- calcd for: C15H14N3O5-: 316.0939; Found: 316.0939.

[0045] Example 6, Synthesis of Gallic Acid Derivatives Containing Urea Structure, Synthesis Test Three: The synthesis method is referred to Examples 4 and 5, and the substituent structure of the compound, reaction time and product yield are shown in the following table:

[0046]

[0047] Example 7, Anti-bacterial activity test of gallic acid derivatives with urea structure:

[0048] The anti-bacterial activity test substance is the synthesized target compound gallic acid derivative with urea structure; the bacterial strain used in the anti-bacterial activity test is Vibrio harveyi and Vibrio parahaemolyticus.

[0049] The anti-bacterial activity of the test substance was determined by the Oxford cup method, which is a drug diffusion method. The anti-bacterial compound diffuses through the medium, killing or inhibiting the bacteria around the drug, thereby forming an inhibition zone. The size of the inhibition zone reflects the degree of inhibition of the test substance on the indicator bacteria. For compounds with good activity, the minimum inhibitory concentration (MIC) was further determined.

[0050] Anti-bacterial activity determination of the test substance: Under sterile conditions, pour about 20 mL of sterilized beef extract peptone agar medium (for Vibrio harveyi and Vibrio parahaemolyticus culture) into a plate, add 200 μL of bacterial suspension after solidification, evenly spread with a spreader, and stand for 10 min. Place the Oxford cups vertically and evenly on the medium, add 200 μL of the test sample to the cups, perform 3 parallel experiments for each sample, and then move to a 37°C constant temperature incubator for incubation for 18 h. Then measure the diameter of the inhibition zone with an electronic digital caliper. The average value of three measurements was taken as the result.

[0051] The inhibition zone diameters of the compounds against Vibrio harveyi and Vibrio parahaemolyticus at a concentration of 1 mg / mL of the test solution are expressed as the average value ± standard deviation, n = 3.

[0052] The compounds in this series were prepared into a 1 mg / mL solution with methanol, and their MICs were determined. The specific test results are shown in the following table:

[0053] Table 1 Test results of the in vitro inhibition of Vibrio harveyi by target compounds

[0054]

[0055]

[0056] Note: The diameter of the Oxford cup is 7.80 mm.

[0057] As shown in Table 1, compared with the positive drug streptomycin sulfate, the inhibitory activity of compound 6 against Vibrio harveyi is better than that of the positive drug, the inhibitory activity of compounds 1 and 7 against Vibrio harveyi is comparable to that of the positive drug, and the inhibitory effect of the seven synthesized compounds against Vibrio harveyi is better than that of the raw material gallic acid.

[0058] Table 2 Test results of the in vitro inhibition of Vibrio parahaemolyticus by target compounds

[0059]

[0060]

[0061] Note: The diameter of the Oxford cup is 7.80 mm.

[0062] As shown by the results in Table 2, compared with the positive drug streptomycin sulfate, the inhibitory activities of compounds 6 and 7 on Vibrio cholerae are equivalent to that of the positive drug, and the inhibitory effects of 4, 5, 6 and 7 on Vibrio cholerae are better than that of the raw material gallic acid.

[0063] As shown by the results in Table 1 and Table 2, the gallic acid derivatives synthesized by the method of the present application have different degrees of inhibitory effects on Vibrio marinus, and the inhibitory effects of the compound 6 with a 3-Cl substituent on Vibrio harveyi and Vibrio parahaemolyticus are the best, with MICs of 0.0078 mg / mL and 0.0313 mg / mL, respectively.

[0064] In summary, the method of the present application is simple and safe, has high economic efficiency, wide application range, and simple post-processing, and is a fast and effective synthesis method; the raw material is easy to obtain and has a low price. The gallic acid derivatives synthesized by the method of the present application have different degrees of inhibitory effects on Vibrio harveyi and Vibrio parahaemolyticus, and are basically better than the raw material gallic acid, so the compounds have great implementation value and potential social and economic value.

[0065] The above description is only an embodiment of the present application, and does not limit the patent scope of the present application, and any changes, modifications, replacements and variations made according to the present application are also included in the patent protection scope of the present application.

Claims

1. A gallic acid derivative containing a urea-like structure, characterized by, The structural formula is as follows: ； In the formula, R is selected from 4-CH3-, 3-CH3-, 2-Cl-, 4-F-, 4-OCH3-.

2. The method for synthesizing the gallic acid derivative containing the urea structure according to claim 1, characterized by, The steps are as follows: (1) Gallic acid, H2SO4 and anhydrous ethanol are reacted to generate 3,4,5-trihydroxybenzoic acid ethyl ester; (2) 3,4,5-trihydroxybenzoic acid ethyl ester is reacted with hydrazine hydrate to obtain 3,4,5-trihydroxybenzohydrazide; (3) 3,4,5-trihydroxybenzohydrazide is reacted with substituted isocyanate to obtain N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide compound; the substituent of the substituted isocyanate is selected from 4-CH3-, 3-CH3-, 2-Cl-, 4-F-, 4-OCH3-.

3. The method of synthesis of claim 2, wherein: In step (1), the reaction pH is 1-5, the reaction temperature is 40-60°C, and the reaction time is 20-22 h.

4. The method of synthesis of claim 2, wherein: In step (2), the reaction solvent is anhydrous ethanol, hydrazine hydrate is excessive, the molar ratio of 3,4,5-trihydroxybenzoic acid ethyl ester to hydrazine hydrate is (1-1.5):(7-10), the reaction temperature is 20-30°C, and the reaction time is 34-36 h.

5. The method of synthesis of claim 2, wherein: In step (3), the reaction is carried out in acetone as the solvent, the molar ratio of 3,4,5-trihydroxybenzohydrazide to substituted isocyanate is 1:(1-2), the reaction temperature is 40-60°C, and the reaction time is 6-8 h.

6. The method of synthesis of claim 2, wherein: In step (1), after the reaction is completed, water is added to the reaction solution, NaHCO3 is used to adjust the pH, ethyl acetate is used for extraction, the organic phase is dried, concentrated, and 3,4,5-trihydroxybenzoic acid ethyl ester is obtained.

7. The method of synthesis of claim 2, wherein: In step (2), after the reaction is completed, a large amount of solid is precipitated, anhydrous ethanol is added, filtration is performed, and the white solid 3,4,5-trihydroxybenzohydrazide is obtained after water washing three times.

8. The method of synthesis as claimed in claim 2, wherein: In step (3), after the reaction is completed, acetone is removed by rotary evaporation, solid is precipitated, the solid is dissolved in ethyl acetate, water is added for extraction, the water phase is combined, and the organic phase is dried and concentrated to obtain N-phenyl-2-(3,4,5-trihydroxybenzoyl)hydrazine-1-carboxamide compound.

9. Use of the urea-containing structural gallic acid derivative of claim 1 or of the urea- containing structural gallic acid derivative synthesized by the method of any one of claims 2 to 8, characterized in that: The use is the use of gallic acid derivatives containing a urea structure in the preparation of antibacterial drugs, and the bacteria inhibited are Vibrio harveyi and Vibrio parahaemolyticus in marine Vibrio.

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